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Meningococcal Disease

Tom Sprong Het onderzoek in dit proefschrift werd mogelijk gemaakt door een beurs van de Nederlandse Organisatie voor Wetenschappelijk Onderzoek, beurs nummer 920-03-176

Colofon Thesis Radboud University Nijmegen Medical Centre, with Summary in Dutch Meningococcal Disease

Printing: Print Partners Ipskamp Cover design and Lay out: Martijn Prins Cover art: Kasper Peter Sprong: ‘impression of meningococci in blue’ Meningococcal Disease

Een wetenschappelijke proeve op het gebied van de Medische Wetenschappen

Proefschrift

ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus, prof. mr. S.C.J.J. Kortmann, volgens het besluit van het college van decanen in het openbaar te verdedigen op woensdag 4 februari 2009 om 15.30 uur precies

door

Tom Sprong

geboren op 20 september 1975 te s´Hertogenbosch Promotor: Prof. dr. J.W.M. van der Meer

Copromotor: Dr. ir. M. van Deuren

Manuscriptcommissie: Prof. dr. R. de Groot Prof. dr. J.G. van der Hoeven Prof. dr. T.W. Kuijpers

Paranimfen: Drs. N. Sprong Drs. W.C.H. Jacobs

Contents

1. Introduction and outline of the thesis 9

2. Contributions of Neisseria meningitidis LPS and non-LPS to proinflammatory cytokine response 15

3. Neisseria meningitidis can induce pro-inflammatory cytokine pro-duction via pathways independent from cD14 and toll-like receptor 4 33

4. Complement activation and complement-dependent inflammation by Neisseria meningitidis are independent of lipopolysaccharide 49

5. Human lipoproteins have divergent neutralizing effects on E. coli LPS, N. meningitidis LPS, and complete gram-negative bacteria. 67

6. N. meningitidis lipid A mutant lipopolysaccharides (LPS) function as LPS antagonists in humans by inhibiting toll-like receptor 4 (TLR4) dependent cytokine production 85

7. Mannose binding lectin enhances IL-1beta and IL-10 induction by non-lipopolysaccharide (LPS) components of Neisseria meningitidis 99

8. Inhibition of C5a-induced inflammation with preserved c5b-9-mediated bactericidal activity in a human whole blood model of meningococcal sepsis 115

9. Deficient alternative complement pathway activation due to factor D deficiency by two novel mutations in the Complement Factor D gene in a family with meningococcal infections 141

10. Mannose-binding lectin is a critical factor in systemic complement activation during meningococcal septic shock 159 11. PCR-Restriction Fragment Length Polymorphism method to detect the X/Y polymorphism in the promoter site of the mannose-binding lectin gene 173

12. The double-edged sword of complement activation during sepsis. 179

13. Vascular endothelial growth factor is increased during the first 48 hours of human septic shock and correlates with vascular permeability 193

14. Macrophage migration inhibitory factor (MIF) in meningococcal septic shock and experimental human endotoxemia. 207

15. PTX3 and C-reactive protein in severe meningococcal disease 223

16. Mannose-binding lectin (MBL) and meningococcal disease, a case-parent study 237

17. innate cytokine production capacity and clinical manifestation of meningococcal disease 253

18. summary and General Discussion 269

nederlandse Samenvatting en Discussie 279

Dankwoord - word of thanks 290

curriculum Vitae 293

list of publications 294

awards and Presentations on national and international conferences 298

Introduction and outline of the thesis 1 Meningococci are new pathogens. Evolutionary speaking brand-and-shining-new pathogens, which emerged probably as recently as 200 years ago. They appeared when harmless Neisserial commensals in our nasopharynx obtained the ability to cause invasive disease, possibly due to accidentally acquiring a polysaccharide capsule from another bacterium. In contrast, our immune systems are old. Very old, probably nearly as old as multicellular life itself. In spite if its age however, our immune system is perfectly fit to deal with the multitude of micro-organisms that threaten us from our environment.

So, how does our ancient immune system battle a new pathogen such as Neisseria meningitidis? Some people, often previously perfectly healthy, young children, may be left utterly defenseless. When they become infected, and get meningococcal septic shock, they develop rapidly progressive disease that kills one out of five patients, leaving another third with severe sequelae. Fortunately, only a minority of people will acquire invasive meningococcal disease. Also, not all people exhibit the same type of illness, there are multiple clinical manifestions associated with meningococcal infections. In sharp contrast to the dreadful manifestation of meningococcal septic shock, there even are some patients that present with chronic 'benign' meningococcal bacteraemia. This is a disease in which patients can have meningococci in their blood for weeks and still recover completely with simple antibiotic treatment.

Although great progression has been made previously, many children are still killed or severely disabled by meningococcal infections. We still cannot prevent children from being infected. Therefore, we need to study pathogenesis. What if we could turn - using our knowledge of pathogenesis - the clinical course of fulminant meningococcal septic shock into benign meningococcal bacteraemia?!

The present thesis studies the interplay between meningococci and the innate immune system. The focus is on the role of cytokines and the complement system. The questions are straightforward and simple: what happens during invasive disease, how does it happen and are there ways to stop it from happening? The answers are complex but will increase our knowledge, hopefully leading to new and better treatments for this dreadful disease.

Lipopolysaccharide (LPS) has always been regarded as the principle cytokine inducing element of N. meningitidis and other Gram-negative bacteria, and LPS is also thought to be important for the activation of complement. In chapters 2 - 4 we made use of a genetically modified meningococcus that was deficient for LPS, addressing the question how important LPS versus non LPS components of

12 1

meningococci are in the induction of cytokines and the activation of complement. Cytokines are crucial mediators of the inflammatory response during meningococcal disease. Excessive production of these cytokines is held responsible for the development of tissue damage and shock. The relative contribution of lipopolysaccharide (LPS) and non-LPS of Neisseria meningitidis to the proinflammatory cytokine response in human cells and to mortality in a murine model of meningococcal sepsis is addressed in chapter 2. In chapter 3 we dealt with the question which role CD14 and TLR4 play in the induction of cytokines by LPS or non LPS components of meningococci.

The complement system is crucial in the initial defense against Neisseria meningitidis. In contrast, uncontrolled activation in meningococcal sepsis contributes to the development of tissue damage and shock. Whether complement activation and complement dependent inflammation in whole blood by meningococci is dependent or independent of LPS is studied in chapter 4.

For E. coli LPS it has been reported that lipoproteins inhibit cytokine production, opening new options for future therapies. However, meningococcal LPS is structurally different from E. coli LPS and intact Gram-negative bacteria are composed of more cytokine inducing elements then solely LPS. In chapter 5 we questioned whether lipoproteins also neutralize meningococcal LPS or complete Gram-negative bacteria.

Another way to inhibit cytokine production by LPS is to use LPS antagonist molecules. Recently, novel variant LPS species derived from N. meningitidis have been created with a lipid A portion consisting of five or four fatty acids in the lipid A part of the LPS. Because penta- or tetra-acylated LPS from other bacterial species is reported to be antagonistic, in chapter 6 we explored whether these meningococcal LPS variants show antagonism for LPS induced TLR4 dependent cytokine production.

A bridge between cytokine production and the complement system is found in chapter 7, where we study the influence of mannose binding lectin (MBL), the key molecule of the lectin pathway of complement activation on cytokine production by non-LPS components of meningococci.

In chapters 8 - 12 we addressed in vitro, in a whole blood model, as well as in vivo, in patients with meningococcal disease, the issue how complement is activated systemically during meningococcal septic shock. In addition, we also investigated ways to stop the consequences of systemic activation of complement. Chapter 8

13 we asked whether an anti-C5a molecule is able to inhibit complement dependent inflammation in whole blood. In addition, in this chapter, the mechanism by which complement is activated by meningococci is studied. In chapter 9, we report a family deficiency for complement factor D with meningococcal infections and addressed the question to what extent factor D deficiency affects complement activation by meningococci. In chapter 10 we dealt with the question to what extent the alternative and lectin pathways of complement contribute to the systemic activation of complement and the ensuing tissue damage. A restriction fragment length polymorphism (RFLP) assay to genotype the X/Y polymorphism in the promotor region of the MBL-gene is presented in chapter 11. Chapter 12 provides a concise review of the current knowledge on complement activation during septic shock.

In chapters 13 - 15 we study so far unexplored new mediators activated in patients with meningococcal infections, in order to find new possible targets for adjuvant treatment and early diagnosis of the disease. In chapter 13 vascular endothelial growth factor (VEGF), a molecule that mediates vascular permeability as well as angiogenesis is investigated. We questioned whether VEGF can contribute to the development of shock by mediating increased capillary leakage. Chapter 14 addresses the role of macrophage migration inhibitory factor (MIF), an important mediator of the inflammatory cytokine response. We asked how this molecule is induced both in a human LPS model of sepsis and during meningococcal sepsis, and investigated whether it could be responsible for the development of shock. The kinetics of pentraxin 3 (PTX3) - a molecule that is important for the activation of complement - and its use as an early marker for shock is investigated in chapter 15.

In chapters 16 and 17 the results of genetic and functional analysis in a large group of patients with meningococcal disease are presented. We asked the question how we can explain disease susceptibility and clinical manifestation of the disease. In chapter 16 the consequences of polymorphisms in gene for MBL for susceptibility to and clinical manifestation of meningococcal disease is investigated. In Chapter 17 we questioned whether innate cytokine production capacity in whole-blood influences clinical manifestation of meningococcal disease and disease severity.

Finally, chapter 18 provides summary, general discussion and recommendations for future research.

14 1

Contributions of Neisseria meningitidis LPS and non-LPS to proinflammatory cytokine response 2

Tom Sprong, Nike Stikkelbroeck, Peter van der Ley, Liana Steeghs, Loek van Alphen, Nigel Klein, Mihai G. Netea, Jos W. M. van der Meer and Marcel van Deuren Journal of Leukocyte Biology. 2001 Aug;70(2):283-8. Abstract

To determine the relative contribution of lipopolysaccharide (LPS) and non-LPS components of Neisseria meningitidis to the pathogenesis of meningococcal sepsis, this study quantitatively compared cytokine induction by isolated LPS, wild-type serogroup B meningococci (strain H44/76), and LPS-deficient mutant meningococci (strain H44/76lpxA). Stimulation of human peripheral-blood mononuclear cells with wild-type and LPS-deficient meningococci showed that non-LPS components of meningococci are responsible for a substantial part of tumor necrosis factor (TNF)-α and interleukin (IL)-1β production and virtually all interferon (IFN)-γ production. Based on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LPS in proteinase K-treated lysates of N. meningitidis H44/76, a quantitative comparison was made between the cytokine-inducing capacity of isolated and purified LPS and LPS-containing meningococci. At concentrations of >107 bacteria/ mL, intact bacteria were more potent cytokine inductors than equivalent amounts of isolated LPS, and cytokine induction by non-LPS components was additive to that by LPS. Experiments with mice showed that non-LPS components of meningococci were able to induce cytokine production and mortality. The principal conclusion is that non-LPS parts of N. meningitidis may play a role in the pathogenesis of meningococcal sepsis by inducing substantial TNF-α, IL-1β, and IFN-γ production.

18 2 Introduction

It is generally accepted that the induction of cytokine synthesis and the subsequent pathophysiological events during Gram-negative septic shock are primarily elicited by the lipopolysaccharide (LPS) component (endotoxin) of the bacterial outer membrane 1. Purified LPS is able to induce a proinflammatory cytokine pattern and a clinical condition that resembles, at a first glance, the symptoms encountered in Gram-negative septic shock. The LPS-molecule is composed of a lipid-A part harboring its toxic properties, a saccharide part, and one or more molecules of 2-keto-3-deoxy-octanate (KDO) connecting the lipid-A and the saccharide part 2. Anti-LPS strategies explored so far have failed to ameliorate the clinical course of Gram-negative sepsis 3-6, which raises the question whether LPS is the sole toxic element in Gram-negative sepsis 7.

Fulminant meningococcal sepsis is considered the prototypical human gram- negative septic shock, characterized by extremely high endotoxin and cytokine concentrations 8-11. Thus, the causative bacterium Neisseria meningitidis is a suitable subject for the study of cytokine induction by gram-negative bacteria. Recently, a viable N. meningitidis mutant was constructed that is devoid of LPS but still contains all other outer membrane constituents 12. This mutant has made it possible to assess the cytokine-inducing potency of the non-LPS parts of this gram-negative bacterium in a quantitative fashion.

The principal aim of the present study is to determine the contribution of LPS and non-LPS components of N. meningitidis to the pathogenesis of meningococcal sepsis. Therefore, we compared quantitatively the cytokine production induced by meningococcal LPS, wild-type meningococci, and LPS-deficient meningococci.

Materials and Methods Bacterial strains and lipopolysaccharides The wild-type serogroup B N. meningitidis H44/76 strain was isolated from a patient with invasive meningococcal disease 13. Meningococcal strain H44/76lpxA is a viable isogenic mutant, completely devoid of LPS in its outer membrane. This mutant was constructed by insertional inactivation of the lpxA gene, essential for the first committed step in biosynthesis of LPS 14,15. The absence of LPS in this strain was confirmed by tricine sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (TSDS-PAGE) with silver staining of LPS 16,17, whole-cell enzyme- linked immunosorbent assay (ELISA) with LPS-specific monoclonal antibodies, and gas-chromatographic/mass-spectrometric detection of LPS-specific 3-OH

19 fatty acids 12. Furthermore, absence of LPS activity in the lpxA batch suspension was confirmed by nonreactivity in the Limulus amebocyte lysate assay. Heat-killed (1 h, 56°C) bacteria washed in phosphate-buffered saline (PBS) were used in all experiments. The amount of bacteria in the suspensions used was measured by spectrophotometry; an optical density (OD) of 0.2 at 620 nm appeared to be equivalent to approximately 109 bacteria/mL.

Outer membrane complexes (OMCs) of both meningococcal strains were prepared by sarcosyl extraction as described previously 18. These OMCs consist primarily of PorA (class 1), PorB (class 3), and the RmpM (class 4) outer membrane proteins. The H44/76lpxA OMCs contain no LPS and express increased amounts of an OpA protein 19, H44/76 OMCs contain 10–20% LPS. Protein content was determined by a bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL) with bovine serum albumin as a standard.

The molecular mass of meningococcal strain H44/76 LPS is 4,044 Da 20; one molecule of LPS contains two molecules of KDO (molecular mass, 238 Da). LPS was isolated by the phenol/water extraction method as described by Westphal and Jann 21. After isolation, LPS was treated with proteinase K (Sigma-Aldrich Co.) and with DNase and RNase (Roche Diagnostics) for additional purification, recovered by ultracentrifugation, freeze-dried, and solved in sterile PBS. The amount of protein contamination in this purified LPS solution was determined by bicinchoninic acid assay. DNA and RNA contamination was determined by spectrophotometry using a Genequant RNA/DNA calculator (Pharmacia Biotech AB, Uppsala, Sweden).

The KDO content of the purified H44/76 LPS solution and of the bacterial suspensions was measured spectrophotometrically by the method described by Weissbach and Hurwitz 22.

TSDS-PAGE followed by silver staining of LPS was used for quantification of LPS in N. meningitidis H44/76. Cell lysates of H44/76 meningococci were made by suspending 1.35 x 1010 bacteria in 500 µL of SDS-buffer (7.5% glycerol, 1.25 M Tris/ HCl, 1.5% SDS) and incubating this for 5 min at 100°C. Proteins in this suspension were digested by incubation with proteinase K (0.5 mg/mL) for 4 h at 56°C 23. Serial dilutions of purified H44/76 LPS were used as a standard. After silver staining, the polyacrylamide gel was analyzed by densitometry.

Human peripheral blood mononuclear cells Blood for the isolation of human peripheral blood mononuclear cells (PBMCs) was drawn in 10-mL EDTA-anticoagulated tubes (Vacutainer System; Beckton Dickinson,

20 2 Rutherford, NJ) from healthy volunteers. PBMCs were isolated by density gradient centrifugation on Ficoll-Hypaque (Pharmacia Biotech AB). The cells from the interphase were aspirated, washed three times in sterile PBS, and resuspended in culture medium RPMI 1640 (Dutch modification; Flows Lab, Irvine, Scotland) supplemented with L-glutamine (2 mmol), pyruvate (1 mmol), and gentamycin (50 mg/mL) and 5% freshly pooled human serum. PBMCs (5x106/well) were incubated with various stimuli in 200-µL 96-well plates (Greiner BV, Alphen a/d Rijn, The Netherlands) at 37°C and 5% CO2 for 4 and 24 h. The supernatant was obtained by centrifugation and stored at -20°C until the cytokine assay was performed.

Murine experiments C57Bl/6J mice, obtained from the Jackson Lab (Bar Harbor, ME), were bred in our local facility. The animals were fed standard laboratory food (Hope Farms, Woerden, The Netherlands) and housed under specific pathogen-free conditions; 6- to 8-week-old mice weighing 20 to 25 g were used for the experiments. Resident peritoneal macrophages were harvested by rinsing the peritoneal cavity aseptically with sterile PBS (4°C), 105 cells/well were incubated with the different stimuli for 24 h. For lethality experiments, mice were pretreated with galactosamine (10 mg/ mouse) 30 min before the pathogen was injected into an orbital vein.

Cytokine assays Tumour necrosis factor (TNF-) and interleukin-1ß (IL-1ß) were determined by radioimmunoassay as described previously 24. Interferon- (IFN-) was determined by enzyme-linked immunosorbent assay with a commercially available kit (Pelikine Compact Human IFN- ELISA kit, Central Laboratory of the Netherlands Red Cross, Amsterdam, The Netherlands). The lower detection limit was 80 pg/mL for both TNF- and IL-1ß and 4 pg/mL for IFN-. Murine TNF- (mTNF-) and murine IL-1ß (mIL-1ß) were determined by radioimmunoassay as described by Netea et al. 25. Detection limits were 40 pg/mL for mTNF- and 20 pg/mL for mIL-1ß.

Statistics Results were compared by a Wilcoxson-Mann-Whitney test for unpaired, nonparametrical data. Spearman’s rank correlation coefficient was calculated to quantify the correlation between study parameters. P values of >0.05 were considered significant.

Results Quantification of LPS in N. meningitidis H44/76 bacteria To enable a quantitative comparison between cytokine induction by isolated LPS

21 and H44/76 meningococci, the purity of N. meningitidis H44/76 LPS was analyzed, and the amount of LPS in N. meningitidis H44/76 bacteria was determined. Protein contamination in the purified H44/76 LPS was <2.5%, and DNA and RNA contamination was 3.0% and 2.7%, respectively. This indicates that the H44/76 LPS used was at least 92% pure. The amount of KDO that was detected in 1 mg/mL of H44/76 LPS solution was 0.091 mg/mL. Assuming an average molecular mass for LPS of 4,044 Da, the amount of LPS in this solution based on the KDO assay was (0.091x4,044)/(238x2) 0.8 mg of LPS/mL.

TSDS-PAGE with silver stain of LPS was used to determine the amount of LPS in cell lysates of N. meningitidis H44/76 (Figure 1) 17,23,26. Density analysis of the silver stain of LPS in different dilutions of cell lysates of N. meningitidis H44/76 bacteria and of serial dilutions of purified LPS showed that 7 x 105 bacteria contain approximately 1 ng of LPS.

As an alternative approach to determine the amount of LPS in the LPS-containing H44/76 strain, the amount of KDO detected in the LPS-deficient lpxA suspension was subtracted from that in the H44/76 suspension. Because the only difference between these isogenic strains is the presence of LPS, the difference in KDO content reflects LPS-associated KDO. In this way, it was calculated that 10 x 105 H44/76 bacteria contain approximately 1 ng of LPS, a result rather similar to that obtained by TSDS-PAGE analysis.

Quantitative comparison of cytokine induction The dose-response relationship of meningococcal LPS, wild-type N. meningitidis H44/76, and LPS-deficient N. meningitidis H44/76lpxA for cytokine production by PBMCs after 24 h is shown in Figure 2. In this figure the x-axis was calibrated for LPS together with the number of H44/76 bacteria that contained an equivalent amount of LPS, based on the above-presented TSDS-PAGE results. Therefore, the effect of all three stimuli could be compared quantitatively.

It can be seen that at concentrations higher than 107 bacteria/mL, LPS-containing meningococci were more potent inductors of TNF- and IL-1β than equivalent amounts of isolated LPS (P<0.05). Below these concentrations, LPS was a more potent inductor of TNF- and IL-1β production. LPS-deficient lpxA meningococci were able to induce substantial amounts of TNF-α, IL-1β, and IFN-γ production. For TNF-α and IL-1β, approximately 10-fold-higher amounts of LPS-deficient bacteria were required to induce the same level of cytokine production as that in wild-type bacteria. Of interest, significant IFN-γ production occurred after stimulation with both the wild-type and the LPS-deficient meningococci, but only minute amounts

22 2 of IFN-γ were produced after stimulation with isolated LPS (P<0.05). Experiments (n=8) performed with 4 h of incubation showed a similar pattern for TNF-α and IL- 1β (data not shown).

To assess whether the IFN-γ production induced by LPS-containing or LPS-deficient meningococci is secondary to the TNF-α or IL-1ß production, a series of induction experiments with 6 x 108/mL of H44/76 and lpxA bacteria was performed with cells from 30 donors. In these experiments, the TNF-α and IL-1β production appeared to be correlated (r=0.061 (P<0.01) and r=0.70 (P<0.001) for wild-type and LPS- deficient meningococci, respectively). However, IFN-γ production did not correlate with the TNF-α or IL-1β production values for r between -0.12 and 0.22 (P=not

Figure 1. TSDS-PAGE silver staining of 0.1 µg (lane 1), 0.19 µg (lane 2), 0.39 µg (lane 3), and 0.77 µg (lane 4) of purified H44/76 LPS and LPS in proteinase K-treated lysates of 5x108 (lane 5) and 2.5 x 108 (lane 6) N. meningitidis H44/76 bacteria. Insert: Standard curve as calculated by linear regression for contour density (OD xmm2) of the purified LPS samples. Contour density of the lysates corresponds with 0.7 µg of LPS in sample 5 and 0.34 µg of LPS in sample

Figure 2. Production of TNF-α (left panel), iL-1ß (middle panel), and IFN-γ (right panel) after 24 h by human PBMCs stimulated with different concentrations of H44/76 LPS (•), wild-type H44/76 meningococci (█), and LPS- deficient H44/76lpxA meningococci (□). Values on the x-axis are calibrated for H44/76 LPS and the number of H44/76 bacteria that contain the same amount of LPS (1 ng of LPS ~7x105 bacteria). Mean values ± SE are presented (n=5).

23 significant). In addition, it could be seen that IFN-γ production showed a striking interindividual variety [range, 56–7,800 pg/mL (median, 555 pg/mL) and 40–6800 pg/mL (median, 660 pg/mL) for H44/76 and lpxA bacteria, respectively]. Thus, IFN-γ is probably not produced in response to TNF-α or IL-1β, and the individual response in IFN-γ production after stimulation with meningococci differs considerably.

The time course for TNF-α and IL-1β production by PBMCs showed a similar pattern for meningococcal LPS and both meningococcal strains. In brief, TNF-α became detectable after 2 h, reached a maximum after 8 h, and declined thereafter whereas IL-1β was detectable after 4 h and reached a plateau phase after 12 h (data not shown).

The relative contribution of non-LPS structures to the total induction of TNF-α and IL-1β induction by N. meningitidis was assessed in a series of combination

Figure 3. IL-1ß production after 24 h by human PBMCs stimulated with 6 x 106 or 6 x 108/mL of wild-type H44/76 meningococci (open bars) (n=25), the same amounts of LPS-deficient H44/76lpxA meningococci (light-gray bars) (n=25), or the same amounts of LPS-deficient lpxA meningococci substituted with equivalent amounts of LPS (0.0085 and 0.85 µg/mL, respectively) as present in wild-type H44/76 meningococci (dark-gray bars) (n=5). Mean values are presented ± SE. **, P < .01 compared with wild-type meningococci as well as LPS-deficient meningococci substituted with LPS

Figure 4. Production of IL-1ß after 24 h by human PBMCs stimulated with different concentrations of OMCs isolated from the wild- type H44/76 meningococci (█) containing 10–20% LPS and from mutant H44/76lpxA meningococci (□) devoid of LPS. OMC concentration is expressed as protein content in micrograms per milliliter. Mean values (n=5) are presented ± SE

24 2 experiments. In these experiments, meningococci were supplemented with approximately the same amount of LPS as present in the wild-type strain. Figure 3 shows the results of this experiment for IL-1β after 24 h of incubation. It can be seen that IL-1β production induced by LPS-deficient bacteria was approximately half of that induced by LPS-containing bacteria, whereas after addition of LPS, the IL- 1β induction by LPS-deficient bacteria was restored to the level of LPS-containing bacteria. Results for TNF-α and for experiments (n=5) with 4 h of incubation showed a similar pattern (data not shown). This additive effect of LPS to the non-LPS- induced cytokine synthesis suggests that both stimuli may use different pathways for the induction of TNF-α and IL-1β.

Cytokine induction by outer membrane complexes To determine whether the non-LPS components responsible for cytokine induction reside in the outer membrane, cytokine induction by OMCs isolated from the wild- type H44/76 meningococci and the LPS-deficient lpxA meningococci was assessed. Figure 4 shows the results for IL-1β after 24 h of incubation. It appeared that the wild-type H44/76 OMCs were potent inductors of cytokine synthesis, whereas the cytokine-inducing capacity of lpxA OMCs was 1,000- to 10,000-fold less than that of H44/76 OMCs. Results for TNF-α and for experiments (n=5) with 4 h of incubation showed a similar pattern (data not shown). Taken together these results indicate that the outer membrane components present in OMCs like PorA, PorB, RmpM, or OpA make a minimal contribution to cytokine induction.

Cytokine induction and lethality in mice In mice we assessed whether the observed cytokine induction by non-LPS components of meningococci coincides with the capacity to provoke disease. Results in Table 1 indicate that LPS-deficient meningococci were able to induce cytokine production in murine peritoneal macrophages in vitro. Table 2 demonstrates

Table 1. Stimulus MTNFα (ng/mL) mIL-1α(ng/mL) N. meningitidis H44/76 (2 x 109/mL) 1.85 ± 0.39 4.67 ± 1.09 N. meningitidis H44/76lpxA (2 x 109/mL) 0.86 ± 0.54 1.96 ± 0.77 Production of murine tumour necrosis factor-α (mTNFα) and murine interleukin-1α (mIL-1α) after 24 hours by murine peritoneal macrophages stimulated with wild-type H44/76 meningococci and LPS-deficient H44/76lpxA meningococci. Mean data ± SD are presented (n = 4 for H44/76 meningococci, n = 5 for H44/76lpxA meningococci).

Table 2. dosage of bacteria / mouse 107 108 109 N. meningitidis H44/76 60 100 100 N. meningitidis H44/76lpxA 0 0 60 Mortality at 24 hours (%) in galactosamine pre-treated mice after i.v. injection of heat-killed N. meningitidis (5 animals per group).

25 that LPS-deficient meningococci were able to induce lethality in galactosamine- sensitized mice in vivo. The dose needed for LPS-deficient meningococci to induce mortality was approximately 100-fold higher than for LPS-containing meningococci.

Discussion

The principal finding of the present study is that LPS is not the sole cytokine- inducing element of N. meningitidis. Using a meningococcal mutant completely devoid of LPS, it was shown that non-LPS components of this bacterium were responsible for a substantial part of the TNF-α and IL-1β production, that non- LPS components induced IFN-γ, and that non-LPS parts could provoke disease. Based on TSDS-PAGE analysis of LPS in proteinase K-treated cell lysates of N. meningitidis H44/76, a quantitative comparison between the cytokine-inducing capacity of LPS and that of LPS-containing bacteria became possible. It was shown that concentrations of >107/mL of bacteria were more potent inductors of cytokine synthesis than equivalent amounts of isolated LPS and that cytokine induction by non-LPS components was additive to that by LPS. Furthermore, it was demonstrated that the non-LPS components responsible for the cytokine induction do not reside in sarcosyl-extracted outer membrane complexes.

Non-LPS components of bacteria can induce cytokine production 7, as has been shown by experiments with gram-positive bacteria 27-31 and with various isolated elements of these bacteria like peptidoglycan 32-34, lipopeptides 28, lipoproteins 28, lipoteichoic acid 35, and capsular polysaccharides 36. However, so far studies trying to assess quantitatively the contribution of these non-LPS structures to cytokine induction by gram-negative bacteria were hampered by the inevitable copresence of LPS, outflanking the cytokine induction by non-LPS structures. To circumvent this problem we used a meningococcal mutant that is entirely deficient of LPS. With this strain we demonstrated that approximately half the amount of TNF-α and IL- 1β induced by meningococci in human PBMCs or murine peritoneal macrophages was elicited by non-LPS structures. Similarly, LPS-deficient meningococci could cause lethal disease in galactosamine-pretreated mice, albeit the dosages of LPS- deficient meningococci required to induce mortality were approximately 100-fold higher than for wild-type meningococci.

TNF-α, IL-1β, and IFN-γ are pivotal mediators in the pathogenesis of Gram-negative septic shock. After infusion of LPS in human volunteers or primates, TNF-α and IL- 1β appear within 1 to 2 h 37, but no IFN-γ seems to appear 38. However, after the infusion of whole bacteria, IFN-γ is induced and can be detected in the circulatory system after 6–8 h 38,39. During meningococcal infections, IFN-γ is increased in

26 2 plasma or cerebrospinal fluid and correlates with the severity of disease 40,41. In our in vitro study, meningococcal LPS stimulated only minimal IFN-γ production, whereas both LPS-containing and LPS-deficient bacteria induced significant amounts of IFN-γ. In addition, IFN-γ was not produced in response to TNF-α or IL-1β, which indicates that non-LPS components of meningococci were primarily responsible for IFN-γ induction. Because the primary source of IFN-γ is the lymphocyte and marked differences occur between individuals, it is tempting to speculate that certain non- LPS components of meningococci can act as superantigens.

In this study we used the widely employed KDO assay to determine the amount of LPS in the H44/76 LPS preparation 22,42,43. With this method (0.8/0.92) x 100% (i.e., 85%) of the LPS was detected. Limitations of this assay are conversion of a fraction of KDO during acid hydrolysis to entities inert to the thiobarbituric acid reaction and incomplete hydrolysis, both leading to an underestimation of the amount of KDO. On the other hand, contaminants in the LPS may coreact in the assay, which leads to overestimation of the amount of LPS 44,45. The relatively accurate yield of KDO in the present study showed that these limitations of the KDO assay are likely to be of minor importance for determination of H44/76 LPS.

By TSDS-PAGE analysis of LPS in lysates of N. meningitidis H44/76, the amount of LPS in H44/76 bacteria was determined. It appears that 7 x 105 bacteria corresponded to approximately 1 ng of LPS, which fits rather well with the results obtained by KDO analysis in H44/76 and lpxA bacteria. This estimate is in good accordance with reported estimates of 1 ng of LPS for 105 bacteria with Escherichia coli 1,46-50, taking into account that the MW of meningococcal LPS is 2- to 10-fold lower than that of E. coli LPS. In addition, our estimate compares fairly well to clinical data of Brandtzaeg et al. 51, Mariani-Kurkdjian et al. 52, and Bingen et al. 53, who detected LPS values up to 500 ng/mL and bacterial numbers up to 5 x 108 CFU/mL in cerebrospinal fluid during meningococcal meningitis.

Based on the estimate that 7 x 105 bacteria correspond to 1 ng of LPS, we could compare the cytokine-inducing potency of isolated LPS with that of meningococci containing the same amount of LPS. It was found that at concentrations below 107 bacteria/mL or equivalent amounts of LPS, LPS induced more TNF-α and IL-1β, but at higher concentrations, complete meningococci were more potent. Because at these higher concentrations the TNF-α and IL-1β-inducing capacities of LPS-deficient meningococci increased in a parallel fashion, the higher activity of bacteria at these higher concentrations is likely to have been caused by the non- LPS components of the bacterium.

27 Fulminant meningococcal sepsis is characterized by high plasma concentrations of endotoxin that range from 0.75 to 170 ng/mL 8,10,54-56. Based on our estimation of 1 ng of LPS per 7 x 105 bacteria, the reported range of endotoxin concentrations corresponds to 5 x 105 to 1.2 x 108 bacteria/mL. We speculate that strategies designed to block LPS-induced cytokine synthesis alone are of limited value, because at this degree of bacteremia, a substantial part of the proinflammatory cytokine response is elicited by non-LPS parts of the meningococcus 57.

Because several outer membrane proteins of N. meningitidis are known to interact with human cell receptors 58,59, we examined whether sarcosyl-extracted outermembrane complexes are able to induce cytokine production. LPS-deficient OMCs primarily composed of the major outer membrane proteins PorA, PorB, RmpM, and OpA did not induce cytokines. Thus, other meningococcal components not retained after sarcosyl extraction, for instance certain lipoproteins, chromosomal DNA, the polysaccharide capsule 60, or the peptidoglycan cell wall 32-34, must have been responsible for the observed cytokine induction by the LPS-deficient mutant. Further research is needed to identify which of the non-LPS components are responsible for cytokine induction and to quantify their relative contribution to the pathogenesis of invasive meningococcal disease

28 2 References 1. Hurley JC. Endotoxemia: methods of detection and clinical correlates. Clin Microbiol Rev. 1995;8:268-292 2. Luderitz O, Tanamoto K, Galanos C, McKenzie GR, Brade H, Zahringer U, Rietschel ET, Kusumoto S, Shiba T. Lipopolysaccharides: structural principles and biologic activities. Rev Infect Dis. 1984;6:428-431 3. Ziegler EJ, Fisher CJ, Jr., Sprung CL, Straube RC, Sadoff JC, Foulke GE, Wortel CH, Fink MP, Dellinger RP, Teng NN, et al. Treatment of gram-negative bacteremia and septic shock with HA-1A human monoclonal antibody against endotoxin. A randomized, double- blind, placebo-controlled trial. The HA-1A Sepsis Study Group [see comments]. N Engl J Med. 1991;324:429-436 4. The J5 Study Group. Treatment of severe infectious purpura in children with human plasma from donors immunized with Escherichia coli J5: a prospective double-blind study. J Infect Dis. 1992;165:695-701 5. Derkx B, Wittes J, McCloskey R. Randomized placebo-controlled trial of HA-1A, a human monoclonal anti-endotoxin antibody, in children with meningococcal septic shock. Clin Infect Dis. 1999;28:770-777 6. Levin M, Quint PA, Goldstein B, Barton P, Bradley JS, Shemie SD, Yeh T, Kim SS, Cafaro DP, Scannon PJ, Giroir BP. Recombinant bactericidal/permeability-increasing protein (rBPI21) as adjunctive treatment for children with severe meningococcal sepsis: a randomised trial. rBPI21 Meningococcal Sepsis Study Group. Lancet. 2000;356:961- 967. 7. Henderson B, Poole S, Wilson M. Bacterial modulins: a novel class of virulence factors which cause host tissue pathology by inducing cytokine synthesis. Microbiol Rev. 1996;60:316-341 8. Brandtzaeg P, Kierulf P, Gaustad P, Skulberg A, Bruun JN, Halvorsen S, Sørensen E. Plasma endotoxin as a predictor of multiple organ failure and death in systemic meningococcal disease. J Infect Dis. 1989;159:195-204 9. Waage A, Halstensen A, Espevik T. Association between tumour necrosis factor in serum and fatal outcome in patients with meningococcal disease. Lancet. 1987;i:355-357 10. Van Deuren M, van der Ven Jongekrijg J, Bartelink AKM, van Dalen R, Sauerwein RW, van der Meer JWM. Correlation between proinflammatory cytokines and antiinflammatory mediators and the severity of disease in meningococcal infections. J Infect Dis. 1995;172:433-439 11. Van Deuren M, Brandtzaeg P, Van der Meer JWM. Update on meningococcal disease with emphasis on pathogenesis and clinical management. Clin Microbiol Rev. 2000;13:144- 166 12. Steeghs L, den Hartog R, den Boer A, Zomer B, Roholl P, van der Ley P. Meningitis bacterium is viable without endotoxin. Nature. 1998;392:449-450 13. Holten E. Serotypes of Neisseria meningitidis isolated from patients in Norway during the first six months of 1978. J Clin Microbiol. 1979;9:186-188 14. Steeghs L, de Cock H, Evers E, Zomer B, Tommassen J, van der Ley P. Outer membrane composition of a lipopolysaccharide-deficient Neisseria meningitidis mutant. EMBO J. 2001;20:6927-6945 15. Galloway SM, Raetz CR. A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis. J Biol Chem. 1990;265:6394-6402 16. Tsai CM, Frasch CE. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982;119:115-119. 17. Lesse AJ, Campagnari AA, Bittner WE, Apicella MA. Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate- polyacrylamide gel electrophoresis. J Immunol Methods. 1990;126:109-117. 18. Van der Ley P, Heckels JE, Virji M, Hoogerhout P, Poolman JT. Topology of outer membrane porins in pathogenic Neisseria spp. Infect Immun. 1991;59:2963-2971 19. Steeghs L, Kuipers B, Hamstra HJ, Kersten G, van Alphen L, van der Ley P.

29 Immunogenicity of outer membrane proteins in a lipopolysaccharide- deficient mutant of Neisseria meningitidis: influence of adjuvants on the immune response. Infect. Immun. 1999;67:4988-4993 20. Pavliak V, Brisson JR, Michon F, Uhrin D, Jennings HJ. Structure of the sialylated L3 lipopolysaccharide of Neisseria meningitidis. J Biol Chem. 1993;268:14146-14152 21. Westphal O, Jann JK. Bacterial lipopolysacharide extraction with phenol- water and further application of the procedure. Methods Carbohydr Chem. 1965;5:83-91 22. Weissbach A, Hurwitz B. The formation of 2-keto-3-deoxyheptonic acid in extracts of Escherichia coli B. J Biol Chem. 1959;234:705-709 23. Hitchcock PJ, Brown TM. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol. 1983;154:269-277. 24. Drenth JP, van Deuren M, van der Ven Jongekrijg J, Schalkwijk CG, van der Meer JW. Cytokine activation during attacks of the hyperimmunoglobulinemia D and periodic fever syndrome. Blood. 1995;85:3586-3593 25. Netea MG, Demacker PN, Kullberg BJ, Boerman OC, Verschueren I, Stalenhoef AF, van der Meer JWM. Low-density lipoprotein receptor-deficient mice are protected against lethal endotoxemia and severe gram-negative infections. J Clin Invest. 1996;97:1366- 1372 26. Tsai CM. The analysis of lipopolysaccharide (endotoxin) in meningococcal polysaccharide vaccines by silver staining following SDS-polyacrylamide gel electrophoresis. J Biol Stand. 1986;14:25-33. 27. Bjork L, Andersson J, Ceska M, Andersson U. Endotoxin and Staphylococcus aureus enterotoxin A induce different patterns of cytokines. Cytokine. 1992;4:513-519 28. Kreutz M, Ackermann U, Hauschildt S, Krause SW, Riedel D, Bessler W, Andreesen R. A comparative analysis of cytokine production and tolerance induction by bacterial lipopeptides, lipopolysaccharides and Staphylococcus aureus in human monocytes. Immunology. 1997;92:396-401 29. Timmerman CP, Mattsson E, Martinez-Martinez L, De Graaf L, Van Strijp JA, Verbrugh HA, Verhoef J, Fleer A. Induction of release of tumor necrosis factor from human monocytes by staphylococci and staphylococcal peptidoglycans. Infect Immun. 1993;61:4167-4172 30. Wakabayashi G, Gelfand JA, Jung WK, Connolly RJ, Burke JF, Dinarello CA. Staphylococcus epidermidis induces complement activation, tumor necrosis factor and interleukin-1, a shock-like state and tissue injury in rabbits without endotoxemia. Comparison to Escherichia coli. J. Clin. Invest. 1991;87:1925-1935 31. Heumann D, Barras C, Severin A, Glauser MP, Tomasz A. Gram-positive cell walls stimulate synthesis of tumor necrosis factor alpha and interleukin-6 by human monocytes. Infect. Immun. 1994;62:2715-2721 32. Schrijver IA, Melief MJ, Eulderink F, Hazenberg MP, Laman JD. Bacterial peptidoglycan polysaccharides in sterile human spleen induce proinflammatory cytokine production by human blood cells. J Infect Dis. 1999;179:1459-1468 33. Kengatharan KM, De Kimpe S, Robson C, Foster SJ, Thiemermann C. Mechanism of gram-positive shock: identification of peptidoglycan and lipoteichoic acid moieties essential in the induction of nitric oxide synthase, shock, and multiple organ failure. J Exp Med. 1998;188:305-315 34. Mattsson E, Rollof J, Verhoef J, Van Dijk H, Fleer A. Serum-induced potentiation of tumor necrosis factor alpha production by human monocytes in response to staphylococcal peptidoglycan: involvement of different serum factors. Infect Immun. 1994;62:3837-3843 35. Bhakdi S, Klonisch T, Nuber P, Fischer W. Stimulation of monokine production by lipoteichoic acids. Infect Immun. 1991;59:4614-4620 36. Soell M, Diab M, Haan-Archipoff G, Beretz A, Herbelin C, Poutrel B, Klein JP. Capsular polysaccharide types 5 and 8 of Staphylococcus aureus bind specifically to human epithelial (KB) cells, endothelial cells, and monocytes and induce release of cytokines. Infect Immun. 1995;63:1380-1386 37. Kuhns DB, Alvord WG, Gallin JI. Increased circulating cytokines, cytokine antagonists, and E-selectin after intravenous administration of endotoxin in humans. J Infect Dis.

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31 spectrometry, ultracentrifugation, and electron microscopy. J Clin Invest. 1992;89:816- 823 57. Uronen H, Williams AJ, Dixon G, Andersen SR, Van der Ley P, Van Deuren M, Callard RE, Klein N. Gram-negative bacteria induce proinflammatory cytokine production by monocytes in the absence of lipopolysaccharide (LPS). Clin Exp Immunol. 2000;22:312 58. Kallstrom H, Liszewski MK, Atkinson JP, Jonsson AB. Membrane cofactor protein (MCP or CD46) is a cellular pilus receptor for pathogenic Neisseria. Mol Microbiol. 1997;25:639- 647 59. Virji M, Watt SM, Barker S, Makepeace K, Doyonnas R. The N-domain of the human CD66a adhesion molecule is a target for Opa proteins of Neisseria meningitidis and Neisseria gonorrhoeae. Mol Microbiol. 1996;22:929-939 60. Stephens DS, Spellman PA, Swartley JS. Effect of the (alpha 2®8)-linked polysialic acid capsule on adherence of Neisseria meningitidis to human mucosal cells. J Infect Dis. 1993;167:475-479

32 2

Neisseria meningitidis can induce pro-inflammatory cytokine production via pathways independent from CD14 and toll-like receptor 4 3

Tom Sprong, Peter van der Ley, Liana Steeghs, Wil J. Tax, Trees J. W. Janssen, Mihai G. Netea, Jos W. M. van der Meer and Marcel van Deuren European Cytokine Network 2002 Oct-Dec;13(4):411-7 Abstract

Fulminant meningococcal sepsis (FMS) is considered the prototypical Gram- negative sepsis. Lipopolysaccharide (LPS) is thought to be the main toxic element that induces pro-inflammatory cytokine production after interaction with CD14 and toll-like receptor 4 (TLR4). However, there is increasing evidence that LPS is not the sole toxic element of meningococci. The aim of the present study was to determine the role of CD14 and TLR4 in pro-inflammatory cytokine induction by meningococci. To this end, cytokine induction by isolated meningoccal LPS, wild- type N. meningitidis H44/76 (LPS+ meningococci) matched for concentrations of LPS and LPS-deficient N. meningitidis H44/76lpxA (LPS- meningococci) was studied in human PBMCs and murine peritoneal macrophages (PMs). Pre-incubation of PBMCs with WT14, a monoclonal antibody against CD14, abolished TNF-α and IL-1β induction by E. coli LPS, while cytokine induction by meningococcal LPS was only partially inhibited. When LPS+ and LPS- meningococci at higher concentrations were used as stimuli, anti-CD14 had a minimal effect. In C3H/HeJ murine PMs, devoid of a functional TLR4, minimal IL-1α, IL-6 and TNF-α production was seen after stimulation with 10 ng/mL E. coli or meningococcal LPS. However, at higher concentrations (1000 ng LPS/mL) the production of TNF-α, but not IL-1α or IL-6, occurred also independently of TLR4. The expression of a functional TLR4 in murine PMs had no effect on the cytokine induction by LPS+ or LPS- meningococci. It is concluded that pro-inflammatory cytokine induction by N. meningitidis can occur independently of CD14 and TLR4.

36 Introduction Invasive infections with the Gram-negative bacterium Neisseria meningitidis are 3 an important cause of morbidity and mortality worldwide 1. Particularly fulminant meningococcal sepsis (FMS), considered to be the prototypical Gram-negative septic shock, is feared for its high fatality rate. Lipopolysaccharide (LPS, endotoxin), an important structural and functional component of the outer membrane of Gram- negative bacteria, is thought to be responsible for the major pathophysiological events during meningococcal septicaemia 2. LPS induces the synthesis of interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α), pro-inflammatory cytokines pivotal in the development of septic shock 3,4.

Recently, the principal pattern-recognition receptors for LPS have been characterised more precisely. After the interaction of LPS with LPS-binding protein (LBP), LPS is shuttled to CD14, a glycophosphatidyl-inositol (GPI) - linked cell- surface glycoprotein present on cells of the myeloid lineage5 6. As CD14 lacks a transmembrane domain, association with the toll-like receptor 4 (TLR4) and its co-factor MD-2 is required for transmembrane signalling 7-9. TLR4 imparts ligand specific recognition of LPS and binding of LPS induces physical proximity between CD14 and TLR4 prior to nuclear translocation of NF-κB 10,11. In addition to its role in the recognition of LPS, CD14 has also been shown to mediate responses after stimulation with other bacterial components 12.

In spite of the importance of LPS in the pathogenesis of FMS, there is convincing evidence that LPS is not the sole toxic element. Anti-LPS strategies have failed to ameliorate the clinical course of FMS 13-16, and it was recently shown in various laboratories that a mutant strain of N. meningitidis devoid of LPS is able to induce cytokine production 17-19. Thus, bacterial components other than LPS, i.e. non-LPS components, may have an important role in the pathogenesis of FMS.

The aim of the present study was to determine the role of CD14 and TLR4 in proinflammatory cytokine induction by the meningococcus.

Materials and methods Neisseria meningitidis strains The wild-type serogroup B N. meningitidis H44/76 strain (designated as LPS+ N. meningitidis) was isolated from a patient with invasive meningococcal disease 20. The meningococcal strain H44/76lpxA (designated as LPS- N. meningitidis) is a viable isogenic mutant, completely devoid of LPS in the outer membrane. This

37 mutant was constructed by insertional inactivation of the lpxA gene, essential for the first committed step of the biosynthesis of LPS. The absence of LPS in this strain was confirmed as described previously 21. Absence of LPS-activity in the LPS- N. meningitidis batch-suspension was assessed by non-reactivity in the Limulus amebocyte lysate assay.

Bacteria were grown overnight in liquid medium, heat-inactivated (1 h, 56°C) and washed in phosphate buffered saline (PBS). Bacteria were quantified by spectrophotometry.

Lipopolysaccharides (LPS) A commercially available TCA extracted E. coli 055:B5 LPS was purchased from Sigma-Aldrich Co (Rutherford, NY). Meningococcal LPS was isolated in our laboratory by phenol/water extraction according to Westphal and Jann 22. After extraction, this meningococcal LPS was treated with Proteinase K (Sigma- Aldrich Co), DNA-ase and RNA-ase (Roche diagnostics) for additional purification, recovered by ultracentrifugation, freeze-dried and solved in sterile PBS.

The commercial E. coli LPS preparation was found to be contaminated with protein (8.5%) (by Lowry-assay) and nucleic acids (6.1%) (by spectrophotometry). The N. meningitidis LPS preparation was free of protein (< 1.0%), nucleic acid content was (4.4%).

Quantification of LPS in N. meningitidis H44/76 bacteria was performed by TSDS- PAGE followed by silverstaining of LPS and assay for 2-keto-3-deoxyoctanate as described previously 17; 1 ng of LPS correlated with 0.7x106 LPS+ N. meningitidis H44/76 bacteria.

Anti-CD14 antibody The monoclonal anti-CD14 antibody WT14 was produced as described previously 23. Binding of LPS to human macrophages was determined by flow cytometry using fluorescein isothiocyanate (FITC) labelled E. coli 055:B5 LPS. FITC labeling of LPS was performed as described by Pollack et al. 24. Purified human monocytes were preincubated with control antibody (which did not bind monocytes), WT-14 antibody or an excess of unlabelled LPS. The cells were washed and FITC-labelled LPS was allowed to bind the monocytes in the presence of 10% pooled human serum (as a source of LPS-binding protein). Monocytes were identified based on size and granularity. In stimulation experiments with PBMCs, WT14 alone did not induce cytokines.

38 Mice Murine C57Bl/6J peritoneal macrophages (PMs) were used to study cytokine induction by meningococcal LPS and LPS+- or LPS- meningococci. The role of 3 TLR4 in cytokine induction by meningococcal LPS and non-LPS components was assessed in PMs isolated from C3H/HeJ or C3H/HeN mice. The mice were obtained from the Jackson Laboratories (Bar Harbour, ME, USA) and were bred in our local facility; 6 to 8 weeks old mice, weighing 20 to 25 grams were used. The animals were fed standard laboratory food (Hope Farms, Woerden, the Netherlands) and housed under specific pathogen-free conditions. C3H/HeJ mice have a nonfunctional TLR4, owing to a base-transversion in the gene coding for TLR4. The parent strain C3H/HeN possesses the normal functional TLR4.

Human and murine cells; culture conditions Blood for the isolation of human peripheral blood mononuclear cells (PBMCs) was drawn in 10-mL EDTA anti-coagulated tubes (Vacutainer System, Beckton Dickinson, Rutherford, NJ) from healthy volunteers. PBMCs were isolated after density gradient centrifugation on Ficoll-Hypaque (Pharmacia Biotech AB). The cells from the interphase were aspirated and washed 3 times in sterile PBS. 5x105 cells per well were incubated in 200 ml 96 wells plates (Greiner BV, Alphen a/d Rijn, the Netherlands) with the various stimuli for 24 hours at 37 °C and 5% CO2. The anti-CD14 antibody WT14 was preincubated (20 mg/mL) with the PBMCs for 2 hours at 37 °C and 5% CO2.

Resident peritoneal macrophages (mPMs) of C3H/HeN or C3H/HeJ mice were harvested by rinsing the peritoneal cavity aseptically with sterile PBS (4 °C). 1x105 cells per well were incubated with the various stimuli. Culture conditions were similar to those for human PBMCs.

RPMI 1640 (Dutch modification, Flows Lab, Irvine, Scotland) supplemented with L-glutamine (2 mMol), pyruvate (1 mMol) and gentamycin (50 mg/mL) was used as culture medium. For experiments with human PBMCs, 5% fresh pooled human serum obtained from 5 healthy volunteers was added.

Cytokine assays Human IL-1β and TNF-α were determined by radioimmunoassay (RIA) as described previously 25. The lower limit of detection was 0.08 ng/mL. Murine IL-1α (mIL-1α) and mTNF-α and were determined by RIA as described 26. mIL-6 was determined by ELISA (Pelikine Compact murine IL-6 ELISA kit, Central laboratory of the Netherlands red cross). The lower limit of detection was 0.04 ng/ml.

39 Calculations and statistics For calculations and graphs, the net cytokine production, i.e. the cytokine concentration in the stimulated sample minus the concentration in the unstimulated sample, was used. Median cytokine production was used to calculate the % decrease in cytokine production. Data were analysed using the Mann-Whitney test for unpaired, non-parametrical data (GraphPad Prism, GraphPad software Inc.); a p-value <.05 was considered significant.

Results Cytokine production after stimulation with meningococcal LPS, LPS+ and LPS- meningococci Cytokine production by human PBMCs and murine C57Bl/6J PMs was studied using a low or a high concentration of meningococcal LPS (6 or 600 ng/mL for human PBMCs and 10 or 1 000 ng/mL for murine PMs) and matched concentrations of LPS- containing (LPS+) or LPS-deficient (LPS-) meningococci. Figure 1 shows the results for IL-1β in human PBMCs. At these concentrations, LPS+ meningococci induced 3 - 4 fold more IL-1β than equivalent amounts of purified meningococcal LPS. The LPS- meningococci induced sizeable amounts of pro-inflammatory cytokines, however less than the wild-type strain. TNF-α production followed a similar pattern (data not shown). In murine PMs, the pattern of the production of IL-1α, TNF-α and IL-6 after stimulation with the three different stimuli was similar to that observed in human PBMCs for IL-1β and TNF-α, although a higher concentration of stimulus was needed to obtain measurable cytokine production.

Effect of anti-CD14 on the binding of E. coli LPS to monocytes and cytokine induction by E. coli LPS Flow-cytometric (FACS) analysis of the binding of FITC-labeled E. coli LPS to human monocytes showed that the anti-CD14 antibody WT14 completely abrogated the binding of E. coli LPS to monocytes (Figure 2). As expected, anti-CD14 inhibited the E. coli LPS (10 ng/mL) induced IL-1β and TNF-α production with > 95% (Figure 3).

Effect of anti-CD14 on cytokine induction by meningococcal LPS, LPS+ and LPS- meningococci Figure 4 shows the effect of anti-CD14 on the IL-1β and TNF-α production by human PBMCs after stimulation with isolated meningococcal LPS, LPS+ meningococci and LPS- meningococci. Meningococcal LPS and both bacterial strains were used at matched concentrations as described above. Anti-CD14 showed a trend towards inhibition of IL-1β and TNF-α production at both low and high concentrations of

40 50 3 A B

Figure 1 β 30 IL-1 production by human PBMCs after stimulation with low (panel A, 6 ng/mL meningococcal LPS or 6 x 106 (ng/mL)

β meningococci/mL ) and high dose (panel 20 B, 600 ng/mL meningococcal LPS or IL-1 6 x 108 meningococci/mL) purified meningococcal LPS (hatched bars), LPS+ 10 N. meningitidis H44/76 (black bars) or LPS- N. meningitidis lpxA (white bars). Median values are presented, error bars indicate inter-quartile range (IQR). n = 0 15 for the LPS experiments, n = 25 or 30 LPS N. men N. men LPS N. men N. men for the experiments with the low or high LPS+ LPS- LPS+ LPS- bacterial concentration respectively.

100 100

80 80

60 60

40 40 Figure 2 % positive% cells

% positive% cells Binding of FITC-labelled E. coli LPS to 20 human monocytes after preincubation with 20 a control antibody, the anti-CD14 antibody WT14 or an excess of unlabelled LPS. Results are given as the mean percentage 0 + 0 of positive cells /- SD, n = 4, performed on control αCD14 LPS the same batch of frozen monocytes. control αCD14 LPS

6 6

4 4 TNF α (ng/mL) (ng/mL)

β Figure 3 β α

IL-1 IL-1 and TNF production by human PBMCs preincubated for 2 hours with 2 2 RPMI (white bars) or 20 µg/mL anti-CD14 (hatched bars) prior to stimulation for 24 hours with 10 ng/mL E. coli LPS. Median P <.01 P =.02 values are presented +/- IQR, n = 5. P-values apply to the comparison between 0 0 preincubation with or without anti-CD14.

41 meningococcal LPS. However, in contrast to E. coli LPS (Figure 3), inhibition was incomplete (64% for IL-1β and 45% for TNF-α) and did not reach statistical significance. After stimulation with the high concentration of meningococcal LPS (600 ng/mL), the production of IL-1β and TNF-α was even less inhibited (53% and 24%, respectively).

IL-1β and TNF-α production after stimulation with the low concentration (6x106/ mL) of LPS+ meningococci was inhibited by anti-CD14 (78% for IL-1β and 61% for TNF-α, p <.05). After stimulation with LPS- meningococci this reduction was 55% for both cytokines (p =.08 and .03 for IL-1β and TNF-α, respecively). At the high concentration of both bacterial strains (6x108 meningococci/mL), no significant inhibition of cytokine production occurred, however, an inhibiting trend on TNF-α production was present.

Cytokine induction by meningococcal LPS, LPS+ meningococci and LPS- meningococci in C3H/HeJ and C3H/HeN mice As a reference, PMs of C3H/HeN and C3H/HeJ mice were stimulated with E. coli LPS (10 ng/mL). Figure 5 shows the results for IL-1α and TNF-α. In accordance with previous studies, production of IL-1α and TNF-α by C3H/HeJ PMs was reduced

20 P =.50 30 ) A B P =.50 15 20

(ng/mL 10 β

P =.03 10 IL-1 5 P =.08 P =.08 P =.11

0 0 10 20 A B 15 P =.21 P =.35 (ng/mL) 5 10 α

P =.03 TNF P =.02 5 P =.08 P =.11

0 0 LPS N. Men N. Men LPS N. Men N. Men LPS+ LPS- LPS+ LPS-

Figure 4 IL-1β and TNFα production in human PBMCs preincubated for 2 hours with RPMI (white bars) or 20 µg/mL anti-CD14 (hatched bars) prior to stimulation for 24 hours with meningococcal LPS, LPS+ N. meningitidis H44/76 and LPS- N. meningitidis lpxa. Panels A : 6 ng/mL meningococcal LPS or 6 x 106 meningococci/ mL, panels B : 600 ng/mL meningococcal LPS or 6 x 108 meningococci/mL. Median values are presented, +/- IQR, n = 5 for all stimuli. P-values apply to the comparison between preincubation with or without anti- CD14.

42 to values approximately equivalent to the blank. The IL-6 production was similarly affected (data not shown). 3 Figure 6 shows the IL-1a and TNF-a production by mPMs of C3H/HeN and C3H/ HeJ mice after stimulation with meningococcal LPS, or matched concentrations of LPS+ or LPS- meningococci. Similar to the pattern for E. coli LPS, minimal IL-1α production occurred in C3H/HeJ mPMs after stimulation with the low or high dose dose of meningococcal LPS. The IL-6 production was similarly affected (data not

1.61.6 1.61.6

1.21.2 1.21.2 Figure 5 α α TNF

ILTNF -1 and TNF production in murine peritoneal macrophages of C3H/HeN α miceα (white bars) and C3H/HeJ mice (ng/mL) (ng/mL) (ng/mL) (ng/mL) 0.80.8 0.80.8 (hatched bars) after stimulation for α α 24 hours with 10 ng/mL E. coli LPS. + IL-1

IL-1 Median values are presented /- IQR, n = 5. Net cytokine production P P=.04=.04 0.40.4 0.40.4 (cytokine level in stimulated sample minus cytokine level in blank) is P P<.01<.01 presented. P-values apply to the comparison between C3H/HeJ and 0 0 0 0 C3H/HeN mPMs.

1.6 4.0 A B 1.2 P =.26 3.0 P =.46 (ng/mL) 0.8 P =.40 2.0

P =.08 IL-1 α 0.4 1.0 P <.01 P =.01 0.0 0.0 1.6 1.6 A B 1.2 P =.33 P =.39 1.2 (ng/mL) 0.8 P =.36 0.8 α P =.14 P =.10

TNF 0.4 P =.02 0.4

0.0 0.0 LPS N. men N. men LPS N. men N. men LPS+ LPS- LPS+ LPS-

Figure 6 IL-1α and TNFα production in murine peritoneal macrophages of C3H/HeN mice (white bars) and C3H/ HeJ mice (hatched bars) after stimulation with meningococcal LPS, LPS+ N. meningitidis H44/76 or LPS- N. meningitidis lpxA. Panels A : 10 ng/mL meningococcal LPS or 107 meningococci/mL, panels B : 1000 ng/ mL meningococcal LPS or 109 meningococci/mL. Median values +/- IQR, n = 10, of net cytokine production (cytokine level in stimulated sample minus cytokine level in blank) are presented. P-values apply to the comparison between C3H/HeJ and C3H/HeN mPMs.

43 shown). However, TNF-α production was only affected after stimulation with the low dose (10 ng/mL) of meningococcal LPS, while after stimulation with the high dose (1000 ng/mL) only 18% reduction occurred as compared with the production in C3H/HeN PMs.

LPS+ meningococci in low concentrations induced 58% less IL-1a in C3H/HeJ mice as compared to C3H/HeN mice (p =.08). IL-6 showed a similar pattern (data not shown). At high concentrations however, virtually equal amounts of IL-1α and IL-6 were produced by C3H/HeJ and C3H/HeN mPMs. Remarkably, TNF-α production was not affected in C3H/HeJ mice. LPS- meningococci induced equal amounts of IL-1α, IL-6 and TNF-α in murine PMs of C3H/HeJ and C3H/HeN mice.

Discussion

In the present study we demonstrated that N. meningitidis can induce cytokines in human PBMCs or murine peritoneal macrophages by pathways independent from CD14 and TLR4.

The finding that anti-CD14 did not completely inhibit the production of proinflammatory cytokines after stimulation with meningococcal LPS is in contrast with the findings for E. coli LPS. In accordance with previous studies we found that IL- 1β and TNF-α induction by E. coli LPS is completely CD14-dependent 6. Because our meningococcal LPS preparation appeared to be less contaminated than the commercially purchased E. coli LPS, the observed CD-14 independent cytokine production in experiments with meningococcal LPS is rather explained by the specific molecular characteristics of meningococcal LPS than contaminants. Indeed, recently Gangloff et al. showed that LPS molecules with a short polysaccharide tail that lacks the typical O antigen, such as meningococcal LPS, may engage CD14- independent pathways for the induction of TNF-α 27.

CD14 has been shown to bind a range of microbial components other than LPS such as peptidoglycan, lipoproteins, lipoteichoic acid, lipoarabinomannan and various other components 12,28. Gram-negative bacteria contain some of these non-LPS components (peptidoglycan, lipoproteins) which are proficient to engage CD14. Recently, Ingalls et al. identified CD14 as a principal ligand for cytokine induction by LPS-deficient meningococci 19. Our finding that, after stimulation with the low dose (6x106 bacteria/mL) LPS-free meningococci, anti-CD14 considerably (55%) inhibited IL-1β and TNF-α production is in accordance with this role for CD14. However, as inhibition was incomplete, the non-LPS components of N. meningitidis may engage also CD14-independent pathways. At higher concentrations (6x108

44 bacteria/mL), we found that pro-inflammatory cytokine production was completely CD14-independent. We presume that at these concentrations, non-specific receptors like the β2-integrins or scavenger receptors are engaged 29,30. 3

TLR4 has been shown to confer ligand-specific recognition of LPS. However, there is recent evidence that TLR2, and not TLR4, is involved in recognition of LPS isolated from Porphyromonas gingivalis or Leptospira spp. Thus, probably various types of LPS may interact differentially with TLRs 31,32. In the present study we showed that in mice, TLR4 is the principal signal transferring receptor for meningococcal LPS at lower concentrations (10 ng/mL). At higher concentrations (1 \000 ng/mL) however, TNF-α - but not IL-1α or IL-6 - was also produced independently of TLR4. Hirschfeld et al. 31 reported that engagement of TLR4 and TLR2 will result in the differential expression of cytokine genes. Combined with our recently published suggestion that the molecular conformation of the lipid-A moiety in meningococcal LPS, with six symmetrically distributed fatty-acids, favors the engagement of TLR2 33, we speculate that the TLR4 independent induction of TNF-α by meningococcal LPS, is mediated by TLR2.

Our experiments with LPS-free and LPS-containing meningococci indicate that TLR4 is not exclusively engaged in cytokine induction by the Gram-negative bacterium N. meningitidis. Using TLR4-nonfunctional C3H/HeJ mPMs, IL-1α production after stimulation with the wild-type LPS+ meningococci was only moderately decreased compared to the parent C3H/HeN murine PMs, and TNF-α production was not decreased at all after stimulation with low dosages of LPS+ meningococci. As cytokine production after stimulation with LPS- meningococci in C3H/HeJ mPMs was not affected, it is likely that the signaling via non-TLR4 pathways by LPS+ meningococci occurs by non-LPS components of the meningococcus. This is underscored by the finding of Massari et al., who found that isolated outer membrane porins of the meningococcus can signal via TLR234.

It should be noted that our data on the role of TLR4 in proinflammatory cytokine induction by N. meningitidis are derived from experiments with murine peritoneal macrophages. However, recent studies with cells transfected with human receptors, i.e. chinese hamster ovary fibroblasts transfected with human TLR4 and TLR2 19 or with HeLa cells transfected with human TLR2 35, suggest that for the response to meningococci non-TLR4 pathways are also of importance in the human situation. Recently, we and others provided evidence for a role of non-LPS components of N. meningitidis in cytokine induction and therefore in the pathogenesis of FMS 17- 19. In the present study we show that pro-inflammatory cytokine induction by N. meningitidis can occur independently of CD14 and TLR4. Therefore, it can be

45 speculated that therapeutic strategies designed to block either LPS, CD14 or TLR4 are likely to be insufficient for the complete abrogation of the harmful proinflammatory cytokine response during FMS.

Acknowledgments

The authors wish to thank Liesbeth Jacobs for assistance with the stimulation experiments in human PBMCs and Ineke Verschueren for assistance with the stimulation experiments in murine peritoneal macrophages.

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47 19. Ingalls RR, Lien E, Golenbock DT. Membrane-associated proteins of a lipopolysaccharide- deficient mutant of Neisseria meningitidis activate the inflammatory response through toll-like receptor 2. Infect Immun. 2001;69:2230-2236. 20. Holten E. Serotypes of Neisseria meningitidis isolated from patients in Norway during the first six months of 1978. J Clin Microbiol. 1979;9:186-188 21. Steeghs L, den Hartog R, den Boer A, Zomer B, Roholl P, van der Ley P. Meningitis bacterium is viable without endotoxin. Nature. 1998;392:449-450 22. Westphal O, Jann JK. Bacterial lipopolysacharide extraction with phenol- water and further application of the procedure. Methods Carbohydr Chem. 1965;5:83-91 23. Bogman MJ, Dooper IM, van de WInkel JG, Tax WJ, Hoitsma AJ, Assmann KJ, Ruiter DJ, Koene RA. Diagnosis of renal allograft rejection by macrophage immunostaining with a CD14 monoclonal antibody, WT14. Lancet. 1989;8657:235-238 24. Pollack MM, Espinoza AM, Guelde G, Koles NL, Wahl LM, Ohl CA. lipopolysaccharide (LPS)-specific monoclonal antibodies regulate LPS uptake and LPS-induced tumor necrosis factor-alpha responses by human monocytes. J Infect Dis. 1995;63:794-804 25. Drenth JP, van Deuren M, van der Ven Jongekrijg J, Schalkwijk CG, van der Meer JW. Cytokine activation during attacks of the hyperimmunoglobulinemia D and periodic fever syndrome. Blood. 1995;85:3586-3593 26. Netea MG, Demacker PN, Kullberg BJ, Boerman OC, Verschueren I, Stalenhoef AF, van der Meer JWM. Low-density lipoprotein receptor-deficient mice are protected against lethal endotoxemia and severe gram-negative infections. J Clin Invest. 1996;97:1366- 1372 27. Gangloff SC, Hijiya N, Haziot A, Goyert SM. Lipopolysaccharide structure influences the macrophage response via CD14-independent and CD14-dependent pathways. Clin Infect Dis. 1999;28:491-496 28. Heumann D, Barras C, Severin A, Glauser MP, Tomasz A. Gram-positive cell walls stimulate synthesis of tumor necrosis factor alpha and interleukin-6 by human monocytes. Infect. Immun. 1994;62:2715-2721 29. Hampton RY, Golenbock DT, Penman M, Krieger M, Raetz CR. Recognition and plasma clearance of endotoxin by scavenger receptors. Nature. 1991;352:342-344 30. Flo TH, Ryan L, Kilaas L, Skjak-Braek G, Ingalss RR, Sundan A, Golenbock DT, Espevik T. Involvement of CD14 and beta2 integrins in activating cells with soluble and particulate lipopolysaccharides and mannuronic acid polymers. Infection and Immunity. 2000;68:6770-6776 31. Hirschfeld M, Weis JJ, Toschakov V, Salowski CA, Cody MJ, Ward DC, Qureshi N, Michalek SM, Vogel SN. Signalling by Toll-Like Receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect Immun. 2001;69:1477-1482 32. Werts C, R.J. T, Mathison JC, Chuang TH, Kravchenko V, Saint Girons I, Haake DA, Godowski PJ, Hayashi F, Ozinsky A, Underhill DM, Kirschning CJ, Wagner H, Aderem A, Tobias PS, Ulevitch RJ. Leptopspiral lipopolysaccharide activates cells through a TLR2- dependent mechanism. Nature Immunology. 2001;2:286-288 33. Netea MG, van Deuren M, Kullberg BJ, Cavaillon JM, van Der Meer JWM. Does the shape of lipid A determine the interaction of LPS with Toll-like receptors. Trends Immunol. 2002;23:135- 34. Massari P, Henneke P, Ho Y, Latz E, Golenbock DT, Wetzler LM. Immune stimulation by neisserial porins is Toll-like receptor 2 and MyD88 dependent. J Immunol. 2002;168:1533- 1537 35. Pridmore AC, Wyllie DH, Abdillahi F, Steeghs L, van der Ley P, Dower SK, Read RC. A lipopolysaccharide-deficient mutant of Neisseria meningitidis elecits attenuated cytokine release by human macrophages and signal via toll-like receptor (TLR)2, but not via TLR4/ MD2. The Journal of Infectious Diseases. 2001;183:89-96

48 3

Complement activation and complement-dependent inflammation by Neisseria meningitidis are independent of lipopolysaccharide. 4

Tom Sprong, Anne-Sophie Møller, Anna Bjerre, Elisabeth Wedege, Peter Kierulf, Jos W.M van der Meer, Petter Brandtzaeg, Marcel van Deuren and Tom Eirik Mollnes. Infection and Immunity. 2004;72(6):3344-9 Abstract

Fulminant meningococcal sepsis has been termed the prototypical lipopolysaccharide (LPS)-mediated gram-negative septic shock. Systemic inflammation by activated complement and cytokines is important in the pathogenesis of this disease. We investigated the involvement of meningococcal LPS in complement activation, complement-dependent inflammatory effects, and cytokine or chemokine production. Whole blood anticoagulated with lepirudin was stimulated with wild-type Neisseria meningitidis H44/76 (LPS+), LPS-deficient N. meningitidis H44/76lpxA (LPS–), or purified meningococcal LPS (NmLPS) at concentrations that were relevant to meningococcal sepsis. Complement activation products, chemokines, and cytokines were measured by enzyme-linked immunosorbent assays, and granulocyte CR3 (CD11b/CD18) upregulation and oxidative burst were measured by flow cytometry. The LPS+ and LPS– N. meningitidis strains both activated complement effectively and to comparable extents. Purified NmLPS, used at a concentration matched to the amount present in whole bacteria, did not induce any complement activation. Both CR3 upregulation and oxidative burst were also induced, independent of LPS. Interleukin-1β, tumor necrosis factor alpha, and macrophage inflammatory protein 1α production was predominantly dependent on LPS, in contrast to IL-8 production, which was also markedly induced by the LPS– meningococci. In this whole blood model of meningococcal sepsis, complement activation and the immediate complement-dependent inflammatory effects of CR3 upregulation and oxidative burst occurred independent of LPS.

52 Introduction

Invasive disease caused by the gram-negative bacterium Neisseria meningitidis is a life-threatening infection in children and young adults. Meningococcal septic shock (or fulminant meningococcal sepsis [FMS]), the prototype of overwhelming gram- negative sepsis, is feared especially because it is able to cause devastating disease with a high case fatality rate. The pathogenic mechanisms leading to FMS are 4 thought to be the uncontrolled growth of meningococci in the circulation, resulting in a massive activation of diverse inflammatory systems, such as the complement system, the cytokine network, and the coagulation cascade 1,2.

In FMS, high lipopolysaccharide (LPS) levels are correlated with an increased bacterial load, high levels of pro- and anti-inflammatory cytokines, increased complement activation, the induction of intravascular coagulation, and a poor outcome. Therefore, meningococcal LPS is regarded as the principal bacterial pathogenic element during FMS 3.

The complement cascade plays a dual role in the pathogenesis of meningococcal infections. A complement deficiency predisposes some individuals to meningococcal infection 4-6, but on the other hand, with FMS, extensive complement activation is correlated with severe disease and a poor outcome 7,8. Thus, the complement system plays an important role in the first line of defense against meningococcal infection, whereas massive activation of the complement system contributes to the development of shock.

Complement is activated on the meningococcal surface by one or more of the three initial complement-activating pathways, i.e., the classical, the lectin, and the alternative pathways (Figure 1). After the activation of complement factor 3 (C3) by any of these pathways, a C5 convertase is formed, which cleaves the pivotal C5 molecule into C5a and C5b. C5b is the initial molecule in the formation of the terminal C5b-9 complement complex (TCC). Membrane-associated TCC, also designated the C5b-9 membrane attack complex, leads to lysis of the bacterium, whereas C5a is a potent anaphylatoxin 9. Experimentally, the role of the complement system in inflammation by N. meningitidis and Escherichia coli was recently given more emphasis by our observation that granulocyte CR3 (CD11b/CD18) upregulation and the formation of reactive oxygen species in human whole blood, key elements in the induction of vascular damage during sepsis, are highly dependent on C5a 10,11.

Recent in vitro studies suggested that components of N. meningitidis other than LPS may also contribute to the inflammatory response of the host. Using a

53 meningococcal mutant that was deficient for LPS in the outer membrane, we and others have shown that proinflammatory cytokines can be induced independent of LPS in cultured human cells 12-14. In addition, we showed that purified meningococcal LPS (NmLPS) is a poor activator of complement, whereas experiments with outer membrane vesicles that were depleted of or completely lacking LPS suggested that non-LPS components in the bacterial outer membrane are the principal complement activators in a whole blood model 15.

For the present study, LPS-containing N. meningitidis, LPS-deficient N. meningitidis, and purified meningococcal LPS were used in a human whole blood model of meningococcal sepsis to determine the role of LPS in complement activation, the complement-dependent inflammatory processes of CR3 upregulation and oxidative burst, and cytokine production.

Materials and Methods

Equipment and reagents. All materials used in the stimulation experiments were endotoxin-free. The polypropylene tubes used were either Nunc cryotubes (Nalgene Nunc, Roskilde, Denmark) or Falcon tubes (Becton Dickinson, Franklin Lanes, N.Y.). Phosphate- buffered saline (PBS) was produced in the laboratory, Dulbecco’s medium was obtained from Invitrogen Corporation (Paisley, Scotland), and lepirudin (Refludan) was purchased from Hoechst (Frankfurt am Main, Germany). Flow cytometry was performed with a FACScalibur instrument (Becton Dickinson San Jose, Calif.), and optical densities were determined with an MRX microplate reader (Dynex Technologies, Denkendorf, Germany).

Bacterial strains. N. meningitidis H44/76 (LPS+) is an isolate from a patient with invasive meningococcal disease 16. This strain is the production strain for the Norwegian group B OMV vaccine and is an international reference strain 17, serologically classified as B:15:P1.7,16, immunotype L3,7,9, as determined by the use of anti- LPS monoclonal antibodies (MAbs) in a dot blotting assay for immunotyping 18. The LPS-deficient strain N. meningitidis H44/76lpxA (LPS–) is a viable isogenic mutant of strain H44/76 that lacks LPS in the outer membrane and was a kind gift from Peter van der Ley and Liana Steeghs (Dutch Vaccine Institute, Bilthoven, The Netherlands). It was constructed by insertional inactivation of the lpxA gene, which is essential for the first committed step of biosynthesis of LPS, as described by Steeghs et al 19. For the present study, batch suspensions of H44/76lpxA were tested and showed no reactivity in a Limulus amebocyte lysate (LAL) assay, a highly

54 specific and sensitive test for LPS. As shown by Steeghs et al., the expression level of the integral outer membrane proteins by the LPS-deficient mutant is similar to that of the wild-type strain; however, the outer membrane phospholipid composition is altered, with a switch to mostly short-chain, saturated fatty acids 20. LPS-deficient mutant meningococci were grown overnight on Kellog’s medium and resuspended in Hanks’ balanced salts solution (Invitrogen). Bacteria were heat inactivated at 60°C for 40 min. The concentration of the bacteria was determined by measuring the 4 optical density at 630 nm. NmLPS was isolated from strain H44/76 by phenol-water extraction 21 followed by DNase, RNase, and proteinase K treatment. The batch of LPS used for this study was previously shown to be highly reactive in the LAL assay and a potent inducer of cytokine production 22. Quantification of the amount of LPS in LPS+ N. meningitidis H44/76 bacteria was performed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining of LPS. The LPS level was determined by digital scanning, with purified H44/76 LPS used as the standard 13,23.

Whole blood model A recently developed whole blood model for the study of N. meningitidis-induced inflammation was used 11. The model is based on anticoagulation with lepirudin, a recombinant hirudin which is a highly specific thrombin inhibitor that minimally influences complement activation or cytokine production. Whole blood from healthy, adult volunteers was collected in polypropylene tubes containing lepirudin (50- µg/ml final concentration). Informed consent was obtained from all donors prior to the experiments, and the human experimentation guidelines of the local ethics committee were followed in conducting the research. Immediately after being drawn, the whole blood (1 ml) was incubated with the stimulants (0.1 ml).

For the determination of granulocyte CR3 (CD11b/CD18) expression and oxidative burst, samples were incubated for 10 min at 37°C in a water bath, followed by immediate processing for flow cytometry.

For the detection of complement activation, samples were incubated for 1 h at 37°C in a Coulter mixer (Coulter, Luton, England). Complement activation was stopped by the addition of EDTA to a final concentration of 20 mM, after which samples were centrifuged at 1,400 x g for 10 min. The plasma was collected and stored at –70°C until samples were assayed in one batch.

Samples for the measurement of tumor necrosis factor alpha (TNF-α), interleukin- 1β (IL-1β), IL-8, and macrophage inflammatory protein 1α (MIP-1α) were processed as described for complement activation, but the incubation time was increased to 2 h.

55 Oxidative burst Oxidative burst was measured by using a commercially available Burst-test kit (Orpegen Pharma, Heidelberg, Germany). After incubation as described above, samples were treated according to the manufacturer’s instructions and assayed by flow cytometry. Granulocytes were identified in a forward scatter-side scatter plot by their typical patterns.

CR3 (CD11b/CD18) expression After being incubated as described above, cells were washed once in sterile PBS and fixed with 0.5% (vol/vol) paraformaldehyde in an equal volume of PBS for 4 min at 37°C. Cells were stained with an anti-CD11b-PE or isotype control (γ2a) (Becton Dickinson) antibody for 15 min at room temperature. An anti-CD14-FITC (Becton Dickinson) antibody and the nuclear dye LDS-751 (Molecular Probes Inc., Eugene, Oreg.) were used to discriminate granulocytes in washed whole blood.

Enzyme immunoassays. Complement activation was measured in plasma by enzyme immunoassays based on MAbs that were highly specific for the activation products; the native, nonactivated components did not interfere in the assays. Activation of the three initial pathways was selectively detected.

(i) C1rs-C1inh complexes. Activation of the classical complement pathway was determined as described previously 24. C1rs-C1 inhibitor (C1rs-C1inh) complexes were measured by using MAb Kok-12, specific for a neoepitope exposed only when C1inh is in complex with its substrates. In brief, microtiter plates were coated with the Kok-12 antibody, the antibody was reacted with plasma or controls, and the complex was detected with a cocktail of anti-C1r and anti-C1s antibodies. Human serum activated with heat-aggregated immunoglobulin G was used as a standard and was defined to contain 1,000 arbitrary units (AU)/ml.

(ii) C4bc. Activation of the classical and lectin pathways was determined by an assay using a MAb specific for a neoepitope that is exposed in activated C4 25. The C4bc assay detects the sum of the activation products C4b, iC4b, and C4c. The same standard was used as for the C1rs-C1inh assay, defined to contain 1,000 AU/ml. Both the MAb for this assay and Kok-12 were a kind gift from C. E. Hack, Department of Immunopathology, Sanquin Research, Amsterdam, The Netherlands.

(iii) C3bBbP complexes. Activation of the alternative pathway was detected by measuring the alternative convertase C3b-Bb-properdin (C3bBbP), as recently described 10. The capture antibody used was an antiproperdin antibody, and the detection antibody was a polyclonal anti-C3 antibody. The standard was normal

56 human serum activated with zymosan and defined to contain 1,000 AU/ml.

(iv) TCC. Activation of the terminal pathway was quantified by using MAb aE11, specific for C9 incorporated in the fluid-phase SC5b-9 complex, as described previously 26. The assay was modified, with a biotinylated anti-C6 antibody used as the detection antibody. Human serum activated with zymosan was used as the standard. 4 An overview of the complement activation markers measured is illustrated in Figure1.

Cytokines and chemokine Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used according to the manufacturers’ instructions to quantify TNF-α, IL-1β, IL-8 (Duo Set ELISA development system [R&D Systems, Minneapolis, Minn.] for all three), and MIP-1α (MIP-1α Cytoscreen [Biosource International, Cammarillo, Calif.]). The lower limits of detection were 8 pg/ml for TNF-α, IL-1β, and IL-8 and 2 pg/ml for MIP-1α.

Classical Lectin Alternative pathway pathway pathway

C1q / C1r / C1s MASP 2 C3/C3b C1rs- D C1inh C4 B P C4bc C2 C3 C3bBbP

C5b-9 C5a

TCC

Figure 1. Schematic overview of the complement system. Boxes indicate specific complement activation products measured in the present study and their relation to specific complement activation pathways. C1rs-C1inh complexes reflect classical pathway activation, C4bc reflects both classical and lectin pathway activation, and C3bBbP reflects alternative pathway activation. TCC is an activation product of the final common and terminal pathways.

57 LAL assay Quantification of the biological activity of LPS in the bacterial strains was performed as described previously 27. E. coli O55:B5 LPS was used as a reference. The lower limit of detection was 0.25 endotoxin units/ml.

Statistics A two-way analysis of variance (ANOVA) for repeated measures was used to test the data for statistically significant differences. P values of <0.05 were considered statistically significant

Results Complement activation by LPS+ and LPS– N. meningitidis or purified NmLPS. Assays for C1rs-C1inh (classical pathway), C4bc (classical and lectin pathways) and C3bBbP (alternative pathway) complexes were used to determine the initial complement activation pathways, whereas soluble TCC, the final product of the terminal pathway, was used as an indicator of total complement activation (Figure 1). The amount of LPS per 106 N. meningitidis cells was estimated by SDS-PAGE analysis; this showed that 106 bacteria contained approximately 5 ng of LPS. In Figures 2 and 3, the x axes are calibrated for the numbers of LPS+ N. meningitidis cells as well as the amounts of LPS that are present in the bacteria. In this way, the activities of the bacteria and purified LPS can be compared quantitatively.

The levels of the markers of initial pathway activation, C1rs-C1inh, C4bc, and C3bBbP, as well as that of TCC from the terminal pathway, were all increased from the baseline after stimulation with LPS+ or LPS– N. meningitidis (Figure 2). The activation of initial complement activation products was seen at concentrations of 106 meningococci/ml or higher, which correspond to approximately 5 ng of NmLPS or more/ml. In contrast, purified NmLPS did not induce complement activation at concentrations up to 1,000 ng/ml. The combination of purified NmLPS and LPS– N. meningitidis induced complement activation to a similar extent as LPS+ or LPS– N. meningitidis (not shown). These data indicate that complement activation by N. meningitidis in human whole blood occurs independently of the LPS outer membrane component.

Granulocyte CR3 upregulation and oxidative burst by LPS+ and LPS– N. meningitidis or purified NmLPS. Recently, the CR3 upregulation and oxidative burst induced by N. meningitidis in

58 35 60 Figure 2. C1rs-c1inh C4bc Complement activation by LPS+ and LPS– N. 30 50 ** * meningitidis and by 40 ** purified NmLPS. 25 The results are for the 30 formation of C1rs-C1inh (classical pathway), AU/mL 20 C4bc (classical and 20 lectin pathways), 15 C3bBbP (alternative 10 pathway), and TCC 4 (final common pathway) 0 0 after the stimulation of 0.01 1 100 10000 0.01 1 100 10000 lepirudin-treated human whole blood for 1 h + 900 25 with LPS (diamonds) C3bBbP TCC or LPS– (squares) N. 800 meningitidis or purified 20 NmLPS (circles). The x 700 ** axes are calibrated for + 15 the numbers of LPS 600 N. meningitidis cells as ** well as the amounts of

AU/mL 500 10 NmLPS that are present in the LPS+ bacteria, 400 according to SDS-PAGE 5 300 analysis. Medians and upper quartiles are 0 0 presented from four 0.01 1 100 10000 separate experiments. *, 0 1 100 10000 P < 0.05; **, P < 0.01, by two-way ANOVA. Dashed LPS (ng/mL) OR N. LPS (ng/mL) OR N. lines indicate baseline 6 meningitidis (0.2 x10 /mL) meningitidis (0.2 x106/mL) complement activation. granulocytes were shown to be complement dependent. Therefore, we investigated the role of LPS in these processes.

Both LPS+ and LPS– N. meningitidis at concentrations of 106/ml induced marked granulocyte CR3 upregulation and oxidative burst. The addition of purified NmLPS to LPS-deficient N. meningitidis did not influence the CR3 upregulation or oxidative burst (not shown). Notably, purified NmLPS did not induce significant CR3 upregulation or oxidative burst at concentrations up to 1,000 ng/ml (Figure 3). These results indicate that the upregulation of CR3 expression and the oxidative burst in whole blood granulocytes after stimulation with N. meningitidis are not dependent on NmLPS.

Cytokine and chemokine induction by LPS+ and LPS– N. meningitidis. Cytokine and chemokine induction in a human whole blood model of meningococcal sepsis was assessed after 2 h of incubation with LPS+ or LPS– N. meningitidis (Figure 4). No cytokine or chemokine production was seen in this whole blood model in the absence of a stimulus. LPS+ N. meningitidis was a potent inducer of IL-1β, TNF-α, IL-8, and MIP-1α production, as it was able to induce cytokine

59 Figure 3. Granulocyte CR3 upregulation and oxidative burst by LPS+ and LPS– N. meningitidis and by purified NmLPS. The results are for CR3 (CD11b/CD18) upregulation 350 30 and oxidative burst in human CR3 Oxidative granulocytes after the 300 25 burst stimulation of lepirudin-treated human whole blood for 10 250 + 20 min with LPS (diamonds) or – 200 LPS (squares) N. meningitidis 15 or purified NmLPS (circles). MFI 150 The x axes are calibrated + 10 for the numbers of LPS N. 100 meningitidis cells as well as 5 the amounts of NmLPS that 50 are present in the bacteria, 0 0 according to SDS-PAGE analysis. One representative 0.01 1 100 10000 10 100 1000 of three separately performed experiments is shown. Dashed LPS (ng/mL) OR N. LPS (ng/mL) OR N. lines indicate baseline CR3 6 6 meningitidis (0.2 x10 /mL) meningitidis (0.2 x10 /mL) expression or oxidative burst.

and chemokine production at concentrations higher than 104 meningococci/ml. IL- 1β, TNF-α, IL-8, and MIP-1α production by LPS– meningococci was significantly reduced compared to that by LPS+ N. meningitidis (P < 0.05 for all cytokines and chemokines measured). However, marked IL-8, and to a lesser extent, TNF-α and MIP-1α production was also induced by LPS– N. meningitidis, suggesting that these mediators can also be induced in the absence of LPS (Figure 4).

4000 30000 IL-1ȕ TNFĮ * 25000 ** 3000 20000

2000 15000 pg/mL 10000 1000 5000

0 0 0.0001 0.01 1 100 0.0001 0.01 1 100

12000 30000 IL-8 MIP-1Į Figure 4. 10000 * 25000 * Cytokines and chemokines induced by LPS+ and LPS– N. 8000 20000 meningitidis. The results are for the 6000 15000 production of IL-1β, TNF-α, pg/mL IL-8, and MIP-1α after the 4000 10000 stimulation of lepirudin-treated human whole blood for 2 h 2000 5000 with LPS+ (diamonds) or LPS– (squares) N. meningitidis. 0 0 Mean cytokine or chemokine 0.0001 0.01 1 100 0.0001 0.01 1 100 production levels from three separate experiments are N. meningitidis N. meningitidis presented. *, P < 0.05, by (x106/mL) (x106/mL) two-way ANOVA

60 Discussion

The principal finding of the present study was that complement activation and the complement-dependent inflammatory events of CR3 upregulation and oxidative burst are independent of LPS in a whole blood model of meningococcal sepsis.

The construction of the LPS-deficient meningococcal mutant H44/76lpxA has 4 made it possible to study the relative importance of the LPS moiety of a gram- negative bacterium in diverse inflammatory processes 19. We used LPS-deficient N. meningitidis to assess the involvement of meningococcal LPS in complement activation, complement-dependent inflammatory effects, and cytokine or chemokine production in a human whole blood model of meningococcal sepsis. The lack of reactivity of the LPS– strain in a LAL assay confirmed the absence of LPS in the batch used for the experiments. The estimate that 106 LPS+ meningococci contained approximately 5 ng of meningococcal LPS was in line with previous estimations 13,22.

N. meningitidis is exclusively a human commensal or pathogen, and none of the animal models available accurately simulate FMS. Therefore, we used an in vitro experimental system approaching, as closely as possible, the human in vivo situation 10,11. The principle of this model is to keep all ambient inflammatory systems intact so that they can be activated and mutually interact but still to avoid coagulation. Because most anticoagulants, such as EDTA, citrate, and heparin, interact with critical steps in the inflammatory network, this model uses the highly specific thrombin inhibitor lepirudin, a recombinant hirudin analogue, as an anticoagulant. CR3 upregulation and oxidative burst were found to be induced to a similar extent by live or heat-inactivated bacteria in this model. Since these processes are highly complement dependent, this indicates that complement activation is also not affected by inactivation of the bacteria. Therefore, heat-inactivated bacteria were used because of practical considerations, as live meningococci constitute a significant health hazard 11.

LPS is present in solution in the form of micellar structures, which is quite distinct from its organization when it is present in the outer membrane. Hypothetically, the physicochemical presentation of LPS could affect the complement-activating ability, favoring membrane-bound LPS as the stronger complement activator. However, the observation that LPS-containing meningococci activate complement to a similar extent as LPS-deficient meningococci suggested that this is not the case, as did the absence of an increase in the complement-activating ability when isolated LPS was added back to the LPS-deficient strain. This is supported by a previous study in

61 which neisserial outer membrane vesicles (OMVs) containing LPS were equipotent to OMVs deficient for LPS 15.

Complement activation by LPS+ or LPS– N. meningitidis was seen at concentrations of 106/ml or higher. This is a concentration of meningococci that is frequently found during FMS 3,28; 106 LPS+ N. meningitidis cells contain approximately 5 ng of LPS. In contrast, complement was not activated by purified LPS at concentrations up to 1,000 ng/ml. This suggests that during meningococcal sepsis, complement activation is not induced by LPS, but by other constituents of N. meningitidis.

Members of our laboratory previously reported that the upregulation of CR3 and the induction of oxidative burst in granulocytes in this whole blood model are dependent on complement activation 10,11. When complement is activated, C5a is formed, and this anaphylatoxin reacts with the C5a receptor on granulocytes and monocytes to induce the upregulation of CR3. CR3 is the principal receptor involved in the phagocytosis of C3b-coated bacteria, and when upregulated, it will enhance phagocytosis of the bacteria and in this way stimulate the oxidative burst process. Our results indicate that CR3 expression and oxidative burst are mediated by non- LPS components of N. meningitidis.

It is commonly accepted that LPS extracted from diverse gram-negative bacteria activates complement readily via classical and alternative pathway-dependent mechanisms 29,30. In addition, LPS has been shown to be able to upregulate CR3 expression and to prime neutrophils for oxidative burst 31-34. However, for the experiments presented in these studies, very high concentrations of LPS were applied (>5 to 10 µg/ml), incubation times of longer than 30 min were employed for granulocyte activation, or isolated cell cultures or plasma were used. In addition, it was recently shown that a contaminating substance of commercially available LPS is, at least in part, responsible for the upregulation of neutrophil responses 35. We claim that the conditions used in our study more closely resemble the in vivo situation than those used in previous studies. In our model, purified NmLPS was tested in whole blood at concentrations that are relevant for the situation encountered during FMS. The relatively short incubation times mimic the function of the complement cascade system, the components of which are present in plasma and able to be immediately activated by an invading pathogen, in this way avoiding the contribution of activation processes secondary to other inflammatory events.

We propose that the observed weak complement-activating property of NmLPS is not specific for this type of LPS, as it has a relatively short saccharide side chain, but that other types of LPS are also weak complement activators at relevant

62 concentrations. Preliminary data from our laboratory suggest that this is the case for E. coli LPS in the same in vitro model. In addition, in primate models, complement is not activated after E. coli LPS infusion, but after whole E. coli cell infusion, marked complement activation occurs within 15 min 36,37. Also, in a human endotoxemia model, no complement activation is observed, whereas significant cytokine production, endothelial cell activation, and activation of the coagulatory 38 and fibrinolytic systems are found . 4

The bacterial components that are involved in the activation of complement, the upregulation of CR3 expression, and oxidative burst remain elusive and will be subjected to further investigation. It was previously reported that OMVs isolated from LPS-deficient N. meningitidis induce substantial complement activation 15. This suggests that the non-LPS components that induce complement activation reside in the outer membrane. The lectin pathway seems to be an important player in complement activation by meningococci when they are grown on Kellog’s medium and studied in the whole blood model 11. Since mannose-binding lectin binding to the bacteria occurs via the outer membrane proteins porin and opacity protein (OpA) independently of LPS 39, it is reasonable to suggest that the porins and OpA are responsible, at least in part, for the observed complement activation and subsequent CR3 upregulation and oxidative burst.

The proinflammatory cytokines TNF-α and IL-1β are primary cytokine mediators during meningococcal sepsis, whereas IL-8 and MIP-1α are important chemotactic proteins that are important for pathogenesis 40-42. We found that LPS– N. meningitidis induced marked IL-8, and to a lesser extent, TNF-α and MIP-1α production in the whole blood model, indicating that bacterial components other than LPS are also proficient at inducing cytokine or chemokine production. However, LPS+ N. meningitidis was more potent in the induction of these proinflammatory mediators, in particular TNF-α and MIP-1α, indicating that LPS is an important factor involved in the regulation of these processes, which is in agreement with previous reports 12- 14. It is tempting to speculate that the observed LPS-independent induction of IL-8, and possibly also TNF-α and MIP-1α, is mediated or potentiated by the activation of complement factor C5a, as has been suggested previously 10,43,44.

We conclude that complement activation occurs independently of the LPS moiety of N. meningitidis. Therefore, CR3 upregulation and oxidative burst, previously shown to be highly complement dependent, are also independent of LPS. Cytokine and chemokine production can be induced via LPS-dependent as well as LPS- independent mechanisms.

63 Acknowledgements

We thank Anne Pharo, Gunni Ulvund, Hilde Fure, Karin Bolstad, and Berit Nyland for their excellent technical assistance. Peter van der Ley and Liana Steeghs are thanked for supplying the LPS-deficient meningococcal strain.

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Human lipoproteins have divergent neutralizing effects on E. coli LPS, N. meningitidis LPS, and complete Gram-negative bacteria. 5

Tom Sprong, Mihai G Netea, Peter van der Ley, Trees J. Jansen, Liesbeth E. Jacobs, Anton F.H. Stalenhoef, Jos W.M. van der Meer and Marcel van Deuren. Journal of Lipid Research. 2004;45(4):742-9 Abstract

The use of lipoproteins has been suggested as a treatment for Gram-negative sepsis because they inhibit lipopolysaccharide (LPS)-mediated cytokine production. However, little is known about the neutralizing effects of lipoproteins on cytokine production by meningococcal LPS or whole Gram-negative bacteria. We assessed the neutralizing effect of LDLs, HDLs, and VLDLs on LPS- or whole bacteria- induced cytokines in human mononuclear cells. A strong inhibition of Escherichia coli LPS-induced interleukin-1β (IL-1β), tumor necrosis factor-α, and IL-10 by LDL and HDL was seen, whereas VLDL had a less pronounced effect. In contrast, Neisseria meningitidis LPS, in similar concentrations, was neutralized much less effectively than E. coli LPS. Effective neutralization of meningococcal LPS required a longer interaction time, a lower concentration of LPS, or higher concentrations of lipoproteins. The difference in neutralization was independent of the saccharide tail, suggesting that the lipid A moiety accounted for the difference. Minimal neutralizing effects of the lipoproteins were observed on whole E. coli or N. meningitidis bacteria under all conditions tested. These results indicate that efficient neutralization of LPS depends on the type of LPS, but a sufficiently long interaction time, a low LPS concentration, or high lipoprotein concentration also inhibited cytokines by the less efficiently neutralized N. meningitidis LPS. Irrespective of these differences, whole bacteria showed no neutralization by lipoproteins.

70 Introduction

Lipopolysaccharide (LPS), a major structural and functional component of the outer membrane of Gram-negative bacteria, is considered to be a major pathogenic element in Gram-negative septicemia 1. LPS interacts with a variety of cells to induce a wide array of inflammatory mediators. Among these, the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) and the antiinflammatory cytokine IL-10 are key factors in the development of shock and disseminated intravascular coagulation 2. 5

Fulminant meningococcal sepsis (FMS) is an important cause of morbidity and mortality worldwide, especially in children and young adults. In addition, it is the prototypical Gram-negative sepsis syndrome, in which LPS concentrations may become extremely high and correlate with cytokine levels, morbidity, and mortality 3,4.

LPS is composed of a polysaccharide tail to which the lipid portion (lipid A) is attached via 2-keto-3-deoxyoctanate (KDO). Lipid A, a glucosamine-derived phospholipid, is considered to be the toxic moiety of LPS and is responsible for many of the biological effects of LPS 5.

In the past, various strategies have been explored to combat the deleterious effects of LPS during sepsis. However, none of these anti-LPS strategies has shown a benefit on outcome 6-9; thus, the search for a LPS-neutralizing treatment modality continues. Lipoproteins have been shown to bind LPS and to neutralize LPS- induced cytokine production 10. Also, in animal models of sepsis, the administration of lipoproteins was found to be beneficial 11,12, and LPS infusion experiments in humans showed that the inflammatory response is inhibited by infusion of lipoproteins 13,14. For these reasons, lipoprotein infusion during septicemia has been advocated as an adjunctive therapy. FMS especially, being the prototypical LPS-mediated disease, has been suggested as a candidate disease for treatment with lipoproteins.

To date, most investigators assessing the effect of lipoproteins have used LPS derived from Enterobacteriaceae species (Escherichia coli or Salmonella typhimurium). However, the LPSs are a heterogeneous group of molecules with interspecies differences in the length and position of the acyl chains in the lipid A portion of the LPS, the length and polarity of the polysaccharide tail, and the formation of supramolecular structures 5,15,16. These differences in LPS have been shown to modulate the capacity to induce cytokines 17-19, and it is likely that these

71 differences also affect the interaction with lipoproteins. In addition, LPS is not the only component of Gram-negative bacteria that induces cytokines 20-22. Therefore, the question is to what extent an adjunctive therapy that targets only LPS, such as lipoproteins, will inhibit cytokine production by complete bacteria.

In the present study, we addressed these questions by comparing the inhibitory effect of lipoproteins on cytokine induction by LPS of E. coli and Neisseria meningitidis in human mononuclear cells. In addition, we assessed the cytokine- inhibiting capacity of lipoproteins on whole E. coli and N. meningitidis bacteria.

Materials and methods LPSs and meningococci E. coli 055:B5 TCA and phenol-extracted LPS were obtained from Sigma Chemical Co. (St. Louis, MO). E. coli K12, D31m4 rough (Re) LPS consisting of lipid A(KDO)2 and lacking the polysaccharide tail was purchased from List Biological Laboratories (Campbell, CA). The lipid A of E. coli LPS is a hexaacyl phospholipid with the fatty acyl acids asymmetrically distributed 23. Meningococcal LPS was isolated by the phenol/water extraction method as described by Westphal and Jann 24. H44/76 LPS was isolated from strain H44/76 25. Meningococcal H44/76rfaC LPS [lipid A(KDO)2] was isolated from the isogenic mutant rfaC of strain H44/76, as described by Stojiljkovic et al. 26. Similar to E. coli LPS, meningococcal LPS has a hexaacyl lipid A; however, in the lipid A of the meningococcal LPS, the fatty acids are symmetrically distributed 27. Whereas E. coli LPS has a polysaccharide tail, meningococcal LPS has an oligosaccharide tail (Table 1).

The commercial E. coli LPS was contaminated with protein (8.5%, by Lowry assay) and nucleic acid (6.1%, by spectrophotometry). The meningococcal LPS was not contaminated with protein, and the nucleic acid content was 4.4%. LPS was suspended in culture medium before use.

E. coli ATCC 35218 is an international reference strain. N. meningitidis H44/76 was isolated from a patient with invasive disease 25. Stationary-phase bacteria grown in liquid medium were used, and bacteria were heat-inactivated (1 h, 56°C).

Table 1. Characteristics of the different LPS-types. Modified from Refs 19,23,27. LPS lipid A - acyl chains Lipid A - molecular saccharide tail conformation E. coli 055:B5 Hexaacyl asymmetrical polysaccharide E. coli K12, D31m4 Hexaacyl asymmetrical absent N. men H44/76 Hexaacyl symmetrical oligosaccharide N. men H44/76rfaC Hexaacyl symmetrical absent

72 Molarity of the LPS solutions Measurement of KDO was used to estimate the molarity of the LPS solutions. KDO was measured by spectrophotometry, as described by Weissbach and Hurwitz 28.

Lipoproteins and lipoprotein-depleted plasma Lipoproteins (VLDL, LDL, and HDL) and lipoprotein-depleted plasma (LPDP) were prepared from fresh EDTA plasma by sequential ultracentrifugation as described previously 29 under pyrogen-free conditions using pyrogen-free materials. Lipoproteins prepared in this manner were previously shown to be endotoxin-free by 5 limulus amebocyte lysate (LAL) assay. Lipoproteins were dialyzed for 24 h against 0.05 M phosphate buffer, pH 7.4, containing 5 µM EDTA, with one exchange of the buffer (LPDP is the fraction of plasma that is obtained after removing the lipid fractions). Lipoproteins were isolated before each experiment from different donors. The concentration of total lipid in the lipoprotein fractions was determined based on total cholesterol as a quantitating unit. Cholesterol in the different lipoprotein fractions was measured by spectrophotometry using a commercially available kit and a Hitachi 747 apparatus (Roche Diagnostics, Almere, The Netherlands). Before use, the lipoproteins were diluted 1:1 in LPDP (unless otherwise stated). Average concentrations of lipoprotein in which the LPS or bacteria were preincubated were 376 mg/l (0.60 mmol/l cholesterol) for LDL, 130 mg/l (0.22 mmol/l cholesterol) for HDL, and 378 mg/l (0.20 mmol/l cholesterol) for VLDL. For experiments using a higher concentration of lipoproteins, the lipoproteins were concentrated approximately five times by additional ultracentrifugation of the separate lipoprotein fractions.

Human peripheral blood mononuclear cells Blood for the isolation of peripheral blood mononuclear cells (PBMCs) was drawn in 10 ml EDTA anti-coagulated tubes (Vacutainer System; Becton Dickinson, Rutherford, NJ) from healthy human volunteers. Informed consent was obtained before each experiment, and the guidelines of the local ethics committee were followed in the conduct of these experiments. PBMCs were isolated by density gradient centrifugation over Ficoll-Hypaque (Pharmacia Biotech AB, Uppsala, Sweden). The cells from the interphase were aspirated, washed three times in sterile PBS, and resuspended in culture medium RPMI 1640 (Dutch modification; Flow Labs, Irvine, UK) supplemented with L-glutamine (2 mmol), pyruvate (1 mmol), and gentamicin (50 mg/ml).

Assay procedure Fifty microliters of the lipoprotein diluted in LPDP or LPDP alone was preincubated with the stimuli (50 µl) for a certain time period. Then, the preincubated mixture

73 was added to 100 µl of 5 x 106 PBMCs on 200 µl 96-well plates at 37°C and 5% CO2 and incubated for 24 h. The supernatant was obtained by centrifugation and stored at -20°C until required for the cytokine assays. LPDP alone significantly enhanced LPS or whole bacteria-induced cytokine production in PBMCs compared with culture medium.

Cytokine assays Levels of IL-1β and TNF-α were determined by radioimmunoassay as described by Drenth et al. 30. The lower limit of detection was 80 pg/ml for both cytokines. IL-10 was determined by a commercially available ELISA kit (Pelikine compact; Sanquin, Amsterdam, The Netherlands).

Statistics and calculations Statistical analysis was performed using an unpaired two-sided t-test; P < 0.05 was considered significant. Two-way ANOVA was performed to compare concentration curves (GraphPad Prism; GraphPad Software, Inc.). To calculate the inhibition of cytokines by the lipoproteins, cytokine production in the presence of the lipoprotein diluted in LPDP was expressed as a percentage of production in the presence of LPDP alone.

Results Cytokine induction by N. meningitidis LPS and E. coli LPS A molarity-based comparison of cytokine induction by N. meningitidis and E. coli LPS (TCA-extracted) in PBMCs, based on assay for KDO, showed that meningococcal LPS is more potent in the induction of IL-1β and TNF-α than is E. coli LPS, especially at concentrations of less than 1 pmol/ml (Figure 1). The minimal concentrations to induce significant IL-1β and TNF-α production were 0.0001 pmol/ ml for meningococcal LPS and 0.001 pmol/ml for E. coli LPS. Phenol-extracted E. coli LPS was approximately as potent in the induction of IL-1β and TNF-α as TCA- extracted LPS (data not shown). Based on these data, in subsequent experiments LPS was used at concentrations of 0.14 pmol/ml (equivalent to ⁓0.6 ng/ml meningococcal LPS and 1 ng/ml E. coli LPS) unless otherwise stated

Effect of lipoproteins on cytokines by E. coli and N. meningitidis LPS The effect of the preincubation of equimolar concentrations of E. coli or N. meningitidis LPS with the lipoproteins LDL, HDL, and VLDL for 8 h on IL-1β, TNF-α, and IL-10 production in PBMCs is shown in Figure 2. Lipoproteins did not induce cytokine production in the absence of stimulus, suggesting minimal contamination

74 30 6

20 4 TNF α α α α (ng/mL) (ng/ml) β β β β IL-1 10 2

Figure 1. Cytokines by E. coli and N. 5 meningitidis LPS. Dose‑response curve of IL-1β and TNFα by E. coli and N. meningitidis 0.0001 0.01 1 100 0.0001 0.01 1 100 LPS in human mononuclear cells based on molar concentrations of LPS (pmol/mL) LPS (pmol/mL) LPS (KDO‑assay). N. meningitidis LPS was more potent in the induction N. meningitidis LPS of Il-1β and TNFα than E. coli LPS. Medians and interquartile ranges E. coli LPS (IQR) are presented, n = 5. of the lipoprotein fractions with LPS. E. coli LPS-induced cytokine production was diminished by LDL to values of 20% of production in LPDP; HDL was slightly less effective in the inhibition of cytokine production, whereas VLDL had only a minimal effect. Notably, meningococcal LPS-induced cytokine production was inhibited to a significantly lesser extent by LDL and HDL than cytokine production by E. coli LPS. LDL reduced the N. meningitidis LPS-induced cytokine production to only 50– LDL HDL VLDL 120 120 120

100 100 100

80 80 80 * 60 60 * 60 40 40 40 * * 20 20 * 20 % % of control production * * 0 0 0 IL-1 TNFααα IL-10 IL-1 TNFααα IL-10 IL-1 TNFααα IL-10

E. coli LPS N. meningitidis LPS

Figure 2. Inhibition of LPS-induced cytokines by lipoproteins. Effect of pre-incubation of LPS (0.14 pmol/ml) with LDL, HDL or VLDL for 8 hours on IL-1β, TNFα and IL-10 by human mononuclear cells. LDL and HDL effectively inhibited E. coli LPS, but not N. meningitidis LPS. Results are expressed as a percentage of production in lipoprotein depleted plasma (LPDP). Means and standard error of the mean (SEM) are presented, n = 5, * = P < .05, for the comparison of E. coli LPS with N. meningitidis LPS, by unpaired t-test.

75 IL-1 120

100

20 Figure 3. Effect of pre-incubation of the LPS (0.14 pmol/mL) % of control% production with LDL (0.6 mmol/L) for various time periods on IL- 0 1β production in human mononuclear cells. 0124824Inhibition of LPS‑induced cytokine by LDL was time- t (hours) dependent, the interaction of N. menigitidis LPS with LDL was slower than that of E. coli LPS, and relatively high LDL concentrations were necessary to inhibit IL- E. coli LPS 1β production. Results are expressed as a percentage N. meningitidis LPS of production in lipoprotein depleted plasma (LPDP). Means and SEM are presented, n = 3.

60% of control production for IL-1β and IL-10, whereas TNF-α production was not inhibited at all. HDL inhibited N. meningitidis-induced cytokines less efficiently than LDL, and the effect of VLDL was minimal.

Next, we investigated the kinetics of LPS neutralization by LDL or HDL using different preincubation times of lipoprotein with LPS and different lipoprotein concentrations (Figure 3) . We found that inhibition of E. coli LPS-induced IL-1β by LDL was dependent on the preincubation time and that for maximal inhibition, 4–8 h of preincubation was required. Of importance, the inhibition of N. meningitidis LPS-induced IL-1β production by LDL, compared with E. coli LPS, required much longer preincubation times to achieve noticeable inhibition, and maximal inhibition was seen only after 24 h of preincubation (Figure 3). The results for TNF-α and HDL showed a similar pattern, although inhibition by HDL was less efficient.

For LDL, we investigated the importance of LPS concentration on lipoprotein neutralization (Figure 4) . The experiments showed that TNF-α production was inhibited significantly better by E. coli LPS than by N. meningitidis LPS (P < 0.05 by two-way ANOVA). However, the effectiveness of inhibition by LDL is dependent on the concentration of LPS; at high concentrations, both LPS types showed minimal inhibition, whereas at the lowest concentration tested, both LPS types were inhibited to a large extent. Results for IL-1α showed a similar pattern

In addition, the influence of preincubation with lipoproteins at concentrations encountered in normal physiological, nonseptic conditions was investigated. By

76 TNF 200

150

Figure 4. Inhibition of TNFα at various LPS 100 concentrations Inhibition of LPS-induced TNFα production in human mononuclear cells by LDL (0.6 mmol/L) after stimulation 50 with a concentration range of E. coli or N. meningitidis LPS (0.014 to 140 5 pmol/mL). LDL was preincubated % % of control production with the different LPS-types for 0 8 hours. Results are expressed as a percentage of production in 0.014 0.14 1.4 14 140 lipoprotein depleted plasma (LPDP). LPS (pmol/mL) N. meningitidis was significantly (P < E. coli LPS .05) better inhibited by LDL than E. coli LPS (Two-way ANOVA). Means N. meningitidis LPS and SEM are presented, n = 3. additional ultracentrifugation, lipid concentrations of 1.1 mmol/l for VLDL, 3.43 mmol/l for LDL, and 1.18 mmol/l for HDL were achieved. Concentrated LDL and HDL inhibited TNF-α by both LPS types effectively and to the same extent, whereas concentrated VLDL also inhibited TNF-α but to a lesser extent than LDL or HDL (Figure 5) . Results for IL-1β showed a similar pattern

Influence of lipid A composition and the saccharide tail To investigate whether the observed differences in lipoprotein-dependent inhibition of E. coli and N. meningitidis LPS-induced cytokines were dependent on the lipid A part or the saccharide tail of the LPS molecule, variants of the LPS lacking the

TNF 120

100

40 Figure 5. Influence of concentrated lipoproteins on LPS-induced cytokine production. 20 Effect of pre-incubation of E. coli or N. meningitidis LPS for

% % of control production 8 hours with concentrated lipoprotein fractions on cytokine induction in human mononuclear cells. Lipoproteins were 0 LDL - C HDL-C VLDL-C concentrated approximately 5 times, lipid concentrations in which the LPS was preincubated after concentration were 1.1 E. coli LPS mmol/L for VLDL (VLDL-C), 3.43 mmol/l for LDL (LDL-C) and 1.18 mmol/L for HDL (HDL-C). Means and SEM are presented, N. meningitidis LPS n = 3.

77 polysaccharide tail were used. E. coli Re K12, D31m4 LPS was approximately equipotent to E. coli 055:B5 LPS, and N. meningitidis H44/76rfaC LPS was approximately equipotent to N. meningitidis H44/76 LPS. IL-1β, TNF-α, and IL-10 produced by E. coli Re K12, D31m4 LPS were inhibited by LDL (Figure 6) and HDL (data not shown) to a similar extent as that produced by E. coli 055:B5 LPS. No difference was found in the weak inhibitory effect of LDL on cytokine production between N. meningitidis H44/76 and N. meningitidis H44/76rfaC LPS. Thus, these data suggest that the differences in lipoprotein-dependent inhibition of cytokine production between E. coli LPS and meningococcal LPS are not caused by the different lengths of the saccharide tails of the LPS but by differences in the lipid A part of the LPS molecule.

Effect of lipoproteins on cytokines by or N. meningitidis whole bacteria Because LPS is not the only cytokine-inducing moiety of Gram-negative bacteria, we assessed whether the lipoprotein fractions were capable of inhibiting the cytokine response by PBMCs to complete Gram-negative bacteria. In these experiments, the E. coli LPS-induced cytokine response was inhibited by LDL or HDL. In contrast, IL-1β, TNF-α, and IL-10 induced by E. coli and N. meningitidis complete bacteria were not, or were only minimally, inhibited by the lipoproteins LDL, HDL, or VLDL (Figure 7) . Because the amount of cytokines induced by these whole bacteria was significantly higher than the amount induced after stimulation with LPS, we tested the capacity of lipoproteins to inhibit a lower concentration of bacteria or the capacity of a higher concentration of LDL to inhibit cytokine production. Even

120 P = n.s. 100 P = n.s. 80 P = n.s.

60 P = n.s. P = n.s. 40 P = n.s. 20

% % of control production 0 IL-1 TNFααα IL-10

E. coli 055:B5 LPS E. coli K12, D31m4 Re LPS N. meningitidis H44/76 LPS N. meningitidis H44/76rfaC LPS

Figure 6. Effect of the saccharide tail of LPS on inhibition by lipoproteins Effect of pre-incubation of various types of LPS (≈ 0.14 pmol/ml) with LDL (0.6 mmol/L) for 8 hours on IL-1β, TNFα and IL-10 by human mononuclear cells. E. coli 055:B5 and E. coli K12, D31m4 Re LPS were inhibited equally by LDL, whereas both meningococcal LPS were less effectively inhibited. Results are expressed as a percentage of production in lipoprotein depleted plasma (LPDP). Means and SEM are presented, n = 5. Differences were tested for significance using unpaired t-test.

78 LDL HDL VLDL 120 120 120

100 100 100 80 80 80 * 60 60 60

40 40 * 40 * * * 20 20 20

% % of control production * 5 * * 0 0 0 IL-1 TNFααα IL-10 IL-1 TNFααα IL-10 IL-1 TNFααα IL-10

E. coli LPS E. coli bacteria N. meningitidis bacteria

Figure 7. Inhibition of whole bacteria induced cytokines by lipoproteins. Effect of pre-incubation of LPS (0.14 pmol/ml) or E. coli and N. meningitidis complete bacteria (0.6 x 106) with LDL, HDL or VLDL for 4 hours on IL-1β, TNFα and IL-10 by human mononuclear cells. LDL and HDL effectively inhibited E. coli LPS, but not E. coli or N. meningitidis whole bacteria. Results are expressed as a percentage of production in lipoprotein depleted plasma (LPDP). Means and SEM are presented, n = 5, * = P < .05 for the comparison of E. coli LPS with the complete bacteria, by unpaired t-test.

TNF 140

120

100

60 Figure 8 α 40 Inhibition of LPS-induced TNF production in human mononuclear cells by LDL (0.6 mmol/L)after stimulation with 20 a concentration range (104 to 107 bacteria/mL) of whole E. % % of control production coli or N. meningitidis bacteria. 0 LDL was preincubated with the different bacteria for 8 104 105 106 107 hours. Results are expressed as a percentage of production Bacteria/mL in lipoprotein depleted plasma (LPDP). No effect of LDL on E. coli bacteria TNFα production by E. coli or N. meningitidis bacteria was N. meningitidis bacteria seen. Means and SEM are presented, n = 3. TNF 120

100

40 Figure 9. Influence of concentrated lipoproteins on whole bacteria- induced cytokine production 20 Effect of pre-incubation of E. coli or N. meningitidis (0.6 x % of control% production 106 bacteria/mL) LPS for 8 hours with concentrated LDL on 0 cytokine induction in human mononuclear cells. LDL was LDL LDL-C concentrated approximately 5 times, LDL concentration in which the LPS was preincubated was 3.43 mmol/l. E. coli bacteria LDL is unconcentrated LDL (0.6 mmol/l), LDL-C indicates concentrated LDL (3.43 mmol/l).Means and SEM are N. meningitidis bacteria presented, n = 3.

79 at the lowest concentration of bacteria tested (104/ml), no effect of VLDL, LDL, or HDL was seen on TNF-, IL-1ß, or IL-10 production (Figure 8 shows the results for LDL and TNF-α). In addition, concentrated LDL (3.43 mmol/l) did not lead to more inhibition of bacteria-induced cytokines (Figure 9)

Discussion

In the present study, we showed that at equimolar concentrations of E. coli or N. meningitidis LPS, the in vitro neutralizing effect of lipoproteins on cytokine production by N. meningitidis LPS was reduced in comparison with that by E. coli LPS. The neutralizing effect on E. coli LPS was shown to be time-dependent and saturable. By increasing the interaction time between the lipoproteins and LPS, increasing the lipoprotein concentration, or increasing the LPS concentration, the dissimilarity in the neutralizing effect of the lipoproteins disappeared. The difference in the extent of inhibition of LDL and HDL on both types of LPS is likely to be caused by the difference in the lipid A portion of the LPS molecule. Of importance, cytokine induction by whole E. coli or N. meningitidis bacteria was not influenced by lipoproteins.

A quantitative comparison of the biological effects of different LPS types on a weight basis may yield incorrect results because LPS from different bacteria have different molecular weights, largely because of the highly variable length of the saccharide tail. To avoid this pitfall, we assessed the molar concentration of the E. coli LPS and meningococcal LPS batches by determination of KDO, a highly conserved part of LPS (molecular mass 238 Da) 5,28, and used equimolar concentrations of LPS in all comparative experiments. In accordance with previously reported data on the bioactivity of LPS 31,32, the molarity-based dose-response curve showed that at concentrations of less than 1 pmol/ml, meningococcal LPS is 10 times more potent in the induction of IL-1β and TNF-α than is E. coli LPS.

The protective effect of the lipoproteins LDL and HDL in models of Gram-negative infection and lethal endotoxemia is well documented, and the inhibition of LPS- induced proinflammatory cytokine production, through binding of LPS by the lipoproteins, is thought to be a key element in this process 10-14. In the present study, we tested whether cytokine induction by the LPS isolated from N. meningitidis, the bacterium that causes the prototypical Gram-negative sepsis syndrome, is inhibited by lipoproteins in a similar manner as the prototypical LPS isolated from E. coli.

The concentrations of LPS used in the current in vitro experiments (0.14 pmol or 0.6 ng/ml) were chosen to be in the range of concentrations of LPS (as determined

80 by LAL assay) seen in the bloodstream during meningococcal sepsis, which range from 0.05 ng/ml to more than 10 ng/ml 4. In E. coli sepsis, LPS levels are generally lower 33,34. As earlier determinations showed that meningococci contain 1 ng LPS/106 bacteria in the outer membrane 20, the concentration of bacteria (0.6 x 106) was chosen to match the concentrations of LPS used. This bacterial concentration is in agreement with the amount of bacteria found during meningococcal sepsis (2.2 x 104/ml to 1.6 x 108/ml) 35.

Lipoprotein plasma concentrations are altered during sepsis or meningitis: LDL and 5 HDL cholesterol decreased (to 0.8–1.4 mmol/l for LDL and 0.2–0.4 mmol/ml for HDL), whereas VLDL cholesterol was increased to a median concentration of 2.3 mmol/ml 36,37. Thus, the concentrations of LDL and HDL used for this study are in range with the concentrations of these lipoproteins as they occur in septic conditions. For some experiments, the lipoproteins were further concentrated approximately five times to also investigate their detoxifying capacity at concentrations that are seen under normal physiological, nonseptic conditions. As VLDL increases during sepsis, we could only study this lipoprotein at concentrations that are seen in healthy individuals.

Confirming earlier observations 38, we found that preincubation of E. coli LPS with LDL as well as HDL inhibited the cytokine response, whereas VLDL had a less pronounced effect. However, at identical molar concentrations and incubation times, N. meningitidis LPS was neutralized much less efficiently than E. coli LPS.

The neutralizing effect of LDL and HDL was dependent on the preincubation time of the LPS with the lipoproteins and the concentration of LPS or lipoproteins. Whereas E. coli was inhibited significantly after relatively short preincubation times of 1–4 h, for meningococcal LPS, maximal inhibition was only seen after 24 h of preincubation. Using a fixed preincubation time (8 h) and a fixed concentration of LDL, the extent of inhibition was influenced by the concentration of LPS. Over the complete range of concentrations tested, E. coli LPS was significantly better neutralized by LDL than was N. meningitidis LPS (as assessed by two-way ANOVA). However, at the highest concentration tested, E. coli LPS also showed minimal inhibition, whereas at the lowest concentration tested, both LPS types were neutralized to a large extent. To investigate the impact of higher concentrations of lipoproteins on LPS detoxification, we maximally concentrated the lipoprotein fractions by additional ultracentrifugation. In this way, lipoprotein fractions approximately five times more concentrated were obtained, and the final concentration of lipoprotein in the PBMC system now equaled concentrations seen under physiological, nonseptic conditions. At these higher concentrations of lipoprotein, both LPS types were inhibited equally

81 and to a large extent by LDL and HDL after 8 h of preincubation. Thus, irrespective of the difference in neutralization seen at low concentrations of lipoproteins, this suggests that at physiological concentrations both E. coli and N. meningitidis LPSs are neutralized by the lipoproteins effectively. VLDL had a less pronounced neutralizing effect on cytokines, although it cannot be excluded that at even higher concentrations, as seen during sepsis, the effect of VLDL is also increased. Taken together, these results indicate that the efficient neutralization of LPS depends on the type of LPS, but a sufficiently long interaction time, a low concentration of LPS, and a high concentration of lipoproteins will also inhibit cytokines by a less efficiently neutralized LPS. Our results emphasize the concept that lipoproteins might be important for E. coli LPS-mediated disease. However, because during severe meningococcal sepsis, LPS concentrations are much higher, the time period from the onset of disease to admission is very short 4, and concentrations of LDL and HDL are decreased, it is unlikely that lipoproteins play an important role in neutralizing LPS during this type of infection.

The main molecular differences between E. coli LPS and N. meningitidis LPS are the size and structure of the saccharide tail and the position and distribution of the six acyl chains on the glucosamine backbone. To investigate which of these differences determine the neutralization by lipoproteins, we measured the effect of lipoproteins on cytokine induction by two types of E. coli and N. meningitidis LPS that are completely devoid of a saccharide tail, i.e., E. coli Re K12, D31m4 LPS and N. meningitidis H44/76rfaC LPS. This showed that the neutralizing action of lipoproteins is independent of the presence of the saccharide tail. Thus, the lipid A part, with its asymmetrical hexaacylated conformation in E. coli LPS and its symmetrical distribution of the six acyl chains in N. meningitidis LPS, accounts for the divergent effect. The bioactivity and receptor affinity of an LPS molecule is suggested to be determined by the three-dimensional conformation of the lipid A, possibly altering the bioactivity and receptor affinity of the LPS molecule 19. For LPS-lipoprotein interactions, Levine and colleagues 12 have proposed the “leaflet insertion” model, in which the fatty acyl acids of the lipid A portion of LPS are inserted into the lipid monolayer of the lipoproteins, inactivating the “toxic moiety” of LPS. We presume that the lower degree of neutralization of meningococcal LPS by lipoproteins is caused by the symmetrical lipid A structure interacting more slowly with the lipoprotein lipid monolayers.

LPS is generally considered to be the pathogenic component of Gram-negative bacteria 1. However, recently, it has been shown that non-LPS components of Gram-negative bacteria also highly influence cytokine production 20-22. Therefore, we studied whether lipoproteins affect cytokine production after stimulation with

82 whole bacteria, even at relatively low bacterial concentrations or high (physiological, nonseptic) lipoprotein concentrations. This was not the case. This finding indicates that whole bacteria, in contrast to isolated LPS, in which neutralization by lipoproteins depends on the interaction time and concentration of LPS or lipoprotein, are impervious to neutralization by lipoproteins. We assume that LPS, anchored tightly in the outer membrane of the Gram-negative bacterium, interacts less easily with the lipoproteins than isolated LPS. In addition, it can be speculated that bacterial components, other than LPS, of E. coli or N. meningitidis that are not inhibited by lipoproteins may account for the absent cytokine-inhibitory effect 5 of lipoproteins on whole Gram-negative bacteria. The data presented seemingly contradict the findings in animal models, in which lipoprotein administration did reduce cytokines and outcome after the administration of whole E. coli or after cecal ligation and puncture 39,40. Therefore, further investigation is needed on the immune-modulating mechanisms of lipoproteins during Gram-negative sepsis.

In conclusion, LDL and HDL inhibit cytokines by E. coli LPS, but N. meningitidis LPS shows reduced neutralization by human lipoproteins. However, a sufficiently long interaction time, a low LPS concentration, or higher lipoprotein concentration also inhibits cytokines by this less efficiently neutralized LPS. In contrast, lipoproteins do not have a neutralizing effect on whole E. coli or N. meningitidis bacteria. This raises questions about the rationale for adjuvant treatment of meningococcal and other types of Gram-negative sepsis with lipoproteins.

Acknowledgements

The authors wish to thank P. Demacker, PhD for advice on the lipoprotein isolation.

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N. meningitidis lipid A mutant lipopolysaccharides (LPS) function as LPS antagonists in humans by inhibiting toll-like receptor 4 (TLR4) dependent cytokine production 6

Tom Sprong, Liana Steeghs, Peter van der Ley, Shahla Abdollahi- Roodsaz, Leo Joosten, Jos van der Meer, Mihai Netea, Marcel van Deuren submitted Abstract

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram- negative bacteria and important in the induction of pro-inflammatory responses. Recently, novel LPS species derived from Neisseria meningitidis H44/76 by insertional inactivation of the lpxL1 and lpxL2 gene have been created with a lipid A portion consisting of five (pentaacylated lpxL1) or four (tetraacylated lpxL2) fatty acids connected to the glucosamine backbone instead of six fatty acids in the wild- type LPS. We show that these mutant LPS-types are poor inducers of cytokines (TNFα, IL-1β, IL-10, IL-RA) in human mononuclear cells. Both penta- and tetra- acylated meningococcal LPS were able to inhibit cytokine production by wild type E. coli or meningococcal LPS. Binding of FITC-labelled E. coli LPS TLR4 transfected Chinese hamster ovary (CHO) cells was inhibited by both mutant LPS-types. Experiments with CHO fibroblasts transfected with human CD14 and TLR4 showed that the antagonizing effect was dependent on the expression of human TLR4. In contrast to the situation in humans, lpxL1 LPS has agonistic activity for cytokine production in peritoneal macrophages of DBA mice, and exacerbated arthritis in murine collagen induced arthritis model. In conclusion, N. meningitidis lipid A mutant lipopolysaccharide (LPS) lpxL1 and lpxL2 function as LPS antagonists in humans by inhibiting toll-like receptor 4 (TLR4) dependent cytokine production but have agonistic activity in mice.

88 Introduction

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram- negative bacteria and is important in the induction of inflammatory responses and a potent adjuvant in vaccine development 1-3. In Neisseria meningitidis, the molecule consists of an oligosaccharide tail, a conserved saccharide inner core and a hexaacylated lipid A portion. The lipid A portion is considered the toxic-moiety of LPS, responsible for the induction of pro-inflammatory signalling, that binds the cellular receptor complex for LPS.

The cellular receptor complex for LPS is well established, consisting of CD14, TLR4 and MD-2 4-8. After binding of LPS to this complex, a cascade of intracellular molecules is engaged ultimately leading to the release of nuclear factor (NF)-κB and the induction of cytokine genes. However, not all LPS function as agonists for TLR4. LPS with a penta or tetra-acylated lipid A such as Rhodobacter spaeroides 6 LPS 9, Porphyromonas gingivalis LPS 10, LPS from the E. coli msbB strain 11 or synthetic LPS such as lipid A-like compound E5531 12 are poor pro-inflammatory stimuli and can even act as antagonists of LPS-induced signals. Curiously, it has been shown previously that some of these LPS-antagonists in humans are LPS agonists in rodent cells, inducing a normal inflammatory response 13.

Recently, by insertional inactivation of the lpxL1 and lpxL2 gene of N. meningitidis H44/76, mutant meningococci have been created expressing mutant LPS with a lipid A portion consisting of five (pentaacylated lpxL1) or four (tetraacylated lpxL2) fatty acids connected to the glucosamine backbone instead of six fatty acids in the wild-type H44/76 LPS. These mutant meningococcal strains have previously been shown to have reduced toxicity in comparison with the wild-type H44/76 strain, as shown by a decreased potency for the induction of TNFα in a human macrophage cell-line 14. In addition, a LPS mutant (msbB) structurally equal to the lpxL1 LPS has been shown to inhibit IL-8 production in TLR4 transfected HEK293 cells 15. In the present study we investigated the ability of the lpxL1 and lpxL2 LPS mutants to inhibit cytokine responses mediated by human TLR4. In addition, we tested these LPS mutants in mouse macrophages and a mouse model of arthritis.

Materials and Methods Bacterial strains and lipopolysaccharides Construction and characterization of the meningococcal H44/76 lpxL1 and lpxL2 mutants have been described in detail elsewhere 14. The parent strain N. meningitidis H44/76 is an international reference strain, isolated from a patient with invasive

89 meningococcal disease 16, serologically classified as B:15:P1.7,16, immunotype L3,7,9 17. As analysed by mass-spectometry, lpxL1, makes lipopolysaccharide (LPS) with penta- instead of hexa-acylated lipid A, in which the secondary lauroyl chain is missing from the nonreducing end of the GlcN disaccharide. Insertional inactivation of the other (lpxL2) gene was not possible in wild-type strain H44/76 expressing full-length immunotype L3 lipopolysaccharide (LPS) but was achieved in a galE mutant expressing a truncated oligosaccharide chain. Structural analysis of lpxL2 mutant lipid A showed a major tetra-acylated species lacking both secondary lauroyl chains and a minor penta-acylated species 14. For isolation of LPS, the N. meningitidis strain H44/76 and its derivatives with inactivated lpxL1 or lpxL2 genes, were grown at 37°C in liquid medium supplemented with IsoVitaleX (Becton

Dickinson) in a humid atmosphere containing 5 % CO2. Bacterial suspensions were heat inactivated for 30 min at 56 °C. LPS was isolated by the phenol/water extraction method as described by Westphal and Jann 18. After extraction, LPS was treated with Proteinase K (Sigma-Aldrich Co), DNA-ase and RNA-ase (Roche diagnostics) for additional purification, recovered by ultracentrifugation, freeze-dried and solved in sterile PBS. LPS purified and isolated in this way has previously been shown to be free of protein contamination (< 1.0 %) (determined by Lowry-assay), whereas nucleic acid contamination is approximately 5 % 19. E. coli 055:B5 LPS and FITC labeled E. coli LPS was obtained from Sigma-Aldrich chemical Co.

Cells and culture conditions

Human peripheral blood mononuclear cells (PBMCs) Blood for the isolation of PBMCs was drawn in 10 mL EDTA anti-coagulated tubes (Vacutainer System, Beckton Dickinson) from healthy human volunteers. Informed consent was obtained before each experiment, and the guidelines of the local ethics committee were followed in the conduct of these experiments. PBMCs were isolated by density gradient centrifugation over Ficoll-Hypaque (Pharmacia Biotech AB). The cells from the interphase were aspirated, washed 3 times in sterile phosphate buffered saline (PBS) and resuspended in culture medium RPMI 1640 (Dutch modification, Flow Labs) supplemented with L-glutamine (2 mMol), pyruvate (1 mMol) and gentamicin (50 mg/mL). Cells were incubated with the different stimuli for 24 hours, unless otherwise indicated, where after the supernatants were collected and stored at -80°C until assay in one batch.

Chinese hamster ovary fibroblasts (CHO) stably transfected with human CD14 (3E10) or CD14 and TLR2 (3E10-TLR2) or CD14 and TLR4 (3E10-TLR4) Chinese hamster ovary fibroblasts (CHO) stably transfected with human CD14

90 (3E10) or human CD14 and hTLR4 (3E10-TLR4), described previously in detail 5, were a kind gift from Dr. Robin Ingalls and Dr. Douglas T Golenbock. It is important to note that 3E10 expresses also hamster TLR4 on the outer membrane, and is capable of LPS-mediated signaling. These cell-lines express inducible membrane CD25 under control of a region from the human E-selectin (ELAM-1) promoter containing NF-κB binding sites; this promoter element is absolutely dependent 13 ° on NFκB . Cells were maintained at 37 C and 5% CO2 in HAM’s F12 medium (Gibco™, Invitrogen) supplemented with 10 % FCS, 0.01 % L-glutamine, 50 μg/mL gentamycin and 400 U/mL hygromycin and 0.05 mg/mL of puromycine for 3E10- TLR4 as additional selection antibiotics. TLR4 expression was confirmed by flow- cytometry (Coulter FACS-scan) using PE-labelled or anti-TLR4 (clone HTA125) (Immunosource). For stimulation experiments, 500 μl of cells in culture medium at a density of 1 x 105/mL were plated in 24-well culture plates. After an overnight incubation cells were incubated with the various stimulants for 20 hours, whereafter the cells were harvested using trypsin/EDTA (Cambrex) and prepared for flow- 6 cytometry (Coulter FACS-scan). CD25 expression of the CHO-cells was measured using FITC labelled anti-CD25 (DAKO).

FITC-LPS binding experiments LpxL1 and lpxL2 LPS (1 μg/mL) were preincubated with CHO 3E10-TLR4 for 15 minutes whereafter FITC labelled E. coli LPS was added at increasing concentrations. Cells were incubated for 1 hour at RT in the dark, harvested using trypsin/EDTA and washed with sterile PBS. Read out was by flow cytometry (Coulter FACS-scan).

Murine model of arthritis Collagen-induced arthritis (CIA) was induced in DBA-1 mice between 10 and 12 weeks of age. Bovine type II collagen was dissolved in 0.05 M acetic acid to a concentration of 2 mg/ml and emulsified in an equal volume of Freund’s complete adjuvant (2 mg/ml of M. tuberculosis strain H37Ra; Difco laboratories, USA). Mice were immunized by intradermal injection of 100 µl of the emulsion at the base of the tail and were given an intraperitoneal booster injection of 100 µl of CII dissolved in phosphate-buffered saline without any adjuvant on day 21. Clinical onset and progression of arthritis was macroscopically evaluated by two blinded observers and scored on a scale between 0 and 2 for each paw as described previously 20. Mice were treated using three i.p. injections of lpxL1 (400 µg/kg body weight) once in two days started on day 22 of immunization before the clinical onset of the disease.

91 Stimulation of murine peritoneal macrophages Murine macrophages were isolated from naïve DBA/1 mice by the lavage of the peritoneal cavity using 10 ml cold medium (DMEM + 10% FCS). Adherent cells were harvested and cultured for four days before use. Cells were incubated with E. coli LPS (10 ng/ml), lpxL1 (100 ng/ml) or a combination of both.

Cytokine assays Human tumor necrosis factor-α (TNFα), interleukin-10 (IL-10) and IL-1RA were determined by ELISA (In-house 4-span ELISA for TNFα 21, Pelikine compact ELISA (Sanquin) for the other cytokines, lower limit of detection 20 pg/mL), interleukin-1β (IL-1β) was determined by radioimmunoassay (RIA) as described by Drenth et al 22. The lower limit of detection was 40 pg/mL. Murine cytokines were measured using the Bioplex cytokine assays (Bio-Rad, USA) following the manufacturer’s instruction.

Results Cytokine induction by N. meningitidis H44/76 LPS and lpxL1 or lpxL2 mutant LPS N. meningitidis H44/76 LPS was a potent inducer of the cytokines TNFα and IL-10 in human PBMC. In contrast, the lpxL1 and lpxL2 mutant LPS types were 100 – 1000 times less potent in the induction of these cytokines, inducing only minimal cytokine production at concentrations of 10 μg/mL (Figure 1). The mutant LPS were also minimal inducers of IL-1β and IL-1RA (data not shown).

Antagonistic properties of lpxL1 and lpxL2 mutant LPS Similar to meningococcal LPS, E. coli LPS was a potent inducer of TNFα and IL- 10 in human PBMCs. Pre incubation of the PBMCs with lpxL1 or lpxL2 LPS (1

400 400 TNFĮ IL-10

300 300

200 200 pg/mL pg/mL Figure 1 ** TNFα and IL-10 production after 100 ** 100 stimulation of human PBMCs for 24 hours with N. meningitidis ** ** H44/76 LPS, and the mutant 0 0 lpxL1 and lpxL2 LPS. 0.0001 0.01 1 100 0.0001 0.01 1 100 Mean values and SD are shown, n = 3. ** indicates P < 0.01 for LPS (µg/mL) LPS (µg/mL) the comparison of lpxL1 and lpxL2 LPS with N. meningitidis H44/76 LPS lpxL1 LPS lpxL2 LPS H44/76 LPS by two way ANOVA.

92 300 800 TNFĮ IL-10 250 600 200 Figure 2 150 400 E.coli induced TNFα and IL-10 pg/mL pg/mL production by human PBMCs in 100 the absence or presence of the 200 mutant lpxL1 and lpxL2 LPS (1μg/ 50 mL). * LpxL1 and lpxL2 LPS were * preincubated with the PBMCs for 0 * 0 * 30 minutes prior to stimulation with 0.0001 0.001 0.01 0.1 1 0.0001 0.001 0.01 0.1 1 E. coli LPS. Mean values and SD are shown, n = 3. * indicates P < E. coli LPS (ȝg/mL) E. coli LPS (ȝg/mL) 0.05 for the comparison of lpxL1 and lpxL2 LPS with the control CONTROL lpxL1 LPS lpxL2 LPS (RPMI) by two way ANOVA

μg/mL) completely inhibited TNFα and IL-10 production induced by E. coli LPS. This antagonistic effect was present at all concentrations of E. coli LPS tested, 6 ranging form 0.1 ng/mL up to 100 ng/mL (Figure 2). N. meningitidis H44/76 LPS was inhibited to a similar extent by the mutant LPS (not shown). The antagonistic effect of lpxL1 and lpxL2 LPS on cytokine induction by 10 ng/mL of E. coli LPS was present in concentrations of 10 ng/mL of lpxL1 or lpxL2 or higher. Complete inhibition was seen at concentrations of 100 ng/mL of lpxL2 LPS and 1 µg/mL of lpxL1 LPS (Figure 3). In addition, we examined whether pre-incubation was necessary for inhibition of LPS-induced cytokines by lpxL1 and lpxL2. We found that LPS-induced cytokine production was inhibited by the lpxL-mutants equally well when they were preincubated with the PBMCs or simultaneously added with the E. coli LPS (Figure 4).

Finally, we tested whether the observed inhibitory effect on cytokine production was dependent on inhibition of LPS-binding to TLR4. Figure 5 shows that binding of FITC-labelled E. coli LPS to CHO cells transfected with hCD14 and hTLR4 was

800 Preincubation with RPMI 800 Preincubation with lpxL1 PreincubationLPS with RPMI 600 PreincubationPreincubation with with lpxL1 lpxL2 LPSLPS 600 400 Preincubation with lpxL2 LPS TNF (pg/mL)

400 200 TNF (pg/mL)

200 0 Figure 3 0 0.01 0.1 1 E. coli (10 ng/mL) induced TNFα production by human PBMC in lpxL1/2 (ȝg/mL) the presence of different concentrations of lpxL1 and lpxL2 LPS. 0 LpxL1 and lpxL2 LPS were preincubated with the PBMCs for 30 minutes prior to stimulation with E. coli LPS. Mean values and 0 0.01 0.1 1 SD are shown, n = 3. lpxL1 and lpxL2 did not significantly differ lpxL1/2 (ȝg/mL) in their ability to inhibit E. coli induced TNFα (two-way ANOVA)

93 completely inhibited by the lpxL1 and lpxL2 mutant LPS. This indicates that the antagonistic effect is dependent on TLR4 binding by the lpxL-mutant LPS.

Expression of human TLR4 is necessary for LPS antagonism To test whether the observed antagonistic effect of lpxL1 and lpxL2 LPS was dependent on differences in TLR4-mediated signalling, we made use of the differences in TLR4 dependent signalling between human and hamster TLR4, employing a CHO cell line stably expressing human CD14 and either hamster TLR4 (3E10) or overexpressing human TLR4 (3E10-TLR4).

Isolated E. coli LPS and wild-type N. meningitidis H44/76 LPS (not shown) induced NF-κB activation in both cell lines. In contrast, lpxL1 and lpxL2 LPS induced a similar response to E. coli LPS in the CD14 transfected CHO cells expressing hamster TLR4, but when these CHO cells overexpress human TLR4, the NF-κB response is blunted (Figure 6A). These results indicate that NF-κB activation by the lpxL1 and lpxL2 mutant LPS differs between species, and its absence in humans is

400 PRE-INCUBATION CO-INCUBATION 300

(pg/mL) 200

Į TNF 100 Figure 4 E. coli-induced TNFα production by human PBMCs in the absence (RPMI) or presence of lpxL1 and lpxL2 mutant 0 LPS, pre-incubated with the PBMCs for 30 minutes or added directly together with the E. coli LPS. medium lpxL1 lpxL2 Mean values and SD are shown, n = 2

100 100 PreincubationPreincubation with with RPMI RPMI Preincubation with lpxL1 PreincubationLPS with lpxL1 75 LPSPreincubation with lpxL2 75 LPS Preincubation with lpxL2 50 LPS

50 binding % CHO-cells 25

0 % CHO-cells binding % CHO-cells 25 0 0.1 1 10 100 FITC-LPS (µg/mL)

0 Figure 5 0 0.1 1 10 100 Inhibition of binding of FITC LPS to CHO 3E10-TLR4 by lpxL1 and lpxL2 LPS. FITC-LPS (µg/mL) Representative experiment of 3 performed is shown.

94 dependent on TLR4.

Antagonistic activity of lpxL1 and lpxL2 LPS was dependent on expression of human TLR4, as the lpxL mutants were able to inhibit E. coli LPS-induced NF-κB activation in hCD14-transfected CHO cells overexpressing human TLR4 but not in hCD14-transfected CHO cells expressing hamster TLR4 (Figure 6B).

Taken together, these results indicate that that lpxL1 and lpxL2 LPS bind hamster and human TLR4, inducing a strong NF-κB response by hamster TLR4, but no NF- κB dependent signaling by human TLR4. In this way, they are proficient to inhibit binding of agonist-LPS to human TLR4, antagonizing the effects of agonist-LPS on NF-κB dependent cytokine production in humans. lpxL1 is not an antagonist in mice, and exacerbates arthritis in a murine model of arthritis 6 Because of the antagonistic nature of the lpxL1 LPS in human cells we investigated whether lpxL1 LPS was able to attenuate arthritis in a murine model of collagen induced arthritis, similar to previously published results for Bartonella quintana LPS 23. However, in contrast to the findings with Bartonella LPS, we found that the lpxL1 LPS exacerbated arthritis in DBA mice, as evidenced by higher arthritis scores in the mice injected with lpxL1 LPS in comparison with control mice injected with saline (Figure 7A). As previous studies have shown that agonist E. coli LPS also causes exacerbation of arthritis, we investigated whether the lpxL1 LPS is an agonist for cytokine production in the DBA mice. Experiments with murine peritoneal macrophages demonstrated that in mice, the lpxL1 LPS induced a potent TNFα, IL-1β and IL-6 response - albeit that lpxL1 LPS was approximately 10 times

60 60 A B 50 50 E. coli LPS 40 40 N. Men lpxL1 LPS 30 30 N. Men lpxL2 LPS

20 20 E. coli LPS + lpxL1 LPS E. coli LPS + lpxL2 LPS CD25expression (%) 10 CD25expression (%) 10

0 0 CD14 CD14-TLR4 CD14 CD14-TLR4

Figure 6 NF-κB dependent CD25 expression on CHO cells stably transfected with human CD14 alone or human CD14 and human TLR4. Cells were stimulated with E. coli LPS lpxL1 LPS or lpxL2 LPS at 1 μg/mL for 20 hours (A) or preincubated with the lpxL1 and lpxL2 LPS for 30 minutes and they were stimulated with E. coli LPS (1μg/mL) for 20 hours (B). average and SD are presented of 3 experiments performed.

95 A 120 B 5 * Saline 100 4 * lpxL1 80 3 (pg/ml) 60  2 40 TNF 1 20

Mean macroscopic0 score 0 0 2 5 7 9 medium lpxL1 LPS E. coli LPS lpxL1 + Days after injection E. coli LPS

Figure 7 Exacerbation of arthritis in a collagen induced arthritis model in DBA mice by lpxL1 LPS (panel A). Mean and SEM of arthritis scores in 11 mice are shown;  = mice challenged with collagen and 3 intraperitoneal injections of lpxL1 LPS at day 0, 2 and 5, ο = mice injected with 0.9 % saline solution. Panel B shows TNFα production by murine peritoneal macrophages 24 hours after stimulation with DMEM, lpxL1 LPS (100 ng/mL), E. coli LPS (10 ng/mL) or a combination of lpxL1 LPS (100 ng/mL) and E. coli LPS (10 ng/mL). Representative experiment of 2 performed is shown. * indicates P < 0.05 by Mann Whitney test

less potent then E. coli LPS - and was not able to inhibit cytokine production by E. coli LPS (Figure 7B, results for TNFα are shown). This indicates that in the DBA mice, the lpxL1 LPS functions as an agonist for TLR4 - in contrast to the situation in humans where lpxL1 LPS is antagonistic.

Discussion

In the present study, we show that lipid A mutant LPS, derived from genetically engineered meningococci expressing penta- or tetra-acylated LPS, have a strongly reduced capacity to induce NFκB activation and cytokine production by human TLR4. In contrast, they function as LPS-antagonists in humans, by binding TLR4 and competitively inhibiting cytokine production by agonist LPS. This effect on TLR4 transmitted signals is species-specific. Pentaacylated mutant lpxL1 LPS exacerbates disease in a murine arthritis model and is an agonist for TLR4 mediated cytokine production in murine peritoneal macrophages and hamster cells.

Recently, Teghanemt et al. reported that the meningococcal msbB LPS-mutant, structurally equal to the lpxL1 LPS mutant used in the present study, bound MD2 and subsequently TLR4 in a similar manner as wild type meningococcal LPS but was a partial antagonist of TLR4 dependent IL-8 production in HEK293 cells 15. We expand these findings with our observations that the antagonistic effect is also present in human primary cells for a range of pro and anti-inflammatory cytokines, that the lpxL2 LPS has a similar antagonistic effect as the lpxL1 LPS and that this effect is species-specific, not present in DBA mice or hamster cells. Teghanemt et al., proposed that the mechanism behind the antagonistic effect of the msbB

96 LPS was an alteration of the interaction of the MD2-LPS complex with the TLR4 receptor 15. For E. coli msbB LPS a similar mechanism was suggested by Coats et al, who found that MD-2 and TLR4 were the only components of the receptor complex necessary to mediate msbB LPS antagonism of TLR4 dependent signalling by wt E .coli LPS 11. Interestingly, the CHO cells we used in the present study only overexpress human CD14 and TLR4, whereas MD-2 is not overexpressed, although it may be present endogenously 24. Thus, the antagonistic property of the lpxL1 and lpxL2 LPS mutants in the CHO cells overexpressing human TLR4 is independent of human MD-2. This suggests that the TLR4 molecule itself is central to the antagonistic effect of the lpxL1 and lpxL2 LPS.

An important feature of LPS molecules that exhibit antagonistic properties is that this effect is species-specific. For instance, R. sphaeroides LPS and lipid IVa are agonists for cytokine production in hamster cells 25, whereas in mouse, R. sphaeroides LPS is an antagonist and lipid IVa is an LPS mimetic 9. In the present 6 study we found that the lpxL1 and lpxL2 LPS are antagonists in humans, are LPS- mimetics in CHO cells expressing hamster TLR4 but are antagonists again in CHO cells overexpressing human TLR4. Interestingly, in DBA mice, lpxL1 LPS was an agonist for cytokine responses, and exacerbated disease in a murine model of arthritis. This is a well know effect of agonistic LPS 20,26 and shows that the in vitro differences in antagonistic properties between species for these LPS types also confer to in vivo functional effects. Our results are in agreement with the differential signaling of lpxL1 LPS through murine and human TLR4 as reported by Steeghs et al. 27, which showed that the reduction in activity of lpxL1 as compared to wildtype meningococcal LPS is much more pronounced in the human than the mouse system.

The antagonistic nature of penta or tetra-acylated LPS has received significant attention as potential therapy for human diseases such as septic shock 28,29, rheumatoid arthritis 23,30, asthma 31 and inflammatory bowel disease 32. The beneficial effects may be caused by inhibition of the interaction of Gram-negative bacteria with cells of our immune system, but there is increasing evidence that LPS is not the only ligand for the TLR4 complex, and it is possible that antagonists for LPS will also be antagonists for these other ligands 33.

In conclusion, N. meningitidis lipid A mutant lipopolysaccharide (LPS) lpxL1 and lpxL2 function as LPS antagonists in humans by inhibiting TLR4 dependent cytokine production. As such, lpxL1 and lpxL2 mutant LPS might be of therapeutical value in TLR4 dependent inflammatory conditions. Differences of human and murine TLR4 however, preclude the use of mouse models in studies addressed to this item.

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98 19. Sprong T, van der Ley P, Steeghs L, Tax WJ, Verver-Janssen TJW, Netea MG, van der Meer JW, van Deuren M. Neisseria meningitidis can induce pro-inflammatory cytokine production via pathways independent from CD14 and toll-like receptor 4. European cytokine network. 2002;13:411-417 20. Joosten LA, Helsen MM, van de Loo FA, van den Berg WB. Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice. A comparative study using anti-TNF alpha, anti-IL-1 alpha/beta, and IL-1Ra. Arthritis Rheum. 1996;39:797-809 21. Grebenchtchikov N, van der Ven-Jongekrijg J, Pesman GJ, Geurts-Moespot A, van der Meer JW, Sweep FC. Development of a sensitive ELISA for the quantification of human tumour necrosis factor-alpha using 4 polyclonal antibodies. Eur Cytokine Netw. 2005;16:215-222 22. Drenth JP, Van Uum SH, Van Deuren M, Pesman GJ, Van der Ven Jongekrijg J, Van der Meer JWM. Endurance run increases circulating IL-6 and IL-1ra but downregulates ex vivo TNF-alpha and IL-1 beta production. J. Appl. Physiol. 1995;79:1497-1503 23. Abdollahi-Roodsaz S, Joosten LA, Roelofs MF, Radstake TR, Matera G, Popa C, van der Meer JW, Netea MG, van den Berg WB. Inhibition of Toll-like receptor 4 breaks the inflammatory loop in autoimmune destructive arthritis. Arthritis Rheum. 2007;56:2957- 2967 24. Medvedev AE, Henneke P, Schromm A, Lien E, Ingalls R, Fenton MJ, Golenbock DT, Vogel SN. Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of Toll-like receptors 6 2 or 4. J Immunol. 2001;167:2257-2267 25. Delude RL, Savedra R, Jr., Zhao H, Thieringer R, Yamamoto S, Fenton MJ, Golenbock DT. CD14 enhances cellular responses to endotoxin without imparting ligand-specific recognition. Proc Natl Acad Sci U S A. 1995;92:9288-9292 26. Yoshino S, Sasatomi E, Ohsawa M. Bacterial lipopolysaccharide acts as an adjuvant to induce autoimmune arthritis in mice. Immunology. 2000;99:607-614 27. Steeghs L, Keestra AM, Van Mourik A, Uronen-Hansson H, van der ley P, Callard R, Klein N, van Putten JP. Differential activation of human and mouse Toll-like receptor 4 by the adjuvant candidate lpxL1 of Neisseria meningitidis. Infect Immun. 2008;76:3801-3807 28. Macagno A, Molteni M, Rinaldi A, Bertoni F, Lanzavecchia A, Rossetti C, Sallusto F. A cyanobacterial LPS antagonist prevents endotoxin shock and blocks sustained TLR4 stimulation required for cytokine expression. J Exp Med. 2006;203:1481-1492 29. Bunnell E, Lynn M, Habet K, Neumann A, Perdomo CA, Friedhoff LT, Rogers SL, Parrillo JE. A lipid A analog, E5531, blocks the endotoxin response in human volunteers with experimental endotoxemia. Crit Care Med. 2000;28:2713-2720 30. Popa C, Abdollahi-Roodsaz S, Joosten LA, Takahashi N, Sprong T, Matera G, Liberto MC, Foca A, van Deuren M, Kullberg BJ, van den Berg WB, van der Meer JW, Netea MG. Bartonella quintana lipopolysaccharide is a natural antagonist of Toll-like receptor 4. Infect Immun. 2007;75:4831-4837 31. Savov JD, Brass DM, Lawson BL, McElvania-Tekippe E, Walker JK, Schwartz DA. Toll- like receptor 4 antagonist (E5564) prevents the chronic airway response to inhaled lipopolysaccharide. Am J Physiol Lung Cell Mol Physiol. 2005;289:L329-337 32. Fort MM, Mozaffarian A, Stover AG, Correia Jda S, Johnson DA, Crane RT, Ulevitch RJ, Persing DH, Bielefeldt-Ohmann H, Probst P, Jeffery E, Fling SP, Hershberg RM. A synthetic TLR4 antagonist has anti-inflammatory effects in two murine models of inflammatory bowel disease. J Immunol. 2005;174:6416-6423 33. Johnson GB, Brunn GJ, Platt JL. Activation of mammalian Toll-like receptors by endogenous agonists. Crit Rev Immunol. 2003;23:15-44

Mannose binding lectin enhances IL-1beta and IL-10 induction by non- lipopolysaccharide (LPS) components of Neisseria meningitidis. 7

Tom Sprong, Dominic L. Jack, Nigel J. Klein, Malcolm W. Turner, Peter van der Ley, Liana Steeghs, Liesbeth Jacobs, Jos W.M. van der Meer and Marcel van Deuren. Cytokine. 2004;28(2):59-66 Abstract

Mannose binding lectin (MBL) is a key molecule in the lectin pathway of complement activation, and likely of importance in our innate defence against meningococcal infection. We evaluated the role of MBL in cytokine induction by LPS or non-LPS components of Neisseria meningitidis, using a meningococcal mutant deficient for LPS. Binding experiments showed that MBL exhibited low, but significant binding to encapsulated LPS+ meningococci (H44/76) and LPS-deficient (LPS−) meningococci (H44/76lpxA). Experiments with human mononuclear cells (PBMCs) showed that MBL significantly augmented IL-1β production after stimulation with LPS+ and LPS− meningococci, in a dose-dependent fashion. In addition, IL-10 production was enhanced after stimulation with LPS− meningococci. In contrast, TNFα, IL-6 and IFNγ productions were unaffected. No effect of MBL was observed on cytokine induction by meningococcal LPS. MBL enhanced cytokine production at concentrations >107 meningococci. It is concluded that MBL interacts with non-LPS components of N. meningitidis and in this way modulates the cytokine response.

102 Introduction

Meningococcal disease, caused by the Gram-negative diplococcus Neisseria meningitidis, is an important cause of mortality and morbidity, particularly in children. The clinical course of the disease ranges from meningitis with a relatively benign clinical picture and a mortality rate of less then 5% to fulminant meningococcal sepsis with a rapidly deteriorating clinical condition and a mortality rate ranging from 20 to 50% 1.

Disease severity and mortality in meningococcal disease are correlated with high plasma concentrations of the pro-inflammatory cytokines TNFα and IL-1β, which induce shock and disseminated intravascular coagulation 2,3. However, cytokines play a complex dual role in the pathophysiology of invasive meningococcal disease, as it also has been shown that a low production capacity for TNFα combined with a high production capacity for the anti-inflammatory cytokine IL-10 or poor regulation of the IL-1 system constitute a risk factor for invasive disease 4,5. Meningococcal lipopolysaccharide (LPS) is generally considered to be the main cytokine-inducing element 6. Using LPS-deficient mutant N. meningitidis we and others have shown 7 however, that non-LPS components of the meningococcus can also induce cytokine production in human monocytes or macrophages 7-9.

Mannose binding lectin (MBL) is a pattern-recognition molecule present in serum that is involved in the innate immune defence by activation of complement, promotion of opsonophagocytosis and modulation of inflammatory mediators 10. Serum concentrations of MBL are determined by structural mutations in the exon 1 region and polymorphism of the promoter region of the MBL-gene (Caucasian median value: 1600 ng/mL) 11,12. Homozygousity for any of the 3 structural mutations in the exon 1 region reduces serum levels of MBL to values <100 ng/mL, whereas heterozygousity for these mutations also profoundly reduces MBL concentrations to levels <400 ng/mL. The presence of promoter polymorphisms contributes to the wide range of MBL concentrations as observed in the general population. In addition, MBL is an acute phase protein and during inflammation levels can increase some three-fold 13. Recent evidence suggests that MBL plays a role in the pathogenesis of meningococcal disease. MBL can bind meningococci, activate complement and increase killing of these organisms 14,15. In addition, genetic deficiency for MBL has been shown to increase susceptibility to meningococcal infection, particularly in children 16-18. However, the exact pathophysiological role of MBL in meningococcal disease is incompletely understood.

MBL has been reported to influence also the cytokine network after stimulation

103 with various microorganisms 19-21. Knowing the crucial role of the cytokine network in meningococcal disease, the principal aim of the present study was to assess the role of MBL in cytokine induction by LPS and by non-LPS components of N. meningitidis.

Materials and methods Meningococcal strains, lipopolysaccharide and mannose binding lectin The wild-type encapsulated serogroup B N. meningitidis H44/76 strain (indicated as LPS+N. meningitidis) was isolated from a patient with invasive meningococcal disease 22. The meningococcal strain H44/76lpxA (indicated as LPS−N. meningitidis) is a viable isogenic mutant completely devoid of LPS in the outer membrane. Absence of LPS in this strain was confirmed as described previously 23. Absence of LPS-activity in the lpxA batch-suspension was confirmed by non-reactivity in the Limulus amebocyte lysate assay. The major outer membrane proteins of the LPS− meningococcus are expressed in similar amounts as compared to the LPS+N. meningitidis. However, decreased amounts of the cell surface-exposed lipoproteins LbpB and TbpB are seen. The outer membrane phospholipids in LPS− meningococci consist of shorter chains and more phospholipids with saturated fatty acids are present 24,25. Heat killed (1 h, 56 °C) bacteria washed once in phosphate buffered saline (PBS) were used for stimulation experiments and binding studies. Encapsulated N. meningitidis B1940 wild-type and the isogenic mutant cpsD− which has a truncated, non-sialylated LPS, kindly provided by Dr. Matthias Frosch (University of Würzburg, Germany), were used as negative and positive MBL- binding controls in the dot blotting experiments.

Meningococcal H44/76 LPS was isolated and purified by the phenol/water extraction method as described by Westphal and Jann 26. After extraction, LPS was treated with proteinase K (Sigma–Aldrich Co), DNA-ase and RNA-ase (Roche diagnostics) for additional purification. Protein contamination of the LPS-suspension after treatment was less than 1.0%, nucleic acid contamination was 4.4%. The amount of H44/76 LPS in the batch-suspension was quantified by assaying for 2-keto-3- deoxyoctanate (KDO) 27. The amount of LPS in LPS+N. meningitidis H44/76 was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) and assay for KDO 28. In the stimulation experiments, N. meningitidis H44/76 LPS was used in concentrations approximately equivalent to the amount present in LPS+ H44/76 meningococci.

Mannose binding lectin (MBL) was isolated from human plasma (donated by C.

104 Dash, Blood products laboratory, Elstree, United Kingdom) by a modification of the method of Kilpatrick, as described previously 29,30. The concentration of MBL was determined by ELISA, and purity was verified by non-reducing SDS-PAGE. MBL prepared in this manner is known to be non-covalently associated with MBL- associated serine protease (MASP).

Binding of MBL to N. meningitidis H44/76 and lpxA Binding of MBL to bacteria was determined by dot blotting technique. Meningococci (10 μL of 2 × 108/mL in PBS) were spotted onto a prewetted Hybond-P membrane (AmershamPharmacia Biotech, Amersham, UK). Membranes were blocked for 30 min with Pierce SuperBlock (Perbio Science, Tattenhall, UK) before 60 min of incubation steps with 1.5 μg/mL MBL, then 1 μg/mL anti-MBL (clone 131-1, Statens Serum Institut, Copenhagen, Denmark) and finally a 1/1000 dilution of horse-radish peroxidase conjugated goat anti-mouse immunoglobulin (Sigma, Poole, UK) before chemiluminiscent detection with ECL reagents (AmershamPharmacia Biotech). All dilutions were in Tris-buffered saline containing 0.002% v/v Tween-20 (TBS-Tw). Between incubations, the membrane was washed 4 times in TBS-Tw. 7 Stimulation of human peripheral blood mononuclear cells (PBMCs) Blood for the isolation of PBMCs was drawn in 10 mL EDTA anti-coagulated tubes (Vacutainer System, Beckton Dickinson, Rutherford, NJ) from healthy human volunteers. PBMCs were isolated by density gradient centrifugation over Ficoll- Hypaque (Pharmacia Biotech AB, Upssala, Sweden). The cells from the interphase were aspirated, washed 3 times in sterile, endotoxin-free PBS and resuspended in culture medium RPMI 1640 (Dutch modification, Flow Labs, Irvine, Scotland) supplemented with L-glutamine (2 mM), pyruvate (1 mM) and gentamycin (50 mg/ mL). 0.5 × 106 PBMCs per well were incubated with the various stimuli in 200 μL 96-well plates at 37 °C and 5% CO2 for 24 h. Before incubation with the PBMCs, MBL was pre-incubated with the various stimuli for 2 h at 37 °C and 5% CO2. As no serum is added to the cell-culture in this way, no exogenous source of complement is present. No C3 was found in the PBMC culture system in a blank sample and after stimulation of the PBMC with LPS- N. meningitidis in the presence or absence of MBL. The supernatant was obtained by centrifugation and stored at −20 °C until required for the cytokine assays.

Cytokine assays Levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) were determined by radioimmunoassay as described by Drenth et al. 31. The lower limit of detection was 80 pg/mL for both cytokines. Levels of interleukin-10 (IL-10), interferon-γ

105 (IFNγ) and interleukin-6 (IL-6) were determined by ELISA (Pelekine Compact, Central laboratory of the Netherlands Red Cross, Amsterdam). The lower limit of detection was 8 pg/mL.

Statistics Data were analysed using the non-parametric Mann–Whitney U test for unpaired data, and by one-way ANOVA when indicated. P < 0.05 was considered significant.

Results MBL-binding LPS+ and LPS− meningococci Dot blotting experiments (Figure 1) showed that both LPS+ (B) and LPS− (D) meningococci were able to bind MBL. Binding of these two strains was at a lower level than to the B1940cpsD− mutant (C), but substantially more than to the B1940 strain (A).

Cytokine induction by MBL Purified MBL did not induce IL-1 and TNFα production in concentrations of MBL ranging from 0.75 to 2500 ng/mL. IL-10, IFNγ and IL-6 inductions by purified MBL were tested at a single concentration (2500 ng/mL), but again we observed no effect on the production of any of these cytokines (data not shown).

The role of MBL in cytokine induction by LPS and non-LPS components of N. meningitidis In Figure 2, IL-1β, IL-10, TNFα and IFNγ production after stimulation with both meningococcal strains or meningococcal LPS in the absence or presence of MBL (2500 ng/mL) is shown. IL-1β and IL-10 production after stimulation with LPS−N. meningitidis was significantly enhanced by MBL. MBL also significantly enhanced IL-1β production after stimulation with wild-type LPS+ meningococci. In contrast to IL-1β and IL-10, MBL did not affect TNFα and IFNγ after stimulation with LPS+ or LPS− meningococci. The

A B

C D Figure 1. Binding of MBL to the meningococcal strains. Blotting membrane showing binding of MBL (1500 ng/mL) to 2 × 108 of H44/76 LPS+ meningococci (quadrant B), LPS− mutant meningococci (quadrant D), N. meningitidis B1940 (quadrant A, negative control) and N. meningitidis B1940cpsD− (quadrant C, positive control).

106 30 P = 0.007 5 TNF P = 0.456 α α 4 α α

20 (ng/mL) 3 (ng/mL) P < 0.001 P β β = 0.345 β β 2 10 P = 0.340 P = 0.111 IL-1 1

P = 0.241 P = 0.082 1.0 0.8 IFN γ γ 0.8 0.6 γ γ P = 0.02 (ng/mL) 0.6 0.4 0.4 P = 0.250 IL-10 (ng/mL) 0.2 0.2 P = 0.143 P = 0.345

LPS+ N. LPS- N. LPS LPS+ N. LPS- N. LPS meningitidis meningitidis meningitidis meningitidis

Figure 2. Effect of MBL on cytokine production by LPS and non-LPS of N. meningitidis. Production of TNFα, IL-1β, IFNγ and IL-10 after stimulation of human PBMCs in the absence (white bars) or presence (hatched bars) of MBL (2500 ng/mL) with wild-type LPS+N. meningitidis (6 × 108/mL), LPS−N. meningitidis (6 × 108/mL) and N. meningitidis LPS (600 ng/mL). MBL was pre-incubated with the various stimuli for 2 h. Median values ± IQR of 15 separate experiments in different control subjects are presented for both bacterial strains, n = 5 for meningococcal LPS.

400 400 400 LPS+ LPS- LPS 7 N. meningitidis N. meningitidis 300 300 300

(%) 200 200 200 β β β β

IL-1 100 100 100 

0 0 0

0 10 1000 100000 0 10 1000 100000 0 10 1000 100000 MBL (ng/mL) MBL (ng/mL) MBL (ng/mL)

Figure 3. MBL enhances non-LPS-induced IL-1β production. Dose–effect relationship for MBL in concentrations ranging from 0.75 ng/mL to 2500 ng/mL on IL-1β production after stimulation with wild-type LPS+N. meningitidis (6 × 108/mL), LPS−N. meningitidis (6 × 108/ mL) and N. meningitidis LPS (600 ng/mL). MBL was pre-incubated with the various stimuli for 2 h. Mean relative change (in %) of IL-1β as compared to control production ± SEM of 10 separate experiments in different control subjects is presented for both meningococcal strains, n = 5 for meningococcal LPS. P < 0.01 for LPS+N. meningitidis and P < 0.001 for LPS−N. meningitidis (one-way ANOVA). production of IL-6 was also not affected by MBL (data not shown). Furthermore, MBL had no effect on the cytokine production induced by meningococcal LPS. To exclude the possibility that the enhancing effect of MBL on cytokine induction could be attributed to a non-specific protein effect, the effect of human serum albumin (HSA) at 2500 ng/mL on the cytokines studied was determined: no effect of HSA could be observed (data not shown).

107 10 1000 with MBL with MBL Figure 4. Effect of MBL on IL-1β at without MBL without MBL 8 * 800 different concentrations of LPS−N. meningitidis. Dose–effect relationship 6 600 * for LPS−N. meningitidis in

(ng/mL) concentrations ranging from β 4 400 6 × 106 to 6 × 108 bacteria/ mL on IL-1β and IL-10 IL-1 IL-10 (ng/mL) production in the absence 2 200 (open circles) or presence (closed circles) of MBL (2500 ng/mL.). MBL was pre- 0 0 incubated with the stimulus 1 10 100 1000 1 10 100 1000 for 2 h. Median values ± IQR

- - of 5 separate experiments in LPS N. meningitidis LPS N. meningitidis different control subjects are (106 bacteria/mL) (106 bacteria/mL) presented, *P < 0.05.

To determine the dose–effect relationship for MBL and IL-1β production, PBMCs were stimulated with LPS+ and LPS− meningococci and purified meningococcal LPS in the presence of increasing concentrations of MBL (range: 0.75 ng/mL–2500 ng/mL). Figure 3 shows that IL-1β induction by LPS+N. meningitidis as well as by LPS−N. meningitidis was significantly enhanced by MBL (P < 0.01 for LPS+N. meningitidis and P < 0.001 for LPS−N. meningitidis (one-way ANOVA)), in a dose-dependent fashion. MBL exerted its greatest effect on IL-1β production after stimulation with LPS− meningococci. An enhancement of 84% ± 24.6% (mean ± sD) was found with LPS+ meningococci when MBL was present (2500 ng/mL) which increased to 298% ± 75% with LPS− meningococci at the same MBL concentration. No effect of MBL could be observed on cytokine production after stimulation with isolated LPS (P = 0.42). TNFα production was not affected at any of the MBL concentrations tested (data not shown).

The enhancing effect of MBL on cytokine production was also dependent on the quantity of bacteria used for stimulation. Figure 4 shows IL-1β and IL-10 production in the absence or presence of MBL (2500 ng/mL) after stimulation with 6×106 to 6×108 LPS− meningococci/mL: MBL enhanced cytokine production at concentrations > 107 meningococci. However, at lower concentrations no enhancing effect occurred. For IL-1β, a similar pattern was present in experiments using LPS+ wild-type meningococci, no significant effect of LPS+N. meningitidis was seen on IL-10 production.

Discussion

The principal findings of the present study are that MBL augmented IL-1β and IL-10 production by human PBMCs induced by non-LPS components of meningococci. In

108 contrast, TNFα, IL-6 and IFNγ production was unaffected, suggesting a differential role of MBL in the signalling pathways for IL-1β and IL-10 on one hand, and TNFα, IL-6 and IFNγ on the other. In addition, we found that the LPS+ as well as the LPS− meningococci were able to bind MBL to a similar extent.

MBL reportedly binds to repeating sugar units present on the cell surface of various human pathogens 32. However, as assessed by flow cytometry, MBL binds poorly to intact wild-type serogroup B N. meningitidis probably because of the extensive sialylation of the capsule and LPS 33,34. We have previously noted that flow cytometry may not identify all of the physiologically relevant MBL-binding to a microorganism 14. Therefore, in the present study we improved the sensitivity of the detection of MBL-binding by using dot blotting of whole organisms. In this way, we showed that binding of MBL does occur to the serogroup B H44/76 LPS+ meningococci and the LPS− meningococci. Interestingly, this low level of binding is sufficient for the modulation of cytokine production 30, as IL-1β production was augmented by MBL after stimulation with the LPS+ and LPS− strains. In addition, the finding that MBL binds LPS+ and LPS− meningococci to a similar extent suggests that MBL does not bind to LPS but to other structures present on the outer membrane. 7 This is supported by the findings of Estabrook et al. who found that MBL binds to outer membrane proteins of the meningococcus and not to LPS 35. Further, we confirmed our previous finding that meningococcal mutants having a truncated LPS bound more MBL than organisms having an untruncated LPS. The B1940 parent strain, which was used as a negative control, did not bind significant amounts of MBL. This indicates that in both the strains belonging to serogroup B (B1940 and H44/76), differences in MBL-binding are present. Possibly, differences in sialylation of the LPS or differences in outer membrane protein expression account for this difference.

Although flow cytometry proved to be insufficiently sensitive to detect MBL- binding to H44/76 meningococci14, it has been shown that MBL does not appear to aggregate bacteria on its own. This is supported by a viable counting methodology which shows no effect of MBL on bacterial numbers, even when MBL-binding is high, such as to the B1940cpsD− strain (D.L. Jack, unpublished data).

We previously found that purified MBL added to MBL-deficient whole blood modified the production of TNFα, IL-1β and IL-6 in a dose-dependent manner 30. In the current study, we investigated whether MBL is directly involved in the cytokine response by PBMC to N. meningitidis by excluding complement and other serum proteins. MBL enhanced IL-1β production but did not influence IL-6, TNFα or IFNγ. Our results suggest that MBL has a direct effect on PBMC by highly selective cytokine

109 induction. When compared with our previous results, it appears that other serum proteins also have an effect on cytokine induction by MBL, which is particularly noticeable for IL-6.

We found that MBL had an enhancing effect on IL-1β and IL-10 production only at relatively high bacterial concentrations (>107 bacteria/mL). However, during meningococcal infection, local concentrations of bacteria at the site of entry are probably within this range. In addition, the number of bacteria in the bloodstream or in cerebrospinal fluid during severe invasive disease can exceed 108 bacteria/mL 36-38. Therefore, the finding in this laboratory study may be clinically relevant.

Serum concentrations of MBL in individuals with a normal wild-type MBL genotype range from 1000 to 5000 ng/ml, which is in range with the highest concentration of MBL used in our experiments 12. IL-1β enhancement was observed at MBL concentrations. These are levels as seen in individuals heterozygous for a structural mutation in exon 1 and suggests that an absence of cytokine modulation by MBL is only likely to be seen in individuals completely deficient for MBL.

The ability of meningococcal components other than LPS to induce cytokines has recently been established by us and several other groups using the same LPS− meningococcal mutant 7,8. We showed that MBL had a more pronounced effect on cytokine induction by LPS− meningococci as compared with the LPS+ meningococci and that MBL had no effect on cytokines induced by meningococcal LPS. This suggests that MBL interacts with the non-LPS components of the meningococci to enhance cytokine production. We have recently determined that MBL binds to 2 outer membrane proteins of the meningococcus, porin B and opacity-associated protein but not to LPS. As porin B is a TLR2 ligand, and contributes as a non-LPS ligand to cytokine production by PBMC 39, it is tempting to speculate that MBL may enhance TLR2 signalling.

MBL had a more pronounced effect on IL-1β and IL-10 induction by LPS− meningococci as compared with the LPS+ meningococci. This is unlikely to be due to an increased expression of MBL-binding outer membrane proteins such as porin and opacity protein A (opA) 25,35, because there is no difference in expression of these outer membrane proteins between the different meningococcal strains. Because binding of MBL to both meningococcal strains was similar, and no effect of MBL on the LPS-induced cytokines was seen, it is more likely that using the LPS+ strain enhancement of IL-1β and IL-10 was overshadowed by the relatively higher LPS-dependent cytokine production. However, as certain lipoproteins are expressed in decreased amounts, and there are differences in composition of the

110 outer membrane phospholipids, it cannot be ruled out that the difference in MBL- dependent cytokine production between the strains is dependent on differences in outer membrane composition.

Whereas IL-1β and IL-10 productions were enhanced by MBL, no effect was seen on the TNFα and IL-6 production. Thus, MBL has a differential effect on the signalling pathways of IL-1β and IL-10 on one hand, and TNFα and IL-6 on the other. Recently, it has been found that LPS− meningococci use other cellular receptors than meningococcal LPS: LPS can employ toll-like receptor (TLR) 4 to induce cytokines, whereas LPS− meningococci use pathways independent of TLR4, such as TLR2 28,40. Various studies showed that the engagement of different cellular receptors results in differential production of cytokines 41-43. The differential modulation of cytokine production by MBL, therefore, is likely to be caused by engagement of cellular receptors other than those for LPS, which favour the production of IL-1β and IL-10. The observation that MBL upregulates IL-10 induction by non-LPS of the meningococcus indicates that, interaction of MBL with non-LPS components also contributes to an anti-inflammatory response 44. 7 In contrast to IL-1β, IL-10, TNFα and IL-6, which are monocyte-produced cytokines, IFNγ is a primarily T-cell- and NK-cell-derived cytokine. The lack of effect of MBL on IFNγ production might imply that MBL interacts predominantly with monocytes to augment monocyte-produced cytokines.

In meningococcal disease, a poor outcome is correlated with high plasma levels of pro-inflammatory cytokines 3,45. For IL-1β in particular, its presence in plasma of a patient predicts a fulminant course of the disease 3. On the other hand, IL- 10 is also upregulated in meningococcal disease and has been suggested to be the most important anti-inflammatory cytokine, implicated in down-regulation of monocyte responses to LPS 3 46. Because MBL enhances the production of both IL- 1β and IL-10, we suggest that these in vitro findings are also of importance in the pathogenesis of meningococcal sepsis. However, the ultimate effect of MBL on the balance between pro- and anti-inflammatory cytokines in meningococcal disease remains elusive.

In conclusion, this study shows that MBL engages with non-LPS components of N. meningitidis to enhance the production of the cytokines IL-1β and IL- 10 by human PBMCs, in addition to its effects on complement activation and opsonophagocytosis.

111 Acknowledgements

The authors thank Trees Verver for assistance with the stimulation experiments and cytokine assays and Marina Johnson for assistance in determining MBL-binding to the bacteria.

112 References 1. Van Deuren M, Brandtzaeg P, Van der Meer JWM. Update on meningococcal disease with emphasis on pathogenesis and clinical management. Clin Microbiol Rev. 2000;13:144- 166 2. Waage A, Halstensen A, Espevik T. Association between tumour necrosis factor in serum and fatal outcome in patients with meningococcal disease. Lancet. 1987;i:355-357 3. Van Deuren M, van der Ven Jongekrijg J, Bartelink AKM, van Dalen R, Sauerwein RW, van der Meer JWM. Correlation between proinflammatory cytokines and antiinflammatory mediators and the severity of disease in meningococcal infections. J Infect Dis. 1995;172:433-439 4. Westendorp RGJ, Langermans JAM, de Bel CE, Meinders AE, Vandenbroucke JP, van Furth R, van Dissel JT. Release of tumor necrosis factor: an innate host characteristic that may contribute to the outcome of meningococcal disease. J Infect Dis. 1995;171:1057- 1060 5. Read RC, Cannings C, Naylor SC, Timms JM, Maheswaran R, Borrow R, Kaczmarski EB, Duff GW. Variation within genes encoding interleukin-1 and the interleukin-1 receptor antagonist influence the severity of meningococcal disease. Ann Intern Med. 2003;138:534-541 6. Brandtzaeg P, Kierulf P, Gaustad P, Skulberg A, Bruun JN, Halvorsen S, Sørensen E. Plasma endotoxin as a predictor of multiple organ failure and death in systemic meningococcal disease. J Infect Dis. 1989;159:195-204 7. Sprong T, Stikkelbroeck N, van der Ley P, Steeghs L, van Alphen L, Klein N, Netea MG, van der Meer JWM, van Deuren M. Contributions of Neisseria meningitidis LPS and non- LPS to proinflammatory cytokine response. J Leukoc Biol. 2001;70:283-288. 7 8. Uronen H, Williams AJ, Dixon G, Andersen SR, Van der Ley P, Van Deuren M, Callard RE, Klein N. Gram-negative bacteria induce proinflammatory cytokine production by monocytes in the absence of lipopolysaccharide (LPS). Clin Exp Immunol. 2000;22:312 9. Ingalls RR, Lien E, Golenbock DT. Membrane-associated proteins of a lipopolysaccharide- deficient mutant of Neisseria meningitidis activate the inflammatory response through toll-like receptor 2. Infect Immun. 2001;69:2230-2236. 10. Jack DL, Klein NJ, Turner MW. Mannose-binding lectin: targeting the microbial world for complement attack and opsonophagocytosis. Immunol Rev. 2001;180:86-99. 11. Jack D, Bidwell J, Turner M, Wood N. Simultaneous genotyping for all three known structural mutations in the human mannose-binding lectin gene. Hum Mutat. 1997;9:41- 46 12. Steffensen R, Thiel S, Varming K, Jersild C, Jensenius JC. Detection of structural gene mutations and promoter polymorphisms in the mannan-binding lectin (MBL) gene by polymerase chain reaction with sequence-specific primers. J Immunol Methods. 2000;241:33-42. 13. Thiel S, Holmskov U, Hviid L, Laursen SB, Jensenius JC. The concentration of the C-type lectin, mannan-binding protein, in human plasma increases during an acute phase response. Clin. Exp. Immunol. 1992;90:31-35 14. Jack DL, Jarvis GA, Booth CL, Turner MW, Klein NJ. Mannose-binding lectin accelerates complement activation and increases serum killing of Neisseria meningitidis serogroup C. J Infect Dis. 2001;184:836-845. 15. Sprong T, Brandtzaeg P, Fung M, Pharo AM, Hoiby EA, Michaelsen TE, Aase A, van der Meer JW, van Deuren M, Mollnes TE. Inhibition of C5a-induced inflammation with preserved C5b-9-mediated bactericidal activity in a human whole blood model of meningococcal sepsis. Blood. 2003;102:3702-3710 16. Hibberd ML, Sumiya M, Summerfield JA, Booy R, Levin M, and, the, Meningococcal, Research, Group. Association of variants of the gene for mannose-binding lectin with susceptibility to meningococcal disease. Lancet. 1999;353:1049-1053 17. Garred P, Madsen HO, Svejgaard A, Michaelsen TE. Mannose-binding lectin and meningococcal disease [letter; comment]. Lancet. 1999;354:336; discussion 337

113 18. Bax WA, Cluysenaer OJ, Bartelink AK, Aerts PC, Ezekowitz RA, van Dijk H. Association of familial deficiency of mannose-binding lectin and meningococcal disease [letter]. Lancet. 1999;354:1094-1095 19. Chaka W, Verheul AF, Vaishnav VV, Cherniak R, Scharringa J, Verhoef J, Snippe H, Hoepelman AI. Induction of TNF-alpha in human peripheral blood mononuclear cells by the mannoprotein of Cryptococcus neoformans involves human mannose binding protein. J. Immunol. 1997;159:2979-2985 20. Soell M, Diab M, Haan-Archipoff G, Beretz A, Herbelin C, Poutrel B, Klein JP. Capsular polysaccharide types 5 and 8 of Staphylococcus aureus bind specifically to human epithelial (KB) cells, endothelial cells, and monocytes and induce release of cytokines. Infect Immun. 1995;63:1380-1386 21. Ghezzi MC, Raponi G, Angeletti S, Mancini C. Serum-mediated enhancement of TNF- alpha release by human monocytes stimulated with the yeast form of Candida albicans. J. Infect. Dis. 1998;178:1743-1749 22. Holten E. Serotypes of Neisseria meningitidis isolated from patients in Norway during the first six months of 1978. J Clin Microbiol. 1979;9:186-188 23. Steeghs L, den Hartog R, den Boer A, Zomer B, Roholl P, van der Ley P. Meningitis bacterium is viable without endotoxin. Nature. 1998;392:449-450 24. Steeghs L, Kuipers B, Hamstra HJ, Kersten G, van Alphen L, van der Ley P. Immunogenicity of outer membrane proteins in a lipopolysaccharide- deficient mutant of Neisseria meningitidis: influence of adjuvants on the immune response. Infect. Immun. 1999;67:4988-4993 25. Steeghs L, de Cock H, Evers E, Zomer B, Tommassen J, van der Ley P. Outer membrane composition of a lipopolysaccharide-deficient Neisseria meningitidis mutant. EMBO J. 2001;20:6927-6945 26. Westphal O, Jann JK. Bacterial lipopolysacharide extraction with phenol- water and further application of the procedure. Methods Carbohydr Chem. 1965;5:83-91 27. Weissbach A, Hurwitz B. The formation of 2-keto-3-deoxyheptonic acid in extracts of Escherichia coli B. J Biol Chem. 1959;234:705-709 28. Sprong T, van der Ley P, Steeghs L, Tax WJ, Verver-Janssen TJW, Netea MG, van der Meer JW, van Deuren M. Neisseria meningitidis can induce pro-inflammatory cytokine production via pathways independent from CD14 and toll-like receptor 4. European cytokine network. 2002;13:411-417 29. Kilpatrick DC. Isolation of human mannan binding lectin, serum amyloid P component and related factors from Cohn fraction III. Transfus Med. 1997;7:289-294. 30. Jack DL, Read RC, Tenner AJ, Frosch M, Turner MW, Klein NJ. Mannose-binding lectin regulates the inflammatory response of human professional phagocytes to Neisseria meningitidis serogroup B. J Infect Dis. 2001;184:1152-1162. 31. Drenth JP, Van Uum SH, Van Deuren M, Pesman GJ, Van der Ven Jongekrijg J, Van der Meer JWM. Endurance run increases circulating IL-6 and IL-1ra but downregulates ex vivo TNF-alpha and IL-1 beta production. J. Appl. Physiol. 1995;79:1497-1503 32. Turner MW. Mannose-binding lectin: the pluripotent molecule of the innate immune system. Immunol. Today. 1996;17:532-540 33. Jack DL, Dodds AW, Anwar N, Ison CA, Law A, Frosch M, Turner MW, Klein NJ. Activation of complement by mannose-binding lectin on isogenic mutants of Neisseria meningitidis serogroup B. J. Immunol. 1998;160:1346-1353 34. Van Emmerik LC, Kuijper EJ, Fijen CA, Dankert J, Thiel S. Binding of mannan-binding protein to various bacterial pathogens of meningitis. Clin. Exp. Immunol. 1994;97:411- 416 35. Estabrook MM, Jack DL, Klien NJ, Jarvis GA. Mannose-binding lectin (MBL) binds to two major outer membrane proteins, opacity protein and porin, of Neisseria meningitidis but not to lipooligosaccharide (LOS). J immunol. 2004;in press 36. Hackett SJ, Guiver M, Marsh J, Sills JA, Thomson APJ, Kaczmarski EB, Hart CA. Meningococcal bacterial DNA load at presentation correlates with disease severity. Arch Dis Child. 2002;86:44-46 37. Mariani-Kurkdjian P, Doit C, Le Thomas I, Aujard Y, Bourrillon A, Bingen E. Concentrations

114 bacteriennes dans le liquide céphalo-rachidien au cours des méningites de l’ enfant. Presse Med. 1999;28:1227-1230 38. Bingen E, Lambert-Zechovsky N, Mariani-Kurkdjian P, Doit C, Aujard Y, Fournerie F, Mathieu H. Bacterial counts in cerebrospinal fluid of children with meningitis. Eur J Clin Microbiol Infect Dis. 1990;9:278-281 39. Massari P, Henneke P, Ho Y, Latz E, Golenbock DT, Wetzler LM. Immune stimulation by neisserial porins is Toll-like receptor 2 and MyD88 dependent. J Immunol. 2002;168:1533- 1537 40. Pridmore AC, Wyllie DH, Abdillahi F, Steeghs L, van der Ley P, Dower SK, Read RC. A lipopolysaccharide-deficient mutant of Neisseria meningitidis elecits attenuated cytokine release by human macrophages and signal via toll-like receptor (TLR)2, but not via TLR4/ MD2. The Journal of Infectious Diseases. 2001;183:89-96 41. Hirschfeld M, Weis JJ, Toschakov V, Salowski CA, Cody MJ, Ward DC, Qureshi N, Michalek SM, Vogel SN. Signalling by Toll-Like Receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect Immun. 2001;69:1477-1482 42. Netea MG, Blok WL, Kullberg BJ, Bemelmans M, Vogels MT, Buurman WA, van der Meer JWM. Pharmacologic inhibitors of tumor necrosis factor production exert differential effects in lethal endotoxemia and in infection with live microorganisms in mice. J Infect Dis. 1995;171:393-399 43. Rabehi L, Irinopoulou T, Cholley B, Haeffner-Cavaillon N, Carreno MP. Gram-positive and gram-negative bacteria do not trigger monocytic cytokine production through similar intracellular pathways. Infect Immun. 2001;69:4590-4599. 44. Moore KW, O’Garra A, de Waal Malefyt R, Vieira P, Mosmann TR. Interleukin-10. Annu Rev Immunol. 1993;11:165-190 45. Waage A, Brandtzaeg P, Halstensen A, Kierulf P, Espevik T. The complex pattern of cytokines in serum from patients with meningococcal septic shock. Association between 7 interleukin 6, interleukin 1, and fatal outcome. J Exp Med. 1989;169:333-338 46. Brandtzaeg P, Osnes L, Øvstebo R, Joø GB, Westvik Å-B, Kierulf P. Net inflammatory capacity of human septic shock plasma evaluated by a monocyte-based target cell assay: identification of interleukin-10 as a major functional deactivator of human monocytes. J Exp Med. 1996;184:51-60

115

Inhibition of C5a-induced inflammation with preserved C 5b-9-mediated bactericidal activity in a human whole blood model of meningococcal sepsis. 8

Tom Sprong, Petter Brandtzaeg, Michael Fung, Anne M. Pharo, Ernst- Arne Hoiby, Terje E. Michaelsen, Audun Aase, Jos W.M. van der Meer, Marcel van Deuren and Tom Eirik Mollnes. Blood. 2003;102(10):3702-10 Abstract

The complement system plays an important role in the initial defense against Neisseria meningitidis. In contrast, uncontrolled activation in meningococcal sepsis contributes to the development of tissue damage and shock. In a novel human whole blood model of meningococcal sepsis, we studied the effect of complement inhibition on inflammation and bacterial killing. Monoclonal antibodies (mAbs) blocking lectin and alternative pathways inhibited complement activation by N. meningitidis and oxidative burst induced in granulocytes and monocytes. Oxidative burst was critically dependent on CD11b/CD18 (CR3) expression but not on Fcγ- receptors. Specific inhibition of C5a using mAb 137-26 binding the C5a moiety of C5 before cleavage prohibited CR3 up-regulation, phagocytosis, and oxidative burst but had no effect on C5b-9 (TCC) formation, lysis, and bacterial killing. An mAb-blocking cleavage of C5, preventing C5a and TCC formation, showed the same effect on CR3, phagocytosis, and oxidative burst as the anti-C5a mAb but additionally inhibited TCC formation, lysis, and bacterial killing, consistent with a C5b-9-dependent killing mechanism. In conclusion, the anti-C5a mAb 137-26 inhibits the potentially harmful effects of N. meningitidis-induced C5a formation while preserving complement-mediated bacterial killing. We suggest that this may be an attractive approach for the treatment of meningococcal sepsis.

118 Introduction

The gram-negative bacterium Neisseria meningitidis is an important human pathogen worldwide. It can cause meningitis or fulminant meningococcal sepsis (FMS); the latter is an overwhelming and often lethal condition that may lead to death within 24 hours. The pathogenic mechanism leading to FMS is a breakdown of homeostasis by a massive activation of diverse inflammatory systems such as the cytokine network and plasma cascades such as the complement, coagulation, fibrinolytic, and kinin/kallikreinin systems 1,2.

The importance of the complement system in meningococcal disease is emphasized by several observations. Complement deficiencies are defined risk factors for meningococcal infections, indicating that complement is crucial in the initial defense against this bacterium 3-5. On the other hand, during FMS, disease severity, tissue damage, and outcome are closely related to the degree of complement activation 6,7. Thus, with respect to the pathogenesis of meningococcal disease, the complement system has been aptly named a double-edged sword 8.

Complement is activated on the surfaces of meningococci by one or more of the 3 initial complement-activating pathways—classical, lectin, and alternative. After the activation of complement factor-3 (C3) by any of these 3 pathways, a C5 convertase (C4b2a3b through the classical and lectin pathways or C3bBbC3b through the alternative pathway) 8 is formed, and the pivotal C5 molecule is cleaved into C5a and C5b. C5b is the initial molecule in the formation of the terminal C5b-9 complement complex (TCC). Membrane- associated TCC, also designated the C5b-9 membrane attack complex (MAC), plays a key role in lysis of the bacterium, whereas the fluid-phase SC5b-9 is lytically inactive after binding to vitronectin and clusterin. C5a is an important anaphylatoxin that has a wide range of proinflammatory effects, including endothelium activation, vascular permeability induction, histamine release, coagulation system activation, chemotaxis, cytokine modulation, CR3 up-regulation, and oxidative burst induction 9-11. A schematic overview of the complement system is shown in Figure 1.

In systemic meningococcal disease, there is a continuous search for a treatment modality that abrogates the deleterious inflammatory response. Unfortunately, to date no effective regimen has been found. Modulation of the complement system may be an attractive approach in the treatment of FMS or other inflammatory diseases 12,13. A main challenge of reducing disease severity by inhibiting complement in FMS is to preserve the beneficial bactericidal effect of complement while attenuating the detrimental systemic inflammatory effects, thus blunting one edge of the double- edged sword while sharpening the other.

119 Classical Lectin Alternative pathway pathway pathway

C1q / C1r / C1s MASP 2 C3/C3b C1rs- D C1inh C4 B P C4bc C2 C3 C3bBbP

C5b-9 C5a

TCC

Figure 1. Schematic overview of the complement system. Boxes indicate specific complement activation products measured by enzyme-linked immunosorbent assay (ELISA) and their relation to specific complement activation pathways. C1rs-C1inh complexes reflect classical pathway activation, C4bc reflects both classical and lectin pathway activation, and C3bBbP reflects alternative pathway activation. C3bc and terminal complement complex (TCC) are activation products of the final common and terminal pathways. MBL indicates mannose-binding lectin; MASP, MBL- associated serine protease.

The aim of the present study was to identify such a strategy. For this purpose we developed a human whole blood model of meningococcal sepsis based on a newly described method 11 to determine the effect of inhibiting N. meningitidis-induced complement activation on bactericidal activity and the inflammatory response.

Materials and Methods Equipment and reagents All materials used in the stimulation experiments were endotoxin free. Polypropylene tubes were either NUNC cryotubes (Nalgene NUNC, Roskilde, Denmark) or Falcon (Becton Dickinson, Franklin Lakes, NJ) tubes. Phosphate-buffered saline (PBS) was produced in the laboratory, Dulbecco medium was obtained from Invitrogen (Paisley, Scotland), and lepirudin (Refludan) was from Hoechst (Frankfurt am Main, Germany). Flow cytometry was performed with FACScalibur (Becton Dickinson, San Jose, CA) and with an Epics XL (Coulter, Hialeah, FL) apparatus. MRX microplate reader (Dynex Technologies, Denkendorf, Germany) was used to determine optical density (OD).

Inhibitory antibodies Mouse immunoglobulin G1 (IgG1) mAbs to human C2 (clone 175-26), factor D

120 (clone 166-32), C5 (clone 137-76), C5a (clone 137-26), and the isotype-matched control (clone G3-519, anti-HIV1 gp 120) were produced and purified under identical conditions in the laboratory of one of the coauthors (M.F.) 14. The anti-C5 antibody 137-76 binds C5 and prevents its cleavage, blocking the formation of C5a and that of C5b-9 15. Anti-C5a 137-26 blocks free C5a directly and preneutralizes its biologic effects by binding to the C5a moiety on native C5 without interfering with C5 cleavage. The anti-C5a antibody 137-26 is highly human specific, and no cross- reactivity has been observed with nonprimate species 16.

Purified mouse IgG1 mAb to human CD11b (clone ICRF 44) was obtained from

Serotec (Oxford, United Kingdom). Mouse mAb (F(ab’)2) to human CD16/FcγRIII (clone 3G8) recognizes both allelic forms of CD16 and blocks the binding of immune- complexed IgG to CD16. Mouse mAb (F(ab’)2) to human CD32/FcγRII (clone 7.3) reacts with the domain 2 epitope of all CD32 isoforms and blocks IgG immune- complexed binding. Mouse mAb (F(ab’)2) to human CD64/FcγRI (clone 10.1) recognizes the CD64 molecule of 72 kDa from gene FcγRIA and blocks binding of FcγRIA to IgG opsonized cells. All were obtained from Ancell Corp (Bayport, MN). Purified mouse IgG1 mAb to human mannose-binding lectin (MBL) (clone HYB131- 01), which reacts with human MBL both in its polymeric conformation and as a single subunit, was obtained from the AntibodyShop (Copenhagen, Denmark) and was documented by the manufacturer to block MBL function. All antibodies were used in concentrations ranging from 6.25 to 25 µg/mL because preliminary experiments 8 showed maximum inhibition in this concentration range, except for the anti-CD11b and anti-MBL antibodies, which had to be used in concentrations up to 50 µg/mL (anti-CD11b) or 100 µg/mL (anti-MBL) to obtain maximum inhibition.

Bacterial strains N. meningitidis H44/76 is an isolate of a patient with invasive meningococcal disease 17. H44/76 is the production strain of the Norwegian group B OMV vaccine and an international reference strain. It has been serologically classified as B:15:P1.7,16, immunotype L3,7,9. N. meningitidis was grown overnight on Kellog medium and resuspended in Hanks balanced salt solution (HBSS) (Gibco Invitrogen). Heat inactivation occurred at 60°C for 40 minutes. Escherichia coli and fluorescein isothiocyanate (FITC)-labeled, opsonized E. coli strain LE 392 (American Type Culture Collection 33572) were obtained from Orpegen (Pharma, Heidelberg, Germany) and were used as controls. The concentration of bacteria was determined by measuring OD at 630 nm. The variant H44/76-1, more strongly expressing the Opc protein, was used in the serum-bactericidal activity and whole blood bactericidal activity assays.

121 N. meningitidis was FITC labeled as described previously 18. In brief, 2 x 109 meningococci/mL was incubated with 0.16 mg/mL FITC (isomer 1, 90% high- performance liquid chromatography [HPLC]; Sigma Chemical, St Louis, MO) for 30 minutes at 37°C. Bacteria were washed 4 times and resuspended in PBS. The concentration of bacteria after washing was determined by measuring OD at 630 nm.

For the experiments using live meningococci, bacteria were grown overnight on brain-heart infusion (BHI) agar, reseeded on BHI agar plates, grown for 4 hours

at 37°C in 5% CO2 into mid-log-phase, and harvested in HBSS with 0.1% bovine serum albumin (BSA).

Donors Seven healthy donors were used throughout the experiments. Concentration of specific antibodies in the sera of the controls against N. meningitidis H44/76 was determined by flow cytometry, as described previously 19. In brief, 5 µL of 109 N. meningitidis/mL was incubated with 50 µL of dilutions of serum for 1 hour at room temperature, washed twice, and developed with FITC-labeled goat antihuman IgG (Cappel; Organon Teknika, Turnhout, Belgium) for 1 hour followed by one washing. A 2-fold dilution series of reference plasma was used for the standard curve. For the serum bactericidal assay and the whole blood bactericidal activity assay, 2 separate donors were used with known high bactericidal titers to N. meningitidis in the serum bactericidal assay and a high concentration of antibodies after vaccination. Informed consent was obtained from all donors before the experiments were performed, and the human experimentation guidelines of the local Ethics Committee of Rikshospitalet University Hospital were followed in the conduct of the research.

Whole blood model of inflammation A recently developed whole blood model to study the role of complement in E. coli-induced inflammation was modified for N. meningitides 11. The model is based on anticoagulation with lepirudin, a recombinant hirudin, which is a highly specific thrombin inhibitor not influencing complement activation. Whole blood was collected in polypropylene tubes containing lepirudin (50 µg/mL). Whole blood was preincubated with PBS or antibody for 4 minutes at 37°C in a water bath. For CR3 (CD11b/CD18) expression, phagocytosis, and oxidative burst, incubation with the stimulants was for 10 minutes at 37°C. After incubation, samples were processed immediately for flow cytometry. For the detection of complement activation, samples were incubated for 1 hour at 37°C, and activation was stopped by adding 20 mM (final concentration) ethylenediaminetetraacetic acid (EDTA).

122 Oxidative burst, phagocytosis, and CR3 (CD11b/CD18) expression Oxidative burst and phagocytosis were measured using commercially available Burst-test and Phago-test kits (Orpegen Pharma, Heidelberg, Germany). FITC- labeled meningococci were used in the phagocytosis assay. Using a forward/side scatter (FSC/SSC) dot plot, granulocytes and monocytes were analyzed separately with regard to median fluorescence intensity (MFI). CR3 expression was measured after incubation with inhibitors and stimulants after cells were fixed with 0.5% (vol/ vol) paraformaldehyde. Cells were stained with anti-CD-14 FITC (Becton Dickinson, San Jose, CA) to distinguish monocytes from granulocytes, the nuclear dye LDS- 751 (FL-3) (Molecular Probes, Eugene, OR), anti-CD11b-PE, or isotype control (γ2a).

Complement activation enzyme immunoassays Activation of the classical complement pathway was determined as described previously 20, using the monoclonal antibody Kok-12 specific to a neoepitope exposed only when the C1 inhibitor (C1inh) is in complex with its substrates. This antibody was a kind gift from Prof Dr C. E. Hack (Amsterdam, the Netherlands). Activation of the alternative pathway was detected by quantifying the alternative convertase C3bBbP, as was recently described in detail 11. Activation of the classical and lectin pathways was determined by an assay using a mAb specific for a neoepitope exposed in activated C4 detecting C4b and C4c (C4bc) 21. The antibody was a kind 8 gift from Prof Dr C. E. Hack. Activation of the final common pathway was quantified using the monoclonal antibody bH6 specific for a neoepitope expressed in C3b, iC3b, and C3c (C3bc) as described previously 22. Activation of the terminal pathway was quantified using the monoclonal antibody aE11 specific for C9 incorporated in the fluid-phase SC5b-9 complex (TCC), as described previously 23. An overview of these complement-activation markers can be seen in Figure 1

Lysis of sensitized SRBCs in human whole blood Lytic activity of the terminal complement complex in human whole blood was established using a modified classical pathway hemolytic assay. Sheep red blood cells (SRBCs) (hemolytic system; Virion/Serion, Würtzburg, Germany) were incubated at 37°C for 30 minutes, spun down at 1400g, and resuspended to a 5% solution in sterile veronal buffered saline (bioMerieux SA, Nancy l’Étoile, France) supplemented with 0.01% BSA (Sigma Chemical). SRBCs (20 µL) were incubated in the whole blood (120 µL) samples for 20 minutes at 37°C in a waterbath. Then 20 mM EDTA was added to stop ongoing complement activation, and the samples were spun down at 1400g. Supernatants were collected and read at 405 nm.

123 Serum bactericidal activity The effect of the anti-C5 137-76 and anti-C5a 137-26 mAbs on serum bactericidal activity was tested using an agar overlay method, as described previously 24,25. In brief, human complement, as plasma, was used at a final concentration of 25%. A 2-fold dilution series, in HBSS with 0.1% BSA of the sera to be examined, were inoculated with approximately 100 colony-forming units (CFUs) per well of logarithmic phase-growth meningococci. This mixture was incubated for 60 minutes at 37°C in air; after that agar was added, and plates were incubated overni at 37°C

in 5% CO2. The number of CFUs was counted with the use of a magnifying glass.

Log2 titer represents the highest dilution of sera or the lowest antibody concentration at which more than 50% of the inoculum was killed.

Whole blood bactericidal activity N. meningitidis H44/76, grown overnight on BHI agar, was subcultured and grown into log-phase for 4 hours. Approximately 5000 CFUs were added to 1.1 mL lepirudin-anticoagulated whole blood samples preincubated for 5 minutes with PBS or antibody. Immediately after inoculation (t = 0) and after certain time periods, 100 µL whole blood was seeded on microbiologic Petri dishes containing blood agar and

incubated for 24 hours at 37°C and 5% CO2. Bacterial growth was expressed as CFU/100 µL whole blood added. Blood was mixed thoroughly before each sampling on a whirl mixer.

Statistics The nonparametric Mann-Whitney U test for unpaired data was used to analyze comparisons for statistical significant differences. P values less than .05 were considered statistically significant.

Results Complement activation by N. meningitidis in human whole blood To determine the relative contributions of the classical, alternative, and lectin pathway on complement activation by N. meningitidis, monoclonal antibodies blocking C2 (classical and lectin pathways), MBL (lectin pathway), and factor D (alternative pathway) were tested for their ability to inhibit the formation of specific complement activation products in lepirudin-anticoagulated human whole blood incubated with heat-inactivated bacteria for 1 hour. C1rs-C1inh complexes (classical pathway), C4bc (classical and lectin pathway), and C3bBbP (alternative pathway) were used as markers of initial pathway activation; TCC was used as a marker of total complement activation (Figure 1) N. meningitidis (1 x 108/mL) induced some classical pathway activation, seen as

124 a modest, but significant, increase in C1rs-C1inh complexes. As expected, no effect of any of the antibodies on C1rs-C1inh could be observed (Figure 2A). A marked and significant increase in C4bc formation was seen after stimulation with N. meningitidis. Anti-C2 had no effect; however, anti-MBL completely inhibited C4bc formation (Figure 2B). Noticeable alternative pathway activation was also seen after stimulation with meningococci, and anti-factor D inhibited all this alternative pathway activation (Figure 2C) N. meningitidis induced a marked increase in fluid-phase TCC, which was completely abolished by anti-factor D, markedly reduced by anti- MBL (75% reduction), and modestly but not significantly reduced by anti-C2 (Figure 2D) There was no relation between antibody titers against N. meningitidis H44/76 in the donors used for these experiments and the degree of complement activation or inhibition. Taken together, these data support mainly lectin and alternative pathway-

A B 100 200 C1rs- C4bc 80 C1inh 150 60 100

AU/mL 40 50 20 *

C D 1000 50 8 C3bBbP TCC 800 40

600 30

AU/mL 400 20 200 * 10 * * N. Meningitidis 1 x 108/mL í + + + + + í + + + + + Anti- Anti- Anti- control Anti- Anti- Anti- control mAb C2 MBL D C2 MBL D

Figure 2. Complement activation by N. meningitidis. Lepirudin-anticoagulated human whole blood was incubated with 1 x 108 heat-inactivated N. meningitidis H44/76 for 1 hour at 37°C in the presence or absence of the following complement-inhibiting mAbs: anti- C2 (25 µg/mL), anti-MBL (100 µg/mL), and anti-factor D (25 µg/mL). C1rs-C1inh complexes (classical pathway; panel A), C4bc (classical and lectin pathways; panel B), and C3bBbP (alternative pathway; panel C) were used as markers of initial pathway activation, and fluid-phase terminal complement complex (TCC) was measured as an indicator of total complement activation (D). Minor spontaneous formation of all complement-activation products occurred in unstimulated blood (hatched columns). Meningococci (1 x 108) induced a minor increase of C1rs-C1inh complex formation but significant C4bc, C3bBbP, and TCC formation (second columns). Anti-C2 had no statistically significant effect on any of the activation products, whereas anti-MBL completely inhibited C4bc formation, and anti-factor D completely inhibited alternative pathway activation. The increase in TCC was completely abolished by anti-factor D and was markedly reduced by anti-MBL. Median ± IQRs of 4 separately performed experiments are presented. *P < .05 compared with N meningitidis-stimulated sample (second columns).

125 CD11b expression Oxidative burst 350 7

300 6

250 5

200 4

MFI 150 3 Figure 3. Comparison of live and heat- 100 2 inactivated bacteria. A dose-dependent increase 50 1 of granulocyte CR3 (CD11b/ CD18) expression and oxidative burst was observed in lepirudin- 0 0 anticoagulated human whole blood B 1 10 100 B 1 10 100 after stimulation for 10 minutes at

7 7 37°C. Results for live log-phase Meningococci (1x10 /mL) Meningococci (1x10 /mL) and heat-inactivated meningococci were similar. MFI indicates median t = 0 (unstimulated) fluorescence intensity; B, baseline t = 10 (unstimulated) (unstimulated sample) at time of incubation (t = 0) and after 10 live N. meningitidis minutes (t = 10). The experiment was repeated once with virtually heat-inactivated N. meningitidis identical results.

Granulocytes Monocytes 40 60

50 30 40 Figure 4. Effect of complement inhibition on 20 30 oxidative burst. N. meningitidis H44/76-induced granulocyte and monocyte 20 oxidative burst was measured in whole human blood after a 10- 10 8 minute incubation with 2 x 10 Oxidative burst (MFI) 10 meningococci/mL in the presence or absence of complement inhibitory mAbs. Antibody 0 0 concentrations ranged from 6.25 B 0 10 20 30 B 0 10 20 30 to 25 µg/mL for anti-C2 and anti-D [mAb] or 0.25 x [anti- [mAb] or 0.25 x [anti- and from 25 to 100 µg/mL for anti- MBL / isotype] (µg/mL) MBL / isotype] (µg/mL) MBL and the isotype control. Note that combining anti-factor D with Controls: t = 10 (unstimulated) Isotype control anti-MBL or anti-C2 enhanced the inhibitory effect. B indicates mAbs: Anti-C2 Anti-MBL Anti-D baseline (unstimulated sample) incubated for 10 minutes (t = Anti-D/anti-MBL Anti-D/anti-C2 10). Median values of 4 separate experiments are presented

126 dependent mechanisms of complement activation by N. meningitidis H44/76 in this whole blood assay.

CR3 up-regulation and induction of oxidative burst by live and heat-inactivated meningococci To validate the use of heat-inactivated meningococci in the inflammatory response experiments, live log-phase N. meningitidis H44/76 and heat-inactivated N. meningitidis H44/76 were compared with respect to CR3 (CD11b/CD18) expression and oxidative burst. A dose-dependent increase in granulocyte CR3 and oxidative burst was observed, with no difference between live and heat-inactivated bacteria (Figure 3). Results in monocytes showed a similar pattern to those seen in granulocytes. Based on these results the following experiments were performed with heat-inactivated meningococci.

Effect of initial complement pathway inhibition on oxidative burst N. meningitidis-induced oxidative burst in human whole blood was essentially dependent on complement activation because mAbs blocking the initial complement CD11b expression Phagocytosis Oxidative burst 600 CD11b expression400 Phagocytosis40 Oxidative burst 600 400 40 500 500 300 30 400 300 30 400 8 300 200 20 MFI 300 200 20 MFI 200 200 100 10 100 100 10 100 0 0 0 B 0 010 2030 40 50 B 0 010 2030 40 50 B 0 010 2030 40 50 AntibodyB 0(µg/mL)10 2030 40 50 AntibodyB 0(µg/mL)10 2030 40 50 AntibodyB 0(µg/mL)10 2030 40 50 Antibody (µg/mL) Antibody (µg/mL) Antibody (µg/mL)

t = 0 (unstimulated) t = 10 (unstimulated) Isotypet control = 0 (unstimulated) Anti-C5 t(137-76) = 10 (unstimulated) Anti-C5aIsotype (137-26) control Anti-C5 (137-76) Anti-C5a (137-26) Figure 5. Effect of anti-C5 and anti-C5a on granulocyte responses. N. meningitidis H44/76-induced CR3 (CD11b/CD18) expression, phagocytosis of FITC-labeled meningococci, and oxidative burst by N. meningitidis were measured in whole human blood after a 10- minute incubation with 2 x 108 meningococci/mL in the presence or absence of the mAbs anti-C5 (clone 137-76, preventing C5 cleavage), anti-C5a (clone 137-26), and an isotype control (6.25-50 µg/mL). Anti- C5 and anti-C5a markedly, and to the same extent, inhibited N. meningitidis-induced granulocyte CR3 up-regulation, phagocytosis, and oxidative burst. B indicates baseline (unstimulated sample) at time of incubation (t = 0) or after 10 minutes of incubation at 37°C (t = 10). Results from 1 of 3 representative experiments are shown.

127 pathways markedly inhibited oxidative burst in granulocytes Figure 4 left panel) and monocytes Figure 4 right panel). Anti-C2 and anti-factor D partially reduced the granulocyte oxidative burst, whereas the monocyte oxidative burst was only marginally affected. However, when anti-C2 and anti-factor D were combined, the granulocyte oxidative burst was completely abolished and the monocyte oxidative burst was markedly reduced. Finally, anti-MBL alone markedly inhibited oxidative burst in both cell types, an effect that was further enhanced when anti-MBL and anti-factor D were combined. These data indicate that oxidative burst induced by the meningococcus in whole blood is dependent on complement activation through the lectin and alternative pathways.

Effect of inhibiting C5 and C5a on CR3 expression, phagocytosis, and oxidative burst The effect of inhibiting C5 and C5a on N. meningitidis-induced CR3 expression, phagocytosis of FITC-labeled meningococci, and oxidative burst by N. meningitidis in granulocytes and monocytes was examined in human whole blood using anti- C5 and anti-C5a mAbs. Both antibodies efficiently abolished, in a dose-dependent fashion, CR3 up-regulation, phagocytosis, and oxidative burst in granulocytes, whereas the isotype control antibody had no effect (Figure 5) Monocyte CR3 expression, phagocytosis, and oxidative burst were also inhibited by the anti- C5 and anti-C5a antibodies, though this inhibition was less pronounced than in granulocytes. The data suggest that the inflammatory responses observed, particularly in granulocytes, were largely dependent on C5 activation, and the

350 300

300 250

250 200 200 150 150 Figure 6. Effect of CR3 and FcγR inhibition on 100 100 granulocyte oxidative burst. The anti-CD11b-blocking antibody

Oxidative burst (MFI) 50 ICRF44 (6.25-50 µg/mL) completely 50 inhibited oxidative burst by N. meningitidis H44/76 (2 x 108/mL, 10- 0 0 minute incubation) (left panel). In B 0 10 2030 40 50 B 0 10 2030 40 50 contrast, blocking of FcγR using anti- CD16 (FcγRIII), anti-CD32 (FcγRII), Antibody (µg/mL) Antibody (µg/mL) and anti-CD64 (FcγRI) (6.25-50 µg/mL) had no effect (right panel). Anti-C5a t = 10 (unstimulated) Anti-CD16 (Fcγ RIII) (clone 137-26) was included in both Isotype control Anti-CD32 (Fcγ RII) experiments for comparison. B indicates baseline (unstimulated sample) after a Anti-C5a Anti-CD64 (Fcγ RI) 10-minute incubation (t = 10). Results from 1 of 3 representative experiments Anti-CD11b Anti-C5a are shown.

128 observation that both antibodies were equally efficient indicates that the response was mediated by C5a and not by TCC (C5b-9).

Effect of inhibition of CR3 and Fcγ receptors on the oxidative burst The substantial complement-dependent increase in the expression of CR3 led us to investigate the relative roles of CR3 and Fcγ receptor (FcγR) in the oxidative burst induced by N. meningitidis. Inhibition of CR3 using a mAb blocking CD11b showed that oxidative burst in granulocytes (Figure 6) left panel) was critically dependent on CR3 because anti-CD11b inhibited oxidative burst to baseline values. In contrast,

F(ab’)2 antibodies blocking the interaction of FcγRI, FcγRII, and FcγRIII (CD64, CD32, and CD16, respectively) with their ligands had no effect on granulocyte oxidative burst (Figure 6 right panel). Results obtained for monocyte oxidative burst showed a similar pattern.

Effect of inhibiting C5 and C5a on red cell lysis and bactericidal activity C3bc 400 The effect of the anti-C5 and anti-C5a antibodies on the formation of fluid-phase C3 activation products (C3bc) and TCC in human300 whole blood was first investigated. As expected, neither of the antibodies had any effect on initial complement pathway activation because C3bc formation was unaffected200 (Figure 7 left panel). Blocking C5 activation by anti-C5 137-76 completelyAU/mL abrogated the formation of TCC, whereas 100 8 anti-C5a 137-26 and the isotype control had no effect on TCC formation (Figure 7 right panel). 0 Isotype Anti-C5 Anti-C5a control (137-76) (137-26)

C3bc TCC 400 500

400 300

300 200

AU/mL 200 AU/mL 100 100 * 0 0 Isotype Anti-C5 Anti-C5a Isotype Anti-C5 Anti-C5a control (137-76) (137-26) control (137-76) (137-26)

TCC Figure 7. 500 Effect of anti-C5 and anti-C5a on N. meningitidis-induced complement activation. Generation of C3bc (left panel) and TCC (right panel) in lepirudin-anticoagulated human whole blood after incubation400 for 1 hour at 37°C with 1 x 108 heat-inactivated N meningitidis H44/76 in the presence of anti- C5 (clone 137-76, 50 µg/mL), anti-C5a (clone 137-26, 50 µg/mL), or isotype control antibody (50 µg/mL). Formation300 of TCC was completely blocked by anti-C5 but unaffected by anti-C5a. Median ± IQRs of 4 separate experiments are presented. *P < .05 compared with isotype control.

AU/mL 200

100 * 129 0 Isotype Anti-C5 Anti-C5a control (137-76) (137-26) Table 1. Effect of anti-C5 and anti-C5a on serum bactericidal activity. Bactericidal No Isotype control Anti-C5a (137- Anti-C5 (137-76) activity antibody 26) L,3,7,9 (positive 14 15 15 < 8 control) L8 (negative < 8 < 8 < 8 < 8 control) Subject 1 6 5 5 < 1 Subject 2 7 8 7 < 1 Bactericidal activity (agar overlay method) of serum of the two control subjects and of anti-LPS L3,7,9 IgG2a antibody (positive control) and anti-LPS L8 IgG1 antibody (negative control) expressed as log2 titre of the last reciprocal serum dilution at which more than 50 % of the inoculum was killed. Both control subjects showed excellent serum bactericidal activity against N. meningitidis H44/76-1. Anti-C5 completely inhibited all bactericidal activity, whereas anti-C5a and isotype control had no effect.

The ability of the anti-C5 mAbs to interfere with the formation of a functional lytic C5b-9 MAC was investigated using an adapted CH-50 hemolytic assay to study complement-lytic activity of human whole blood on sensitized SRBCs. Anti-C5 137- 76 inhibited the lysis of SRBCs in whole blood by 89% (interquartile range [IQR], 96%-73%), whereas anti-C5a 137-26 had no effect. Furthermore, in a standard test for serum bactericidal activity (SBA), anti-C5 137-76 completely inhibited SBA of human sera and isolated anti-meningococcal antibody supplemented with exogenous complement,40 whereas anti-C5a 137-26 and the isotype control antibody showed no effect (Table 1). 30

/mL)

Finally, live N. meningitidis2 H44/76 was incubated in human whole blood. Samples from the same 2 control20 subjects used in the SBA measurements were used. After 5 minutes of incubation no growth of meningococci was seen (Figure 8). Anti-C5a CFU CFU (10 10 137-26 and the isotype control antibody had no effect on the bactericidal activity. Inhibition with anti-C5 137-76, however, completely abrogated the ability of human 0 whole blood to eliminate meningococci. Here, bacterial counts after shorter

periods of incubation remained0 20equivalent 40 60 to amounts at the start of incubation, time (min) No antibody 40 Isotype control Anti-C5a (137-26) 30 Anti-C5 (137-76) /mL) 2 20 Figure 8. Effect of anti-C5 and anti-C5a on bacterial killing in whole blood.

CFU CFU (10 10 Bacterial counts on blood-agar plates 24 hours after seeding 100 µL whole blood incubated for different time intervals with live log-phase N. meningitidis H44/76 in the 0 absence or presence of anti-C5 (clone 137- 76), anti-C5a (clone 137-26), and isotype control antibody. Median results of duplicate 0 20 40 60 experiments with 2 different donors are time (min) presented. No antibody Isotype control

130 Anti-C5a (137-26) Anti-C5 (137-76) whereas after 60 minutes bacterial counts had increased almost 2-fold, indicating active bacterial growth in these samples (Figure 8). These data indicate that the elimination of serogroup B N. meningitidis from this human whole blood model at the concentration tested is dependent on the lytic C5b-9 pathway of complement and that this pathway is kept functionally open in the presence of the anti-C5a mAb 137-26.

Discussion

N. meningitidis is exclusively a human pathogen, and no animal model accurately simulating fulminant meningococcal sepsis is available. This mandates the use of an in vitro experimental system approaching as closely as possible the in vivo situation. Recently, an in vitro whole human blood model was developed to study the role of complement in E coli-induced inflammatory processes 11. The principle of this model is to keep all ambient inflammatory systems intact to be activated, and to mutually interact, but still avoid coagulation. Because most anticoagulants such as EDTA, citrate, and heparin interact with critical steps in the inflammatory network, the model uses the highly specific thrombin inhibitor lepirudin, a recombinant hirudin analog, as anticoagulant. In the present study the model was used to evaluate the in vitro inflammatory responses to N. meningitidis. The main limitations of the model with regard to the in vivo situation are the absence of a vascular endothelium and the effect of lepirudin on thrombin. Thrombin is known to be an inflammatory 8 mediator 26, and hirudin-based peptides were found to inhibit thrombin-induced inflammatory effects on endothelial cells 27. However, because whole blood models need anticoagulation, it is impossible to circumvent this problem completely. We suggest that lepirudin is the best alternative in complement activation studies because its effect is limited to thrombin inhibition, the final step of coagulation. Hirudin was found to be superior in studying platelet and monocyte activation in whole blood, and it did not modulate complement activation, interleukin-8 (IL-8) release, or tissue-factor expression, supporting lepirudin as the anticoagulant of choice for this model 28,29.

For safety reasons heat-inactivated bacteria were used whenever possible. Because heat inactivation may influence the antigen exposure on bacteria, the interaction with the immune system may be altered 18,30. Therefore, we compared live and heat- inactivated bacteria with respect to inflammatory responses of importance for this study and showed that they elicited virtually identical responses. This validates the use of heat-inactivated N. meningitidis to investigate the inflammatory response induced by these microorganisms. In the whole blood model, a relatively high bacterial concentration (2 x 108) was

131 used for the induction of CR3 up-regulation and oxidative burst. The central mechanism in the pathogenesis of fulminant meningococcal septic shock (FMS) is an unimpeded—and unmatched by any other infectious organism—outgrowth of meningococci within the bloodstream. Bacterial concentrations in blood during FMS can exceed 108 bacteria/mL,31-33 and these high bacterial loads are seen only in the most severe cases 34. The concentrations we used in the in vitro model resemble the concentration of meningococci seen in the bloodstream during severe FMS and reflect the levels of bacteremia that induce the deleterious systemic inflammatory response. The number of bacteria used in the serum and whole blood bactericidal assays (2.5 x 102) was much lower than the amount used to study CR3 up-regulation and oxidative burst. After invasion in the bloodstream by N. meningitidis, the first line of defense is formed by complement-mediated, C5b-9-dependent direct bacterial lysis. Thus, this line of defense is engaged already at low concentrations of bacteria. When this defense fails, meningococci will multiply unimpeded, reaching concentrations (107-108 bacteria/mL) at which massive activation of the complement system causes tissue damage by granulocyte activation. For these reasons, we studied killing at relatively low concentrations, whereas the harmful effect of complement activation was studied at high concentrations of bacteria.

The aim of the present study was to evaluate with this model an anticomplement strategy that inhibits complement-mediated harmful processes and leaves bactericidal activity intact as a possible treatment option for meningococcal disease. It was shown that mAbs blocking the lectin pathway and alternative pathway, alone or in combination, inhibited complement activation by N. meningitidis. In addition, it was found that blocking complement activation in this way abolished the oxidative burst in granulocytes and attenuated it in monocytes. The oxidative burst was dependent on CR3 (CD11b/CD18) expression, whereas the inhibition of Fcγ- receptors had no effect on oxidative burst, as previously demonstrated for E coli.11 The mAb 137-76, which blocks the cleavage of C5, averted CR3 up-regulation and inhibited phagocytosis and associated oxidative burst, but also inhibited the formation of TCC and bactericidal activity. However, the inhibition of C5a by the mAb 137-26, which binds both free C5a and the C5a moiety of native C5 without interfering with the cleavage of C5,16 had the same inhibitory effect on CR3 upregulation, phagocytosis, and oxidative burst, but it preserved TCC formation and bactericidal activity.

Theoretically, the inflammatory response induced by C5 activation could be attributed to the potent anaphylatoxin C5a or to the C5b-9 complex given that the latter has been shown not only to be a lytic, bactericidal structure but is also able to induce inflammatory reactions by sublytic attack on nucleated cells 35. However,

132 the inflammatory responses we observed, as caused by C5 activation, were solely mediated by C5a and not by the C5b-9 complex because exactly the same degree of inhibition was observed using either the anti-C5 or the anti-C5a antibody.

The initial pathways of complement activation after stimulation of whole blood by N. meningitidis were studied by measuring specific complement activation products and by using monoclonal antibodies blocking specific complement components essential for each of these pathways. N. meningitidis induced significant increases in all activation products, but they were more pronounced for C4bc and C3bBbP than for C1rs-C1inh complexes. We found that anti-MBL blocked C4bc formation and that anti-factor D blocked C3bBbP formation. Both anti-MBL and anti-factor D inhibited TCC formation by N. meningitidis. This indicates that both the lectin and the alternative pathway are involved in complement activation by meningococci. The alternative pathway may be activated directly or as an amplification of the lectin pathway activation. These results also imply a limited role for the classical pathway in complement activation by N. meningitidis, further underscored by the finding that the antibody titer against H44/76 meningococci did not correlate with the magnitude of classical pathway activation in this assay system. Our findings are supported by the findings of Bjerre et al 36, who suggested that complement activation by neisserial OMVs is largely dependent on the lectin and alternative pathways, and by Brandtzaeg et al 8, who found that during meningococcal septic shock, systemic complement activation is predominantly dependent on the alternative pathway. The 8 latter study was performed before the contribution of the lectin pathway could be assessed, and it might be that the alternative pathway activation observed was partly the result of amplification from the lectin pathway. The observation that complement activation by the meningococcus is mostly lectin- and alternative-pathway- mediated seemingly contrasts with the increased SBA seen by specific bactericidal antibodies. Drogari-Apiranthitou et al 37, who also found that complement activation and assembly of the C5b-9 (MAC) on the meningococcal surface are for the most part independent of classical pathway activation, suggest that it is likely that proper MAC insertion mediated by antibody rather than the quantity of MAC formation is of importance for efficient bacterial killing 38,39. The observation that anti-C2 inhibited complement activation to a lesser extent than anti-MBL may be explained by a direct activation of C3 by MBL-associated serine proteases (MASPs), bypassing C2, as was suggested recently 40,41, though recent data contrast such a direct activation under physiologic conditions42.

Incubation of whole blood with N. meningitidis induces the formation of reactive oxygen species such as the superoxide anion and hydrogen peroxide. These reactive oxygen species are reported to activate the nuclear transcription factor

133 NFκB and are highly toxic to host tissues 43,44. When microbes intrude the body, granulocyte activation and oxidative burst—aimed at eliminating the invading agent—occur in a localized area with limited tissue damage. However, if systemic activation of the granulocytes occurs, this contributes to the development of organ failure, including acute respiratory distress syndrome (ARDS) and irreversible septic shock. Importantly, we showed that after incubation of whole blood with N. meningitidis, the production of reactive oxygen species, as reflected by the oxidative burst, is completely dependent on complement activation because it could be fully inhibited by blocking complement. Remarkably, anti-MBL alone inhibited oxidative burst better than one would expect based on the effects of anti-MBL on complement activation. An additional function of MBL, the reported direct effect of this protein on opsonophagocytosis, may explain this observation 45.

The oxidative burst process was dependent on C5a-mediated up-regulation of CR3, leading to increased phagocytosis of meningococci and increased oxidative burst, 2 closely related processes. This expands for N. meningitidis our earlier observation that showed C5a and C5aR play essential roles in the E. coli-induced up-regulation of CR3, phagocytosis, and oxidative burst 11. The divergent effect of C5a inhibition on CR3 expression, phagocytosis, and oxidative burst between granulocytes and monocytes, with a relatively decreased effect of C5a inhibition on these processes in the monocyte, suggests that alternative mediators are also of importance in the regulation of monocyte CR3 expression.

Invasive gram-negative bacteria can be cleared from the blood by C3b- or IgG- dependent phagocytosis or by lysis of the bacterium by the MAC 46. In the present study we showed that in whole human blood with strong anti-meningococcal SBA, meningococci are rapidly and completely killed. Inhibition of C5a, with potent inhibitory effects on CR3-mediated phagocytosis, had no effect on TCC formation, SBA, or bactericidal activity in whole blood. Of importance, however, the inhibition of C5 completely prevented TCC formation and the killing of meningococci. This implies that in the presence of a functional MAC, meningococci are rapidly killed, whereas in the absence of the MAC unimpeded growth occurs. Thus, the MAC is the main determinant of meningococcal killing in this human whole blood assay using a low inoculum of bacteria. Theoretically, the inhibition of phagocytosis by the anti-C5a antibody at high intravascular bacterial concentrations could be deleterious because this is also a primary host defense mechanism. However, if the anti-C5a antibody were used in the treatment of patients with FMS, the situation would differ from the one studied in our in vitro whole blood model. First, the mainstay of treatment of meningococcal infection is bactericidal antibiotic therapy, which kills the meningococci rapidly. Second, the main clearance mechanisms of

134 gram-negative bacteria from the circulation in mammals are Kupffer cells in the liver and, to a lesser extent, spleen macrophages 47,48. The class A scavenger receptor of the macrophages has been implicated as a major receptor for phagocytosis of meningococci 49. Granulocytes appear to play a minor role in the intravascular clearance of meningococci 47, and the phagocytosis of meningococci by granulocytes intravascularly is a pathologic condition that presumably occurs only at high concentrations of bacteria because it is associated with fatal meningococcal septic shock 50. Therefore, the inhibition of systemic phagocytosis-related oxidative burst is likely to be advantageous in clinically severe sepsis.

In meningococcal disease, excessive activation of the complement system is closely related to disease severity and patient death, implicating a possible beneficial effect of complement inhibition on disease severity in FMS 6,7. In contrast to other mediators in FMS such as proinflammatory cytokines that immediately decline after the initiation of effective antibiotic therapy 51-53, complement activation is sustained and reaches a maximum at 12 to 15 hours after the initiation of treatment 8,34. Attempts to influence disease severity by modulating complement activation therefore have an increased time span for possible therapeutic intervention.

By preference, adjunctive treatment for FMS should be aimed at inhibiting the negative effects of inflammatory substances without decreasing the capacity of the immune system to eliminate the infectious organism. In addition, primary 8 mediators that induce secondary inflammatory reactions, enhancing the systemic breakdown of homeostasis, are particularly important candidates for intervention. Inhibition of C5a is a treatment modality that could perform such a task. Previous studies have shown that neutralizing the effect of C5a, either by antibodies or by receptor antagonists, has beneficial effects on survival in a range of animal models of gram-negative septic shock 54-57. These favorable effects may be explained by the preservation of neutrophil function, attenuation of CR3 expression, and reduced production of reactive oxygen species, as we show for N. meningitidis in the present study. In addition, other secondary C5a-mediated effects that may contribute to the degree of septic shock might be attenuated, such as the release of histamine, chemotactic proteins, and proinflammatory cytokines leading to vasodilation and capillary leakage 9,10,58 and to direct effects on coagulation and fibrinolysis 57. In conclusion, the present study supports a beneficial effect of complement modulation on potentially harmful systemic inflammatory responses such as CR3 expression and oxidative burst induced by N. meningitidis. This was achieved by selective inhibition of C5a with preserved bactericidal activity using the anti-C5a mAb 137-26. We suggest this is an attractive approach to the arduous treatment of meningococcal septic shock.

135 Acknowledgements

The authors wish to thank Hilde Fure, Gunni Ulvund and Mohammed R. Mirlashari for excellent technical assistance.

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139 a rat model of sepsis. Am J Pathol. 2002;160:1867-1875 58. Cavaillon JM, Fitting C, Haeffner-Cavaillon N. recombinant C5a enhances interleukin 1 and tumor necrosis factor release by lipopolysaccharide-stimulated monocytes and macrophages. Eur J Immunol. 1990;20:253-257

140 8

141

Deficient alternative complement pathway activation due to factor D deficiency by two novel mutations in the Complement Factor D gene in a family with meningococcal infections. 9

Tom Sprong T, Dirk Roos, Corrie Weemaes, Chris Neeleman, Christel L. Geesing, Tom Eirik Mollnes and Marcel van Deuren. Blood. 2006; 107(12):4865-70 Abstract

The complement system is an essential element in our innate defense against infections with Neisseria meningitidis. We describe 2 cases of meningococcal septic shock, 1 of them fatal, in 2 children of a Turkish family. In the surviving patient, alternative pathway activation was absent and factor D plasma concentrations were undetectable. Concentrations of mannose-binding lectin (MBL), C1q, C4 and C3, factor B, properdin, factor H, and factor I were normal. Mutation analysis of the factor D gene revealed a T638 > G (Val213 > Gly) and a T640 > C (Cys214 > Arg) mutation in the genomic DNA from the patient, both in homozygous form. The consanguineous parents and an unaffected sister had these mutations in heterozygous form. In vitro incubation of factor-D–deficient plasma of the boy with serogroup B N. meningitidis showed normal MBL-mediated complement activation but no formation of the alternative pathway C3-convertase C3bBbP, and severely decreased C3bc formation and terminal complement activation. The defect was restored after supplementation with factor D. In conclusion, this is the second report of a factor D gene mutation leading to factor D deficiency in a family with meningococcal disease. This deficiency abolishes alternative-pathway dependent complement activation by N. meningitidis, and leads to an increased susceptibility to invasive meningococcal disease.

144 Introduction

As stated in 1969 by Goldschneider et al 1,2 and confirmed by large scale vaccination campaigns at the start of this century 3,4, specific antibodies offer complete protection against invasive meningococcal disease. Without antibodies, the primary humoral defense against Neisseria meningitidis invasion relies solely on the complement system.

Complement activation contributes to the clearance of meningococci by C3b and the membrane attack complex (MAC). C3b(i), fixed to meningococci, opsonizes the bacterium. In addition, C3b-coated bacterial particles are shuttled to and cleared by the reticuloendothelial system in liver and spleen by binding to complement receptor type 1 (CR1; CD35) on erythrocytes 5,6. MAC induces bacteriolysis through insertion in the meningococcal outermembrane of a macromolecular complex composed of the terminal complement components C5b, C6, C7, C8, and 1 or more C9 molecules, which become activated in a relay race fashion after adherence of C3b. Deficiency of 1 of the terminal complement factors leads to inadequate MAC formation and a 1000-fold increased risk for meningococcal infections 7,8. Interestingly, most of these infections are caused by relatively rare serogroups and run a mild course 9-11. In cases of insufficient proximal complement activation, not only MAC formation but also C3b-mediated clearance is missing. In these cases, the defense against meningococci is more seriously affected and a more severe course may be expected. However, extensive complement activation in meningococcal infections also has deleterious effects to the host by the generation of excess amounts of anaphylatoxin C3a and C5a 12, which are known to induce the generation of reactive oxygen species by phagocytes 13, up-regulate production of 9 proinflammatory cytokines 14, increase disseminated intravascular coagulation 15, and aggravate cardiodepression 16.

Initial complement activation, aimed to activate C3 and to bind C3b(i) to a microbial or cellular surface, can be started by the classical pathway, the mannose-binding lectin (MBL) pathway, or the alternative pathway. Engagement of the classical pathway requires specific antibodies or C-reactive protein (CRP) attached to the bacterium for binding and activation of C1qrs. In the very early stage of meningococcal disease, specific antibodies and CRP are still absent 17,18. Therefore, only the lectin and the alternative pathway will play a role at this stage. The relative contribution of these pathways is still a subject of study 19,20. As we recently showed in vitro, maximal complement activation by meningococci requires involvement of both pathways 13, with an initiating role for the lectin pathway and an amplifying role for the alternative pathway 21.

145 Clinically, MBL deficiency is associated with a higher risk for invasive meningococcal disease although it runs a less severe course in MBL-deficient patients 22- 24. Deficiency of factor B, the primary alternative pathway initiator, has never been found. However, deficiency of properdin (P), the protein that stabilizes the alternative pathway C3-convertase C3bBb, is associated with a high risk for severe disease 25-27. In 2001, we reported a factor D gene mutation leading to complete deficiency of factor D, in a Dutch family with 2 cases of meningococcal disease 28. Combined with the earlier observation of Hiemstra et al 29, this finding suggests that factor D deficiency also predisposes to invasive meningococcal disease.

In the present study, we describe the clinical course of meningococcal disease in 2 children of a Turkish family with a novel factor D gene mutation leading to undetectable factor D plasma concentrations. The family had a normal genetic makeup for MBL. Therefore, we were able to explore, by additional in vitro experiments, the relative importance of the lectin and the alternative pathway in complement activation by N. meningitidis.

patients, Materials and methods Patient A A 9-month-old girl was admitted in 1996 to a local hospital because of purpura for 2 hours and 45 minutes. She had a history of high fever for 6 days, coughing, diarrhea, and vomiting. At admission the temperature was 40°C, she was tachypneic, and had signs of a poor peripheral circulation and moderate nuchal rigidity. The pulse rate was 220 beats per minute and blood pressure was 141/80 mm Hg. Laboratory examination at admission showed: CRP level, 53 mg/L; leukocyte count, 3.9 x 109/L; platelet count, 234 x 109/L; creatinine, 78 µM; arterial pH, 7.19; PCO2, 4.2 mM; bicarbonate, 11 mM; and base excess, –15.2 mM. Blood cultures were negative. Cerebrospinal fluid (CSF) examination revealed only mild signs of inflammation: leukocytes, 20 x 109/L; glucose, 3.8 mM; and protein, 280 mg/L. CSF cultures grew N. meningitidis serogroup B:P1:15. The chest x-ray showed an infiltrate in the right upper quadrant. Shortly after lumbar puncture, generalized seizures occurred and treatment was started with phenobarbital, ampicillin, dexamethasone, bicarbonate, and paracetamol. Unfortunately, the severity of disease was not sufficiently recognized and the patient was admitted to a regular pediatric ward.

After 1.5 hours, blood pressure fell to 79/52 mm Hg. In spite of 200 mL/kg fluid resuscitation during the first 24 hours, blood pressure remained low and complete renal failure and thrombopenia (38 x 109/L) developed. No inotropics, ventilatory support, or fresh plasma were given. Thirty-six hours after admission, blood

146 pressure decreased suddenly to 49/29 mm Hg followed by respiratory arrest. Cardiopulmonary resuscitation was needed and transfer to our Pediatric Intensive Care Unit (PICU) was requested. At arrival, 36 hours after hospital admission, the patient had poorly perfused extremities and was persistently hypotensive in spite of high dosages of inotropic and vasopressive medication. The electroencephalogram was compatible with postresuscitation cerebral damage. The girl died 11 hours later because of refractory shock. Autopsy was not allowed.

Patient B Four years later, the 13-month-old brother of patient A was admitted to the same local hospital because of sudden onset of fever and a petechial rash. At the age of 4 months he had been hospitalized for a respiratory syncytial virus infection complicated by bacterial superinfection. Later, he suffered several times from recurrent upper or lower respiratory tract infections that required frequent antibiotic treatment. Now, at admission, he was covered with small purpuric lesions, had decreased capillary refill, and no signs of meningismus. His body temperature was 39.7°C, pulse rate 180 beats per minute and a respiration rate of 36 breaths per minute. Laboratory results showed: CRP level, 119 mg/L; leukocyte count, 10.9 x 109/L; platelet count, 324 x 109/L; creatinine, 52 µM, activated partial thromboplastin time (APTT), 46 seconds (normal, 25-35 seconds); and plasma D-dimers, 1.35 mg/L (normal, < 0.5 mg/L). The chest X-ray was normal. Blood cultures grew N. meningitidis B.1.P1-4.

Directly after admission, treatment was started with ceftriaxone, dexamethasone, and fluid challenges (2 x 23 mL/kg 0.9% NaCl). After consultation with our PICU staff by telephone, 4 hours later, dobutamine (5 µg/kg/min) and 15 mL/kg fresh- 9 frozen plasma (FFP) was given. In the first 18 hours, the situation remained stable. Laboratory analysis after 18 hours showed creatinine at 45 µM, a leukocyte count of 40.6 x 109/L, a normal platelet count, and signs of compensated disseminated intravascular coagulation (APTT, 41 seconds; fibrinogen, 7.9 g/L; and D-dimers, 4.47 mg/L). During the following day he received another 2 x 15 mL FFP. Recovery was uneventful. Two weeks after admission he was discharged in good condition.

Because the boy was the second patient with severe meningococcal disease in the same family and his parents appeared to be consanguineous, further immunologic analysis was performed. The concentrations of immunoglobulin classes and subclasses were normal, as were the antibodies against polysaccharide capsule of Streptococcus pneumoniae serogroup 3, 4, and 9 and Haemophilus influenza type B. Analysis of the complement pathway showed normal concentrations of MBL, C1q, C4, and C3, but a complete absence of alternative pathway activation in the

147 Table 1. Sequence of genomic PCR primers used in this study Primer Complement factor D CFD-ex1-fw 5’-GAGTCTGGCAGGAGGTAACCCAGTC-3’ CFD-ex1-rev 5’-GCGTTCAGAGCCTTCCATTAGTGAG-3’ CFD-ex2-fw 5’-GAGAGCTGGGATCCCGTCAGGCAGC-3’ CFD-ex2-rev 5’-GAGTCCGCGGTCGGTGCCAGCCGACTC-3’ CFD-ex3-fw 5’-GAGTCGGCTGGCACCGACCGCGGACTC-3’ CFD-ex3-rev 5’-CATGCAGCAGGAGAGGTCGAGGCTGG-3’ CFD-ex4-fw 5’-CTCCCCGAGCCTAGCGGCATTCTCC-3’ CFD-ex4-rev 5’-TCATGCTCCGCCCATCTTCCAGTTC-3’ CFD-ex5-fw 5’-ACTAGTGAAGACCAAATTAACACGG-3’ CFD-ex5-rev 5’-GCAGGAGTGGATGACTTCATTGCTCG-3’ MBL MBL-L-fw 5’-TTAGCACTCTGCCAGGGCCAACG-3’ MBL-L-rev 5’-CCCATCTTTGTATCTGGGCAGCTGA-3’ MBL-X-fw 5’-TCTTTGGATCACCAAAAGCTTTCAGCTC-3’ MBL-X-rev 5’-GAGGGGTTCATCTGTGCCTAGAC-3’ MBL-ABCD-fw 5’-AGTTTTCTCACACCAAGGTG-3’ MBL-ABCD-rev 5’-ATCCCCAGGCAGTTTCCTCTGGAAG-3’

alternative pathway hemolytic activity 50 (AP50) test (AP50 < 5%; normal, 75%- 125%). Factor B level was 84 IU/mL (normal, 49-129 IU/L). Factor H level was 0.31 g/L (normal, 0.2-0.6 mg/L). Properdin as well as factor I, determined qualitatively, were present. However, factor D was undetectable at less than 0.03 mg/L (normal, 1.0-2.0 mg/L). Because of this deficiency the boy was put on antibiotic prophylaxis (1 x 100 mg clarythromycin daily) and was vaccinated with the conjugated N. meningitidis serogroup C vaccine and the unconjugated serogroup A plus C vaccine. With informed consent of his parents, the patient’s factor D gene defect was analyzed and additional in vitro complement activation tests were performed.

Informed consent statement Studies were conducted in accordance with the local guidelines for human experiments (Commissie Mensgebonden Onderzoek regio Arnhem-Nijmegen, Radboud University Nijmegen Medical Centre). Informed consent (according to the Declaration of Helsinki) was obtained from the patient, his mother, and the healthy controls prior to performing the experiments.

Factor D protein determination and factor D gene sequencing The activity and the protein concentration of factor D were measured as described

148 previously 28. The factor D gene polymerase chain reaction (PCR) was performed with primers annealing to intron sequences close to each exon (Table 1). The PCR conditions and the sequence reactions were as described by Hiemstra et al 29.

MBL protein determination and gene analysis MBL protein was determined in a solid-phase enzyme-linked immunosorbent assay (ELISA) with mannan coated to a 96-well plate and detection with monoclonal antibody (mAb) MBL-1 (biotinylated mouse anti–human MBL, immunoglobulin G1 [IgG1], 10 µg/mL; Sanquin, Amsterdam, the Netherlands). Briefly, microtiter plates were coated with 100 µg/mL mannan in 0.1 M NaHCO3 (pH 9.6) overnight at room temperature. The microtiter plates were washed 5 times with H2O. Plasma or serum samples and MBL standards (standard serum, 1.5 µg/mL MBL) were diluted in TTG/Ca2+ (20 mM Tris [pH 7.4]/150 mM NaCl/0.02% Tween-20/0.2% gelatin/10 mM CaCl2), with 10 U/mL heparin, added to the plates, and incubated by shaking at room temperature for 1 hour. After washing, the plates were incubated for 1 hour with biotinylated MBL-1 in TTG/Ca2+, washed with H2O, and incubated by shaking at room temperature for 30 minutes with streptavidin poly–horseradish peroxidase (HRP) at a ratio of 1:10 000 in TBS/Ca2+/2% milk (20 mM Tris [pH 7.4]/150 mM NaCl/10 mM CaCl2/2% milk). After washing, the color was developed with tetramethyl-3,3’,5,5’-benzidine (TMB)/0.01% H2O2 in 0.1 M Na acetate (pH 5.5), stopped with 2 M H2SO4, and measured spectrophotometrically at 405 nm (BioAssay Reader Sunrise; Tecan, Salzburg, Austria).

MBL gene analysis for mutations in exon 1 and polymorphisms in the promoter region was performed by sequencing of PCR products obtained with primers described in Table 1. The PCR and sequencing conditions were as for factor D. 9

Complement activation experiments Lepirudin (Refludan; Pharmion, Tiel, the Netherlands)–anticoagulated (50 µg/mL final concentration) blood from patient B, his mother, and 4 healthy volunteers was drawn in polyproplylene tubes on ice (2-4 years after presentation with meningococcal sepsis) and centrifuged immediately. The plasma was stored at –80°C until further experiments. Complement activation was tested by adding heat- killed serogroup B N meningitidis H44/76. This strain is an isolate of a patient with invasive disease and an international reference strain, serologically classified as B:15:P1.7,16, immunotype L3,7,9 30.

For the complement activation experiments, 300 µL plasma was incubated in polypropylene 96-well plates (NUNC, Roskilde, Denmark) with 75 µL phosphate- buffered saline (PBS) or N. meningitidis (final concentration, 1 x 108/mL) suspended

149 Table 2. Results of family investigations Factor D Factor D gene MBL protein, MBL gene‡ protein, mg/L* mutation mg/L† Patient B < 0.03 Homozygous 2.6 HYPA/HYPA Mother 0.44 Heterozygous 1.3 HYPA/HYPA Father 0.14 Heterozygous 1.4 HYPD/HYPA Sister 0.34 Heterozygous 2.6 HYPA/HYPA * Normal range, 1.0-2.0 mg/L. †Normal range, > 0.8 mg/L. ‡Normal, HYPA/HYPA

in PBS (75 µL) and purified human factor D at 75 µL to a final concentration of 2 mg/L (QUIDEL, San Diego, CA) or murine mAb against human factor D (clone 166-32) at 75 µL in a final concentration of 25 µg/mL (kindly provided by M. Fung, Tanox, Houston, TX) 31. Sample preparation was always on ice, and incubation was for 1 hour at 37°C. To stop complement activation after 1 hour, the plates were put on ice and 10 mM EDTA was added. Thereafter, samples were stored at –80°C until complement analysis. Complement factor 1 rs (C1rs)–C1-inhibitor (C1rs-C1inh) complexes, C4bc, C3bBbP, C3bc, and soluble terminal complement complex (TCC) was assayed by ELISA as described previously in detail 13,32. For comparison, plasma of 2 healthy adult controls was incubated in all experiments in the same run.

Results Factor D and MBL values in the patient, his parents, and his sister Patient B had an undetectable level of factor D protein in his blood; his mother, father, and unaffected sister had strongly decreased levels of factor D (Table 2). The circulating MBL protein levels of these individuals were also measured to exclude increased susceptibility for Neisseria infection due to MBL deficiency. All members of this family had normal MBL levels, although the father of the patient was heterozygous for an Arg52 > Cys mutation (allele D) (Table 2).

Genetic analysis of the factor D gene in the patient and his family Mutation analysis of the factor D gene revealed a T638 > G (Val213 > Gly) and a T640 > C (Cys214 > Arg) mutation in the genomic DNA from the patient, both in apparently homozygous form. Both parents and the unaffected sister had these mutations in a heterozygous form. These mutations are in conserved residues, close to Ser208 in the catalytic center of the enzyme (Figure 1). Moreover, the Cys214 > Arg mutation destroys an internal disulfide bond between 2 conserved cysteines 33.

150 1 Signal peptide 11 amino acids 5’UTR M H S W E R L A V L V nucleotides GAG TGT CTC AGC CAC AGC GGC TTC ACC ATG CAC AGC TGG GAG CGC CTG GCA GTT CTG GTC

Activation 12 Exon 1 Exon 2 peptide Active factor D 31 amino acids L L G A A A C A A P P R G R I L G G R E nucleotides CTC CTA GGA GCG GCC GCC TGC GCG GCG CCG CCC CGT GGT CGG ATC CTG GGC GGC AGA GAG

32 51 amino acids A E A H A R P Y M A S V Q L N G A H L C nucleotides GCC GAG GCG CAC GCG CGG CCC TAC ATG GCG TCG GTG CAG CTG AAC GGC GCG CAC CTG TGC

52 ≠ Exon 2 71 amino acids G G V L V A E Q W V L S A A H C L E D A nucleotides GGC GGC GTC CTG GTG GCG GAG CAG TGG GTG CTG AGC GCG GCG CAC TGC CTG GAG GAC GCG

72 Exon 3 91 amino acids A D G K V Q V L L G A H S L S Q P E P S nucleotides GCC GAC GGG AAG GTG CAG GTT CTC CTG GGC GCG CAC TCC CTG TCG CAG CCG GAG CCC TCC

92 111 amino acids K R L Y D V L R A V P H P D S Q P D T I nucleotides AAG CGC CTG TAC GAC GTG CTC CGC GCA GTG CCC CAC CCG GAC AGC CAG CCC GAC ACC ATC

112 ≠ Exon3 Exon 4 131 amino acids D H D L L L L Q L S E K A T L G P A V R nucleotides GAC CAC GAC CTC CTG CTG CTA CAG CTG TCG GAG AAG GCC ACA CTG GGC CCT GCT GTG CGC

132 151 amino acids P L P W Q R V D R D V A P G T L C D V A nucleotides CCC CTG CCC TGG CAG CGC GTG GAC CGC GAC GTG GCA CCG GGA ACT CTC TGC GAC GTG GCC

152 171 amino acids G W G I V N H A G R R P D S L Q H V L L nucleotides GGC TGG GGC ATA GTC AAC CAC GCG GGC CGC CGC CCG GAC AGC CTG CAG CAC GTG CTC TTG

172 191 amino acids P V L D R A T C N R R T H H D G A I T E nucleotides CCA GTG CTG GAC CGC GCC ACC TGC AAC CGG CGC ACG CAC CAC GAC GGC GCC ATC ACC GAG

192 Exon 4 Exon 5 ≠ 211 amino acids R L M C A E S N R R D S C K G D S G G P nucleotides CGC TTG ATG TGC GCG GAG AGC AAT CGC CGG GAC AGC TGC AAG GGT GAC TCC GGG GGC CCG

212 Sites of mutations T638>G, T640>C (Val213>Gly, Cys214>Arg) 231 amino acids L V C G G V L E G V V T S G S R V C G N nucleotides CTG GTG TGC GGG GGC GTG CTC GAG GGC GTG GTC ACC TCG GGC TCG CGC GTT TGC GGC AAC

232 251 amino acids R K K P G I Y T R V A S Y A A W I D S V nucleotides CGC AAG AAG CCC GGG ATC TAC ACC CGC GTG GCG AGC TAT GCG GCC TGG ATC GAC AGC GTC

252 9 amino acids L A stop 3’ UTR nucleotides CTG GCC TAG GGT GCC GGG GCC TGA AGG TCA GGG TCA CCC AAG CAA CAA AGT CCC GAG CAA

Figure 1. Amino-acid and cDNA nucleotide sequence of human complement factor D. The sequence data are from White et al. 45 and Biesma et al 28. 5’UTR indicates 5’untranslated region; first arrow, start of protein synthesis; second arrow, start of circulating protein sequence; asterisks, positions of catalytic triad Asp114, His66, and Ser208; arrowheads, positions of mutations; and 3’UTR, part of 3’untranslated region. A disulfide bond exists in the wild-type factor D between Cys148 and Cys214, which is indicated in bold on a gray background (gray boxes).

N. meningitidis–induced complement activation Upon stimulation with 108/mL N. meningitidis, factor D–deficient plasma of the patient produced clearly less C3bc and TCC compared with healthy individuals (see Figure 2). Plasma of the heterozygous mother had a C3bc and TCC production approximately similar to that of the healthy controls. Addition of a monoclonal antibody against factor D inhibited complement activation in the mother and the controls, but had no effect on complement activation in the patient. Thus, factor D is important for the activation of C3 as well as the activation of the terminal

151 500 70

C3bc 60 TCC 400 50

300 40

200 30 AU/mL 20 100 10

0 0 N.meningitidis - - + + + - - + + + Factor D - + - + - - + - + - Anti-factor D - - -- + - - -- +

Figure 2. Proximal and terminal complement pathway activation by N. meningitidis in plasma from the patient and his mother. The formation of C3bc and sTCC, reflecting proximal complement activation and terminal pathway activation, respectively, in plasma of the homozygous factor D-deficient patient B (white bars), his heterozygous mother (black bars), and the median ± interquartile range (IQR) of 4 healthy controls (dashed bars). The plasma was incubated for 1 hour at 37°C without stimulus or with 108/mL meningococci in the absence or presence of recombinant factor D or anti–factor D, as indicated. The experiment was repeated once, yielding virtually identical results (not shown).

100 100 2500 C1rs-C1inh C4bc C3bBbP 80 80 2000

60 60 1500

40 40 1000 AU/mL

20 20 500

0 0 0 N.meningitidis - - + + + - - + + + - - + + + Factor D - + - + - - + - + - - + - + - Anti-factor D - - -- + - - -- + - - -- +

Figure 3. Classical, lectin, and alternative complement pathway activation by N. meningitidis in plasma from the patient and his mother. The formation of C1rs-C1inh complexes (marker of classical pathway activation), C4bc (marker of classical or lectin pathway), and C3bBbP (marker of the alternative pathway) in plasma of the homozygous factor D–deficient patient (white bars), his heterozygous mother (black bars), and the median (± IQR) of 4 healthy controls (dashed bars). The plasma was incubated for 1 hour at 37°C without stimulus or with 108/mL meningococci, in the absence or presence of recombinant factor D or anti–factor D, as indicated. The experiment was repeated once, yielding virtually identical results (not shown).

complement cascade.

Addition of purified human factor D alone in the absence of other stimuli had no effect on complement activation in plasma of the controls and in plasma of the mother. However, some C3bc and TCC were induced in the factor D–deficient plasma of the patient. Factor D supplementation to plasma stimulated with meningococci restored C3bc and TCC activation in the patient’s plasma and that of his mother, for the patient even to values higher than that seen in controls.

152 Next, we assayed complement intermediates indicative for initial pathway complement activation. As can be seen in Figure 3, meningococci did not induce C1rs-C1inh complexes in amounts exceeding that of the baseline. This shows that no classical pathway activation occurred.

However, C4bc, reflecting lectin pathway activation (because classical pathway activation was absent), was induced after stimulation with meningococci in the patient and his mother, possibly to a higher extent than in the controls. As expected, addition of purified human factor D or anti–factor D had no effect on C4bc formation.

Finally, after stimulation with meningococci, C3bBbP reflecting alternative pathway activation was increased in the mother and the controls but not in the factor D–deficient patient. Addition of purified factor D to the patient’s plasma, not stimulated or stimulated with meningococci, increased the production of C3bBbP in the patient to much higher values than in the mother or the controls. Anti–factor D with meningococcal stimulation abolished C3bBbP in the mother and the controls. Taken together, these results indicate that meningococci can induce complement activation via the lectin and alternative pathways, and that complement activation by meningococci is reduced in factor D deficiency, owing to a defect in the alternative pathway

Discussion

In the present study, we report 2 patients, one of them with a fatal course, with invasive meningococcal serogroup B disease in a family of consanguineous parents. 9 The surviving child, a boy of 13 months, had no alternative pathway complement activation as measured by the AP50 assay. This was explained by 2 novel homozygous mutations in the factor D gene, leading to undetectable factor D protein plasma concentrations. Mother, father, and unaffected sister were heterozygous for the mutated gene and had a partial factor D protein deficiency. The genetics of the deceased sister are not known. In vitro incubation of the factor D–deficient plasma of the boy with meningococci showed normal MBL-mediated complement activation but no formation of the alternative pathway C3-convertase C3bBbP, and severely decreased C3bc formation and terminal complement activation. The defect was restored in an overcompensated fashion after supplementation with factor D at a physiologic concentration.

Factor D is a serine protease that 34, in contrast to most other complement proteins, does not require enzymatic cleavage for the expression of its proteolytic activity

153 35. Instead, factor D activity is attained by reversible conformational changes occurring after contact with its exclusive substrate; that is, factor B attached to C3b 33,36. Factor D catalyzes the cleavage of factor B to Bb. Subsequently, the complex C3bBb is stabilized by properdin (P) forming the alternative pathway C3-convertase: C3bBbP. Factor D activity is the rate-limiting step in the formation of C3bBbP. The gene for factor D is located on chromosome 19p13.333,37. Factor D is produced as a preproprotein with a signaling polypeptide of 23 amino acids. The proprotein is formed by amino acids 24-253. The mature protein consists of amino acids 26-253. The 2 mutations found in the patient lead to 2 amino-acid substitutions: Val213 > Gly and Cys214 > Arg. Heterozygosity for these missense mutations leads to protein concentrations in plasma—assayed by a polyclonal ELISA—less than 50% of the normal concentration. Homozygosity leads to complete absence of the protein. Because Cys214 in the wild-type protein forms an S-S bond with Cys148, we presume that the absence of detectable protein is caused by an abnormal folding of the peptide with impeded cellular secretion and/or increased instability.

Pivotal in the cascade of complement activation is cleavage, by the classical (C4b2b) or the alternative pathway C3-convertase (C3bBbP), of the -chain of C3, which generates C3b. This cleavage results in exposure of an internal thioester that enables C3b to bind covalently to hydroxyl or amino groups present on (meningococcal) surfaces. Because classical pathway activation on meningococci does not occur during meningococcal septic shock, as there is no antibody or CRP yet 17,18, the C3-convertase required for C3b generation will be delivered by the lectin pathway or by the alternative pathway. As reported, MBL binds to outer-membrane proteins of serogroup B meningococci 38. The MBL-associated serine protease MASP2 subsequently cleaves C4 and C2, which will bind to the surface as C4b and C2a and form the classical/lectin pathway C3-convertase C4b2a 39. Factor D induces the formation of the alternative pathway C3-convertase C3bBbP. Alternative route activation is under tight control by various membrane-bound regulatory proteins on human cells and by fluid-phase regulatory proteins such as factor I, factor H, and C4 binding protein (C4BP). Factor I degrades C3b in the presence of the cofactor molecules C4BP or factor H 40,41. Factor H also accelerates the dissociation of Bb from active C3bBb. Both the classical/lectin- and the alternative pathway C3-convertase function, in the presence of an additional C3b molecule, as C5-convertase. Cleaving of C5 is the initiating step of MAC formation.

We recently described a whole blood in vitro system for the evaluation of complement activation by meningococci 13,32. In the present study, this model was slightly modified by the use of plasma instead of whole blood. Using this model, we found that the factor D–deficient patient had severely reduced C3 activation

154 (measured as C3bc) and terminal complement activation (measured as TCC). Analysis of the intermediates showed no classical route activity (no C1rs-C1inh), confirming previous results Sprong, 2003 #1800}32,42. Thus, the C4 activation (measured as C4bc) that occurred in the patient’s plasma was mediated solely by the lectin pathway. Notably, as can be seen in the factor-D–deficient serum of the patient and in the experiments with anti–factor D, MBL-induced C4 activation induces only minimal amounts of C3bc and TCC when the alternative pathway activation is switched off. Thus, the alternative route functions as an amplification mechanism for MBL-initiated complement activation by meningococci.

Interestingly, supplementation of factor D to the factor D–deficient plasma of the patient, in the absence of another stimulus, induced some spontaneous alternative route activation (C3bBbP) with C3b formation (C3bc) and terminal complement activation (TCC). Furthermore, in the presence of meningococci, supplementation with factor D gave more complement activation in comparison with the mother and the controls. This may be explained by a greater intrinsic activity of the purified factor D. However, it may also reflect a lower concentration of factor H, factor I, or another regulatory protein in the plasma of the factor D–deficient patient. In our patient, factor H was 0.31 g/L, a normal value, and factor I was present, rendering it unlikely that a lower amount or deficiency of these proteins explains these findings.

Clinically, invasive meningococcal disease presents with varying severity. One side of the spectrum is formed by benign meningococcemia and meningitis, both characterized by a moderate outgrowth of meningococci in the bloodstream. On the opposite side fulminant meningococcal septic shock (MSS) is found, characterized 9 by the rapidly overwhelming intravascular outgrowth of meningococci, high concentrations of endotoxin and massive induction of proinflammatory mediators 43. In case of complement deficiency, the outgrowth of meningococci in the bloodstream is not inhibited and high concentrations of bacteria may develop with massive release of proinflammatory mediators, until start of antibiotic treatment. However, as soon as meningococcal proliferation is stopped by the timely start of antibiotics, the generation of lower amounts of anaphylatoxins may be beneficial. Therefore, the ultimate outcome of meningococcal disease in a complement-deficient individual is determined by the interplay between virulence of the bacterium, the initial host response, and early recognition with timely start of adequate therapy. Reportedly, deficiency of terminal complement factors or MBL is found in patients with mild disease, while properdin deficiency leads to severe disease. This underscores the indispensability of an intact alternative pathway. We have not been able to analyze the complement status in patient A but suspect that she also was factor D deficient.

155 Her fatal outcome may be explained by this defect and by the failure to recognize the severity of disease in time with installation of appropriate antishock therapy. Treatment in patient B was guided by an intensive care physician experienced in the treatment of MSS. Interestingly, this patient received as a part of the initial treatment, before factor D deficiency was known, FFP that contained factor D as well as the alternative pathway regulatory proteins in balanced amounts. The question whether the infusion of FFP has contributed to the favorable outcome— next to early antibiotic treatment and adequate resuscitation—requires further investigation 44.

In conclusion, we describe in a Turkish patient of consanguineous parents, 2 novel homozygous mutations of the factor D gene leading to the complete absence of detectable factor D protein in plasma. This deficiency leads to reduced alternative- pathway–dependent complement activation by meningococci, and explains the increased susceptibility to invasive meningococcal disease.

Acknowledgements

The authors thank André Hannema (Sanquin Research at CLB, Amsterdam, the Netherlands) for the factor D assay, Martin de Boer (Sanquin Research at CLB, Amsterdam, the Netherlands) for the mutation analyses and Grethe Bergseth (Nordlandssykehuset, Bodo, Norway) for the assay of the complement intermediates. M. Fung (Tanox, Houston, TX) is thanked for providing the anti-factor D monoclonal antibody.

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157 in fulminant meningococcal septicemia is predominantly caused by alternative pathway activation. J. Infect. Dis. 1996;173:647-655 20. Hazelzet JA, de Groot R, van Mierlo G, Joosten KFM, van der Voort E, Eerenberg A, Suur MH, Hop WCJ, Hack CE. Complement activation in relation to capillary leakage in children with septic shock and purpura. Infect. Immun. 1998;66:5350-5356 21. Harboe M, Ulvund G, Vien L, Fung M, Mollnes TE. The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation. Clin Exp Immunol. 2004;138:439-446 22. Hibberd ML, Sumiya M, Summerfield JA, Booy R, Levin M, and, the, Meningococcal, Research, Group. Association of variants of the gene for mannose-binding lectin with susceptibility to meningococcal disease. Lancet. 1999;353:1049-1053 23. Bax WA, Cluysenaer OJ, Bartelink AK, Aerts PC, Ezekowitz RA, van Dijk H. Association of familial deficiency of mannose-binding lectin and meningococcal disease [letter]. Lancet. 1999;354:1094-1095 24. Kuipers S, Aerts PC, Cluysenaer OJ, Bartelink AK, Ezekowitz RA, Bax WA, Salimans M, Vandyk H. A case of familial meningococcal disease due to deficiency in mannose- binding lectin (MBL). Adv Exp Med Biol. 2003;531:351-355 25. Sjöholm AG, Kuijper EJ, Tijssen CC, Jansz A, Bol P, Spanjaard L, Zanen HC. Dysfunctional properdin in a Dutch family with meningococcal disease. N. Engl. J. Med. 1988;319:33-37 26. Sjoholm AG, Braconier JH, Soderstrom C. Properdin deficiency in a family with fulminant meningococcal infections. Clin Exp Immunol. 1982;50:291-297 27. Cunliffe NA, Snowden N, Dunbar EM, Haeney MR. Recurrent meningococcal septicaemia and properdin deficiency. J Infect. 1995;31:67-68 28. Biesma DH, Hannema AJ, van Velzen-Blad H, Mulder L, van Zwieten R, Kluijt I, Roos D. A family with complement factor D deficiency. J Clin Invest. 2001;108:233-240 29. Hiemstra PS, Langeler E, Compier B, Keepers Y, Leijh PC, van den Barselaar MT, Overbosch D, Daha MR. Complete and partial deficiencies of complement factor D in a Dutch family. J Clin Invest. 1989;84:1957-1961 30. Holten E. Serotypes of Neisseria meningitidis isolated from patients in Norway during the first six months of 1978. J Clin Microbiol. 1979;9:186-188 31. Fung M, Loubser PG, Undar A, Mueller M, Sun C, Sun WN, Vaughn WK, Fraser CD. Inhibition of complement, neutrophil, and platelet activation by an anti-factor D monoclonal antibody in stimulated cardiopulmonary bypass circuits. J Thorac Cardiovasc Surg. 2001;122:113-122 32. Sprong T, Moller ASWM, Bjerre A, Wedege E, Kierulf P, van Der Meer JW, Brandtzaeg P, van Deuren M, Mollnes TE. Complement activation and complement-dependent inflammation by Neisseria meningitidis occurs independent of lipopolysaccharide. Infect Immun. 2004;72:3344-3349 33. Volanakis JE, Narayana SV. Complement factor D, a novel serine protease. Protein Sci. 1996;5:553-564 34. Niemann MA, Bhown AS, Bennett JC, Volanakis JE. Amino acid sequence of human D of the alternative complement pathway. Biochemistry. 1984;23:2482-2486 35. Lesavre PH, Muller-Eberhard HJ. Mechanism of action of factor D of the alternative complement pathway. J Exp Med. 1978;148:1498-1509 36. Narayana SV, Carson M, el-Kabbani O, Kilpatrick JM, Moore D, Chen X, Bugg CE, Volanakis JE, DeLucas LJ. Structure of human factor D. A complement system protein at 2.0 A resolution. J Mol Biol. 1994;235:695-708 37. Arlaud GJ, Rossi V, Thielens NM, Gaboriaud C, Bersch B, Hernandez JF. Structural and functional studies on C1r and C1s: new insights into the mechanisms involved in C1 activity and assembly. Immunobiology. 1998;199:303-316 38. Estabrook MM, Jack DL, Klien NJ, Jarvis GA. Mannose-binding lectin (MBL) binds to two major outer membrane proteins, opacity protein and porin, of Neisseria meningitidis but not to lipooligosaccharide (LOS). J immunol. 2004;in press 39. Hajela K, Kojima M, Ambrus G, Wong KH, Moffatt BE, Ferluga J, Hajela S, Gal P, Sim RB. The biological functions of MBL-associated serine proteases (MASPs). Immunobiology.

158 2002;205:467-475 40. Blom AM, Villoutreix BO, Dahlback B. Complement inhibitor C4b-binding protein-friend or foe in the innate immune system? Mol Immunol. 2004;40:1333-1346 41. Rodriguez de Cordoba S, Esparza-Gordillo J, Goicoechea de Jorge E, Lopez-Trascasa M, Sanchez-Corral P. The human complement factor H: functional roles, genetic variations and disease associations. Mol Immunol. 2004;41:355-367 42. Bjerre A, Brusletto B, Mollnes TE, Fritzsonn E, Rosenqvist E, Wedege E, Namork E, Kierulf P, Brandtzaeg P. Complement activation induced by purified Neisseria meningitidis lipopolysaccharide (LPS), outer membrane vesicles, whole bacteria and an LPS-free mutant. J Infect Dis. 2002;15:220-228 43. Van Deuren M, Brandtzaeg P, Van der Meer JWM. Update on meningococcal disease with emphasis on pathogenesis and clinical management. Clin Microbiol Rev. 2000;13:144- 166 44. Lehner PJ, Davies KA, Walport MJ, Cope AP, Würzner R, Orren A, Morgan BP, Cohen J. Meningococcal septicaemia in a C6-deficient patient and effects of plasma transfusion on lipopolysaccharide release. Lancet. 1992;340:1379-1381 45. White RT, Damm D, Hancock N, Rosen BS, Lowell BB, Usher P, Flier JS, Spiegelman BM. Human adipsin is identical to complement factor D and is expressed at high levels in adipose tissue. J Biol Chem. 1992;267:9210-9213

159

Mannose-binding lectin is a critical factor in systemic complement activation during meningococcal septic shock 10

Tom Sprong, Tom Eirik Mollnes, Chris Neeleman, Dorine Swinkels, Jos W.M.van der Meer, Marcel van Deuren Submitted Abstract

Systemic activation of complement during meningococcal disease is associated with severe disease and poor outcome. The exact mechanism of activation of complement is unknown, but important for future therapies aimed at modulating the complement system in this disease. We studied complement activation in 22 patients, eighteen with meningococcal septic shock and four with meningococcal disease without shock. Two of the shock patients were MBL-deficient: one patient was homozygous and one patient was compound heterozygous for exon 1 mutations in the gene for MBL. These MBL-deficient patients had relatively low disease severity and mild disseminated intravascular coagulation (DIC). At admission, the MBL-deficient patients had much lower circulating values of C3bc (indicating common pathway activation) and TCC (terminal pathway) than MBL- sufficient patients that presented with meningococcal septic shock. C4bc (classical or lectin pathway) and C3bBbP (alternative pathway) were also decreased in the MBL-deficient patients. Systemic activation of complement excellently correlated with disease severity and parameters of DIC. Testing of convalescent blood of one of the MBL-deficient patients in a model of meningococcal sepsis showed absent lectin pathway activation leading to a reduced activation of complement. This indicates that MBL is critical for the systemic activation of complement seen during meningococcal septic shock.

162 Introduction

Meningococcal septic shock (MSS) is a lethal disease that can slay previously completely healthy children within 24 hours 1. The complement system plays a complex dual role in the pathogenesis of meningococcal septic shock. It is essential for the early defence against Neisseria meningitidis 2,3. On the other hand, extensive systemic complement activation is associated with severe disease and poor outcome 4,5.

Complement is activated on the surfaces of meningococci or meningococcal outer membrane blebs by one or more of the three initial complement-activating pathways - the classical-, the lectin- and the alternative pathway. Antibody, C-reactive protein and complement factor (C) C1q mediate classical pathway activation. Mannose- binding lectin (MBL) is the central molecule in the lectin pathway of complement activation. MBL binds to meningococci 6 and, as was recently shown by us, MBL is crucial for the activation of complement in a whole blood model of meningococcal sepsis 7. Deficiency for MBL (i.e. undetectable plasma levels) occurs when a person is homozygous (or compound heterozygous) for any of the three structural mutations in exon 1 of the MBL-gene; heterozygousity for these mutations leads to reduced plasma MBL concentrations. MBL deficiency is relatively frequent, and is linked to increased susceptibility to certain infectious diseases8,9, although its exact role is still subject to debate. The alternative pathway is activated on bacterial surfaces both by pattern recognition and imbalanced regulatory control, but is particularly important for amplification of complement activation after initiation by the classical or lectin pathway.

After engagement of any of the three initial pathways, the activation converges at the level of C3 and subsequently C5 is activated, leading to the formation of 10 the terminal C5b-C9 complement complex (also called TCC) and the anaphylatoxin C5a. Membrane associated C5b-C9 or membrane attack complex (MAC) is critically important in Neisserial killing, whereas C5a is a powerful pro-inflammatory mediator and harmful to the host during septic shock 10. The mechanism of complement activation as well as the exact role of systemic complement activation in the pathogenesis of tissue damage during MSS is still subject to debate.

We studied complement activation, cytokine production and disease severity in 22 patients with invasive meningcoccal disease. Two of these patients were deficient for MBL, the central molecule in the lectin pathway of complement activation.

163 Methods

From 2001 until 2006, 25 children with meningococcal disease were admitted to our the PICU of the Radboud University Nijmegen Medical Centre. The PICU of the Radboud University Nijmegen Medical Centre receives patients either as direct admissions from the emergency department or after referral by other hospitals. From 22 patients material was available for analysis. Informed consent was given for each patient from the parents, and the study was approved by the local ethics committee and performed according to local guidelines. Diagnosis of meningococcal disease was made based on positive culture or Gram-stain of blood, cerebrospinal fluid or skin biopsy (82%) or typical presentation (18%). All isolated meningococci were serogroup B (the study was started after the national vaccination campaign against group C meningococci). 18 patients presented with septic shock (shock group), and 4 patients had no shock (1 had meningoccal bacteraemia and 3 had meningocccal meningitis). Shock was defined according to the Consensus Conference Definitions for Sepsis and Organ Dysfunction in Pediatrics 11. All patients received prompt antibiotic therapy (ceftriaxone i.v.) and dexamethasone during the first 3 days of admission. Fresh Frozen Plasma (FFP) was given to 14 patients with septic shock and signs of DIC. One of the two MBL-deficient patients (MBL-P1) received no FFP whereas the other MBL-deficient patient (MBL-P2) received FFP before admission to our PICU. Among the patients without MBL-deficiency, three received recombinant tissue plasminogen activator (rTPA) as adjunctive treatment. PRISM score was calculated retrospectively by one of the researchers (TS)12,13.

For determination of complement activation, EDTA-anticoagulated blood was sampled at admission to PICU and at t = 2, t = 8, t = 24 and t = 48 hours after admission. Heparin-anticoagulated blood was obtained for determination of cytokine concentrations. Samples were immediately put on ice, centrifuged for 15 min at 2800 x g, and frozen at -80°C until analysis in batch.

One of the MBL-deficient patients (MBL-P1) and an age and disease matched control that had had meningococcal meningitis were invited, more than 1 year after their episode of meningococcal disease, for an additional in vitro whole blood stimulation experiment 7. In brief, lepirudin anticoagulated whole blood was incubated for 1 hour with 108 / mL serogroup B Neisseria meningitidis at 37°C, whereafter 25 mM EDTA was added, put on ice, centrifuged and frozen at -80°C until assay for the complement intermediates. Patient (MBL-P2) and his parents did not give permission to draw blood for additional experiments after convalescence due to extreme fear for venapuncture in this patient.

164 Complement activation products were measured by enzyme-linked immunosorbent assay (ELISA) as described previously in detail. C1rs-C1inh complexes (classical pathway) 14, C4bc (classical and lectin pathway) 15, and C3bBbP (alternative pathway) 16 were used as markers of initial pathway activation, C3bc 17 and terminal complement complexes (TCC) 18 indicate final common pathway or total complement activation. cytokines (TNFα, IL-1β and IL-10) were determined using commercially available Bio-Plex protein array system (Bio-Rad Laboratories, Inc.) 19. MBL plasma concentration was measured by ELISA (AntibodyShop, Copenhagen, Denmark). MBL-genotype was determined using restriction fragment length polymorphism (RFLP) analysis for the structural mutations in exon 1 of the MBL-gene 20,21.

Results

Eighteen patients presented with shock: One patient (MBL-P1) was identified homozygous for a point mutation in codon 54 of exon 1 of the MBL-gene, and had undetectable plasma concentrations of MBL at admission. A second patient (MBL- P2) was compound heterozygous for mutations in codon 52 and 54 of exon 1, and due to the infusion of FFPs before admission the patient had a plasma concentration of MBL of 840 ng/mL at admission. 7 patients were heterozygous (A/O) for a mutation in exon 1 of the MBL-gene and had MBL-plasma concentrations at admission of median 360, (range 30 - 680 ng/mL). 9 patients had a normal genotype (A/A) (MBL- plasma concentrations at admission median 1750, range 1110 - 9000 ng/mL). 4 patients presented without shock: two had a normal MBL-genotype and two were heterozygous for an exon 1 mutation in the MBL gene (MBL-plasma concentrations at admission median 2351, range 266 - 4350).

The A/A and A/O patients with shock suffered from more fulminant and severe 10 disease, reflected by a higher mortality or more sequelae, a shorter time from disease onset to admission and a higher PRISM score in comparison with the patients without shock. In addition, plasma concentrations of the cytokines TNFα, IL-1β and IL-10 in these shock patients was also increased compared to the no shock patients. Characteristics of the patients MBL-P1 and MBL-P2 (O/O) with shock were in range with that of the other shock patients, although they had a relatively low disease severity and mild DIC (Table 1).

In the A/A and A/O patients with shock considerably higher plasma concentrations of C3bc (reflecting systemic common pathway activation) and TCC (reflecting systemic terminal pathway activation) were seen at admission than in the in the O/O patients and the patients without shock (Figure 1). C4bc (reflecting classical

165 or lectin pathway activation) was higher in the A/A and A/O patients with shock compared to O/O patients. No differences in activation of the classical pathway (C1rs-C1inh complexes) were seen for these patients (data not shown). C3bBbP (reflecting alternative pathway activation) was highest in the A/A ‘shock’ patients, followed by the A/O patients and lowest in patients MBL-P1 and MBL-P2 (O/O) and the patients without shock. Taken together, these results indicate that MBL is of vital importance for the systemic activation of the complement system seen during meningococcal septic shock.

Time-course analysis showed that increased values of complement activation products could be found until 8 hours after admission in the shock patients, hereafter decreasing to values comparable to that in patients without shock (not shown).

Table 1. Disease characteristics for the different patient groups SHOCK and SHOCK and SHOCK and NO SHOCK A/A (n=9) A/O (n=7) O/O (n=2) (n = 4) Male / female 5/4 3/4 1/1 3/1

Age (years) 1.1 3.5 5.5 - 3.6 1.8 (0.5 - 7.3) (0.6 - 5.6) (0.6 - 4.7) Sequelae 6/9 3/7 0/2 0/4

Mortality 0/9 2/7 0/2 0/2

Time (hours) 12 16 14 - 20 38 (9 - 17)** (12 - 17)** (23 - 96) PRISM 13 17 6 - 10 3 (5 - 23)** (7 - 35)** (0 - 5) MAP1 69 50 67 - 77 84 (55 - 87) (30 - 93) (64 - 93) Lactate1 3.3 6.1 2.8 - 3.3 1.9 (2 - 7.4)*, # (4 - 12)** (0.7 - 2.4) Platelet nadir 31 42 198 - 73 259 (13 - 205)** (10 - 129)** (233 - 291) Fibrinogen1 1790 1770 2370 - 3620 3220 (490 - 3760)* (330 -4500)* (2790 - 6420) TNF1 40 17 15 – 38 8 (8 - 8) (8 - 93)* (8 - 125)* IL-11 39 42 59 – 8 16.5 (8 – 515) (32 – 304) (8 – 58) IL-101 5566 2087 3771 - 734 168 (956 - 30732)** (886 - 13571)* (14 - 1140) CRP1 91 93 57 - 64 178 (27 - 108)* (37 - 121)* (99 - 307) A/A = MBL-WT, A/O = heterozygous exon 1 mutation, O/O = homozygous exon 1 mutation. Shown are medians and ranges. P values are by χ2 or Mann-Whitney test when appropriate. * = P < .05 for the comparison with the NO SHOCK group, ** = P < .01 for the comparison with the NO SHOCK group, # = P < .05 for the comparison with the SHOCK and exon 1 heterozygous group. Time indicates time from onset of symptoms to admission to the referring hospital. PRISM = pediatric risk of mortality. MAP = mean arterial pressure. Platelet nadir indicates the lowest value of platelets obtained in the first 48 hours after admission. 1 indicates values obtained at PICU admission.

166 Complement activation correlated significantly with disease severity R (Spearman) = 0.79, P < 0.0001 for the correlation between TCC and Paediatric Risk of Mortality (PRISM)). In addition, high plasma D-dimer concentration at admission and low platelet nadir (i.e., the lowest value in the first 48 hours after admission) 22 - reflecting DIC - significantly correlated with plasma concentration of TCC (R (Spearman) = 0.80, P < 0.0001 for D-dimers, and R (Spearman) = -0.88. P < 0.0001 for platelet nadir).

P = 0.02 P = 0.01 300 10 P = 0.006 250 P = 0.07 8 200 6 150 4 100 TCC (AU/mL) C3bc C3bc (AU/mL) 50 2

0 0 A/A A/O O/O NO SHOCK A/A A/O O/O NO SHOCK

SHOCK SHOCK P = 0.71 P = 0.0056 150 250

125 P = 0.23 200 P = 0.01 100 150 75 100 50 C4bc C4bc (AU/mL)

25 C3bBbP (AU/mL) 50

0 0 A/A A/O O/O NO SHOCK A/A A/O O/O NO SHOCK

SHOCK SHOCK

Figure 1. Complement activation products at admission to PICU in patients with meningococcal disease A/A = MBL-WT, A/O = heterozygous exon 1 mutation, O/O = homozygous exon 1 mutation. C4bc reflects classical or lectin pathway activation, C3bBbP reflects alternative pathway activation, C3bc and TCC 10 reflect total complement activation. Lines indicate medians, differences between groups were tested for significance using Mann-Whitney test.

1000 50

800 40 Um (TCC) AU/mL

600 30 Figure 2. Complement activation in a whole blood model of meningococcal sepsis Lepirudin anti-coagulated whole blood from 400 20 AU/mL the patient deficient for MBL (white bars) and an age and disease matched MBL- 200 10 sufficient control (grey bars) was stimulated with 108 / mL serogroup B meningococci for 1 hour. Left Y-axis is AU/mL for C1rs/C1inh, 0 C4bc, C3bBbP, C3bc, right Y axis is AU/mL C1inh/1rs C4bc C3bBbP C3bc TCC for TCC.

167 Additional in vitro experiments with convalescent blood from one of the MBL- deficient patients (MBL-P1) and an age matched control that had experienced meningococcal meningitis were performed. patient MBL-P1 had normal classical and alternative pathway haemolytic activity. In a whole blood model of meningococcal sepsis, significant C4bc and C3bBbP activation was seen in the control patient, whereas no lectin pathway activation and markedly reduced alternative pathway activation was found in the MBL-deficient patient (Figure 2). Similarly, substantial total complement activation, reflected by formation of C3bc and TCC, was found in the control patient, but was largely abolished in the MBL-deficient patient (Figure 2).

Discussion

This is the first report describing complement activation during meningococcal septic shock in relation to genotype for MBL. In the A/A and A/O patients with meningococcal septic shock substantially higher plasma concentrations of the complement activation products C3bc, TCC, C4bc and C3bBbP were seen than in the in the MBL0deficient (O/O) patients and the patients without shock. This indicates that there is an important role for MBL in the systemic activation of complement during meningococcal septic shock.

The mechanism of complement activation during meningococcal septic shock has been debated, and classical- as well as alternative pathway-dependent routes of activation have been proposed 5,23. However, these studies were performed before the contribution of the lectin pathway could be assessed. We previously found that complement activation in a whole blood model of meningococcal sepsis is inhibited by anti-MBL antibodies, indicating that in whole blood complement activation by meningococci is, at least in part, dependent on MBL 7. The present in vivo results are support the previous in vitro findings. In addition, by using convalescent blood samples of one of the patients homozygous for a mutation in exon 1 of the MBL gene, we confirmed in vitro that MBL-deficiency leads to the absence of lectin pathway activation and decreased common pathway activation, in comparison to an MBL-sufficient control patient.

One of the MBL-deficient patients (MBL-P2) had a plasma concentration of MBL at admission similar to that of heterozygous patients, consistent with the transfusion of large amounts of FFP before admission. We have observed this phenomenon also in other patients that were given FFP and were heterozygous for an exon 1 mutation. For instance, MBL plasma concentration at admission of 495 ng/mL in

168 one patient increased to 2160 ng/mL after FFP transfusion and decreased again to 1135 (48 hours after admission) and 915 ng/mL (144 hours after admission). In addition, this phenomenon was also observed in MBL-deficient patients given FFPs after cardiac surgery 24. Interestingly, the infusion of FFP in patient MBL-P2 did not lead to measurable complement activation. We suggest that this might be due to lack of complement activation during the disease course before FFP was given, combined with a rapid clearance of meningococci and complement activation intermediates after start of treatment.

During meningococcal septic shock and in the whole blood model, the alternative pathway was considerably activated in MBL-sufficient patients, but reduced in the patients with MBL deficiency. This is consistent with the view that the role of the alternative pathway lies in amplification of complement activation; in this case trough MBL initiated lectin pathway activation7,25.

Heterozygousity for an exon 1 mutation in the MBL gene had a limited effect on complement activation during meningcoccal septic shock, although median values of C3bc, TCC and C3bBbP were somewhat lower in this group compared to the A/A group. This suggests that MBL-dependent complement activation is sufficiently maintained in plasma with reduced MBL levels, as in A/O genotypes, but strongly affected when it falls below a certain limit and is lost in O/O plasma 26. Three A/O patients had MBL-plasma concentrations below 200 ng/mL; these very low concentrations may be related to consumption of MBL during septic shock, reflecting binding of MBL to microbes in order to activate complement 27.

The two MBL-deficient patients had a relatively low disease severity and mild DIC. This parallels the findings in patients with terminal complement component deficiencies 3, and is in line with the study by Hibberd who showed that although 10 susceptibility to meningococcal disease was increased by MBL-deficiency, disease severity seemed to be reduced 9. In contrast, patients with alternative pathway deficiencies (Properdin or factor D) who also have increased susceptibility to meningococcal disease, are reported to have a more fulminant disease course 3,28,29. Apparently, there is a slender balance between susceptibility for meningococcal infections and the course of disease.

Crucial for the development of tissue damage and limb-ischemia in meningococcal septic shock is the severity of DIC. Experimentally, C5a is a potent inducer of tissue factor expression, critical in the development of DIC 30-32. We found that the MBL-deficient patients had limited signs of DIC, and that the degree of systemic complement activation was strongly correlated to DIC-parameters. This suggests

169 that MBL-dependent complement activation, which leads to the formation of C5a, is important for development of DIC.

Activation of the cytokine network is considered to be one of the main pathological events during meningococcal septic shock 1. The extent of the inflammatory cytokine response in the MBL-deficient patients was similar to that of the other shock patients. This indicates that the lower disease severity and the absence of DIC in the MBL-deficient patient compared to the other patients, was not caused by a reduction in cytokine activation.

To date, no adjunctive immunomodulatory treatment is available that is proven to influence the course of disease in meningococcal septic shock. Modulation of the complement system is postulated to be an attractive approach, based on in vitro research and data in animal experiments 7,33,34. New therapies that aim to modulate the complement system during meningococcal septic shock should at least also inhibit activation of complement via the lectin pathway.

Acknowledgements

The authors thank Grethe Bergseth (Nordland Hospital, Bodø) and Johanna van der Ven-Jongekrijg (University Medical Centre St Radboud, Nijmegen) for determination of complement activation products and cytokines, and Rianne Roelofs for determination of the MBL-genotypes.

170 References 1. Van Deuren M, Brandtzaeg P, Van der Meer JWM. Update on meningococcal disease with emphasis on pathogenesis and clinical management. Clin Microbiol Rev. 2000;13:144- 166 2. Ross SC, Densen P. Complement deficiency states and infection: epidemiology, pathogenesis and consequences of neisserial and other infections in an immune deficiency. Medicine (Baltimore). 1984;63:243-273 3. Figueroa JE, Densen P. Infectious diseases associated with complement deficiencies. Clin.Microbiol. Rev. 1991;4:359-395 4. Brandtzaeg P, Mollnes TE, Kierulf P. Complement activation and endotoxin levels in systemic meningococcal disease. J Infect Dis. 1989;160:58-65 5. Hazelzet JA, de Groot R, van Mierlo G, Joosten KFM, van der Voort E, Eerenberg A, Suur MH, Hop WCJ, Hack CE. Complement activation in relation to capillary leakage in children with septic shock and purpura. Infect. Immun. 1998;66:5350-5356 6. Jack DL, Dodds AW, Anwar N, Ison CA, Law A, Frosch M, Turner MW, Klein NJ. Activation of complement by mannose-binding lectin on isogenic mutants of Neisseria meningitidis serogroup B. J. Immunol. 1998;160:1346-1353 7. Sprong T, Brandtzaeg P, Fung M, Pharo AM, Hoiby EA, Michaelsen TE, Aase A, van der Meer JW, van Deuren M, Mollnes TE. Inhibition of C5a-induced inflammation with preserved C5b-9-mediated bactericidal activity in a human whole blood model of meningococcal sepsis. Blood. 2003;102:3702-3710 8. Turner MW. Mannose-binding lectin: the pluripotent molecule of the innate immune system. Immunol. Today. 1996;17:532-540 9. Hibberd ML, Sumiya M, Summerfield JA, Booy R, Levin M, and, the, Meningococcal, Research, Group. Association of variants of the gene for mannose-binding lectin with susceptibility to meningococcal disease. Lancet. 1999;353:1049-1053 10. Ward PA. the dark side of C5a in sepsis. Nat Rev Immunol. 2004;4:133-142 11. Goldstein B, Giroir B, Randolph A. International pediatric sepsis consensus conference: definitions for sepsis and organ dysfunction in pediatrics. Pediatr Crit Care Med. 2005;6:2-8 12. Pollack MM, Ruttimann UE, Getson PR. Pediatric risk of mortality (PRISM) score. Crit Care Med. 1988;16:1110-1116. 13. van Brakel MJ, van Vught AJ, Gemke RJ. Pediatric risk of mortality (PRISM) score in meningococcal disease. Eur J Pediatr. 2000;159:232-236. 14. Fure H, Nielsen EW, Hack CE, Mollnes TE. A neoepitope-based enzyme immunoassay for quantification of C1-inhibitor in complex with C1r and C1s. Scand J Immunol. 1997;46:553-557 15. Wolbink GJ, Bollen J, Baars JW, Tenberge RJM, Swaak AJG, Paardekooper J, Hack CE. 10 Application of a monoclonal antibody against a neoepitope on activated C4 in an ELISA for the quantification of complement activation via the classical pathway. J Immunol Methods. 1993;163:67-76 16. Mollnes TE, Brekke OL, Fung M, Fure H, Christiansen D, Bergseth G, Videm V, Lappegard KT, Kohl J, Lambris JD. Essential role of the C5a receptor in E. coli-induced oxidative burst and phagocytosis revealed by a novel lepirudin-based human whole blood model of inflammation. Blood. 2002;100:1869-1877 17. Garred P, Mollnes TE, Lea T. Quantification in enzyme linked immunosorbent assay of a C3 neoepitope expressed on activated human complement factor C3. Scand J Immunol. 1988:329-335 18. Mollnes TE, Lea T, Froland SS, Harboe M. Quantification of the terminal complement complex in human plasma by an enzyme-linked immunosorbent assay based on monoclonal antibodies against a neoantigen of the complex. Scand J Immunol. 1985:197- 202 19. de Jager W, te Velthuis H, Prakken BJ, Kuis W, Rijkers GT. Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells.

171 Clin Diagn Lab Immunol. 2003;10:133-139 20. Madsen HO, Garred P, Thiel S, Kurtzhals JA, Lamm LU, Ryder LP, Svejgaard A. Interplay between promoter and structural gene variants control basal serum level of mannan- binding protein. J. Immunol. 1995;155:3013-3020 21. Roelofs R, Sprong T, de Kok J, Swinkels D. PCR-restriction fragment lenght polymorphism method to detect the X/Y polymorphism in the promoter site of the mannose-binding lectin gene. Clin Chem. 2003;49:1557-1558 22. Van Deuren M, Neeleman C, van ‘t Hek LGFM, van der Meer JWM. A normal platelet count at admission in acute meningococcal disease does not exclude a fulminant course. Intensive Care Med. 1998;24:157-161 23. Brandtzaeg P, Høgåsen K, Kierulf P, Mollnes TE. The excessive complement activation in fulminant meningococcal septicemia is predominantly caused by alternative pathway activation. J. Infect. Dis. 1996;173:647-655 24. Bilgin YM, Brand A, Berger SP, Daha MR, Roos A. Mannose-binding lectin is involved in multiple organ dysfunction syndrome after cardiac surgery: effects of blood transfusions. Transfusion. 2008 25. Harboe M, Ulvund G, Vien L, Fung M, Mollnes TE. The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation. Clin Exp Immunol. 2004;138:439-446 26. Kilpatrick DC. Mannan-binding lectin: clinical significance and applications. Biochim Biophys Acta. 2002;1572:401-413 27. Dumestre-Perard C, Doerr E, Colomb MG, Loos M. Involvement of complement pathways in patients with bacterial septicemia. Mol Immunol. 2007;44:1631-1638 28. Biesma DH, Hannema AJ, van Velzen-Blad H, Mulder L, van Zwieten R, Kluijt I, Roos D. A family with complement factor D deficiency. J Clin Invest. 2001;108:233-240 29. Sprong T, Roos D, Weemaes C, Neeleman C, Geesing CL, Mollnes TE, van Deuren M. Deficient alternative complement pathway activation due to factor D deficiency by 2 novel mutations in the complement factor D gene in a family with meningococcal infections. Blood. 2006;107:4865-4870 30. Laudes IJ, Chu JC, Sikranth S, Huber-Lang M, Guo RF, Riedemann N, Sarma JV, Schmaier AH, Ward PA. Anti-C5a ameliorates coagulation/fibrinolytic protein changes in a rat model of sepsis. Am J Pathol. 2002;160:1867-1875 31. Ikeda K, Nagasawa K, Horiuchi T, Tsuru T, Nishizaka H, Niho Y. C5a induces tissue factor activity on endothelial cells. Thromb Haemost. 1997;77:394-398 32. Markiewski MM, Nilsson B, Ekdahl KN, Mollnes TE, Lambris JD. Complement and coagulation: strangers or partners in crime? Trends Immunol. 2007;28:184-192 33. Mollnes TE, Kirschfink M. Strategies of therapeutic complement inhibition. Mol Immunol. 2006;43:107-121 34. Bohle D, Stagl GL. therapeutic potential of targeting the complement cascade in critical care medicine. Crit Care Med. 2003;31:S97-S104

172 10

173

PCR-Restriction Fragment Length Polymorphism method to detect the X/Y polymorphism in the promoter site of the mannose-binding lectin gene 11

Rian W. Roelofs,Tom Sprong, Jacques B. de Kok, Dorine W. Swinkels Clinical Chemistry 2003;49(9):1557-8 Introduction

Mannose-binding lectin (MBL), a pattern-recognition molecule produced by the liver and present in serum, is an important player in the innate immune system. MBL acts by binding various carbohydrate structures on microbial surfaces, after which it activates the complement system via the lectin pathway. In addition, MBL can promote direct opsonophagocytosis of microorganisms and modulate diverse inflammatory mediators 1.

Deficiency of MBL was first identified in association with a common defect of opsonization in children. Additional studies have identified MBL deficiency as a risk factor for diverse infectious diseases 1. In addition, MBL deficiency has been found to be associated with certain autoimmune diseases 1 and, recently, atherosclerosis 2. MBL deficiency is caused by mutations in the coding and promoter regions of the MBL gene, which have a profound effect on plasma concentrations of the MBL protein.

Three point mutations have been found in the structural region of the MBL gene: at codons 54, 52, and 57 in exon 1. These mutations cause allelic variants designated B, C, and D, respectively. In addition to these exon 1 mutations, there are three major polymorphisms in the promoter region of the MBL gene, which are expressed as four haplotypes called HYP, LYP, LXP, and LYQ. The X/Y polymorphism at position -221 is important because the X haplotype is highly prevalent (24% in Caucasian populations 3) and is associated with low circulating MBL concentrations, comparable to those of the structural variants B, C, and D 4. Therefore, determination of the exon 1 point mutations and the X/Y promoter polymorphism is adequate to assess MBL deficiency in a certain individual.

Several methods have been described for the detection of mutations in the structural region, such as PCR followed by restriction fragment length polymorphism (RFLP) analysis 4. This method is simple and gives unambiguous results, but such a method has not been published for the detection of the X/Y promoter polymorphism mentioned above. Here we describe a PCR-RFLP method for the X/Y polymorphism. The method fits well with the PCR RFLP method we implemented for the detection of the three point mutations in exon 1.

Materials, methods, results and conclusion

PCR was carried out in a final volume of 50 µL containing 1 µL (30–100 ng) of

176 genomic DNA, 2.5 mM MgCl2, 50 µM deoxynucleotide triphosphates, 0.4 µM upstream primer (5'-GTTTCCACTCATTCTCATTCCCTAAG-3'), 0.4 µM downstream primer (5'-GAAAACTCAGGGAAGGTTAATCTCAG-3'), and 1 U of AmpliTaq Gold (Applied Biosystems) in a buffer containing 100 mM Tris-HCl (pH 8.3) and 500 mM KCl.

The PCR was initiated by a 10-min denaturation and enzyme activation step at 95 °C and completed by a 10-min extension step at 72 °C. The temperature cycles were as follows: 35 cycles of 30 s at 95 °C, 30 s at 60 °C, and 45 s at 72 °C. The amplification product, with a length of 350 bp, was digested by BsaJI according to the manufacturer’s instruction (New England Biolabs Inc.).

The PCR product contained one nonpolymorphic BsaJI restriction site that served as an internal control for enzyme digestion, producing two fragments of 242 and 108 bp. In the Y variant, the 242-bp fragment was cleaved into two fragments of 166 and 76 bp. Because of the substitution of the base cytosine for guanine at position -221 of the MBL gene, this does not occur in the X variant. PCR restriction fragments were separated by electrophoresis in 2% Response Research (Biozym) agarose gels (Fig. 1).

To assess the validity of the method, we tested a series of 17 different DNA samples (partially provided by Prof. Dr. M. Turner, Institute of Child Health, London, UK) with our newly developed PCR-RFLP method. Haplotype frequencies were 1 XX, 6 XY, and 10 YY. The observed haplotypes were confirmed by a heteroduplex method using PCR-amplifiable synthetic DNA 5.

In conclusion, our newly developed PCR-RFLP method for the X/Y promoter

Figure 1. Detection of the X/Y polymorphism in the promoter site of the MBL gene by the PCR- RFLP assay. Lane M, molecular weight marker (50-bp DNA ladder; Invitrogen) with sizes in bp. Lane 1, haplotype X/X; lane 2, haplotype Y/Y; lane 3, haplotype X/Y; lane 4, undigested PCR- fragment; lane 5, negative control.

177 polymorphism of the MBL gene works with the previously described PCR-RFLP method for the three exon 1 variants to detect the mutations in the MBL gene that cause MBL deficiency.

178 References 1. Turner MW. Mannose-binding lectin (MBL) in health and disease. Immunobiology. 1998;199:327-339 2. Madsen HO, Videm V, Svejgaard A, Svenning JL, Garred P. Association of mannose- binding lectin deficiency with severe atherosclerosis. Lancet. 1998;352:959-960 3. Madsen HO, Garred P, Thiel S, Kurtzhals JA, Lamm LU, Ryder LP, Svejgaard A. Interplay between promoter and structural gene variants control basal serum level of mannan- binding protein. J. Immunol. 1995;155:3013-3020 4. Madsen HO, Satz ML, Hogh B, Svejgaard A, Garred P. Different molecular events result in low protein levels of mannan-binding lectin in populations from southeast Africa and South America. J Immunol. 1998;161:3169-3175 5. Turner MW, Dinan L, Heatley S, Jack DL, Boettcher B, Lester S, McCluskey J, Roberton D. Restricted polymorphism of the mannose-binding lectin gene of indigenous Australians. Hum Mol Genet. 2000;9:1481-1486

179

The double-edged sword of complement activation during sepsis. 12

Tom Sprong, Tom Eirik Mollnes and Marcel van Deuren Netherlands Journal of Critical Care. 2006; 11(1):19-23 Introduction

Sepsis and septic shock are major killers world-wide. Antibiotic treatment and early- goal directed therapy in the intensive care unit are essential elements of care, but no adjunctive immuno-modulatory treatment to alter the outcome has proven to be effective. The complement system plays a protective role in infectious diseases acting locally to kill the microbe, but during sepsis, complement can be activated systemically to induce an undesired excessive inflammation with subsequent tissue damage. This review discusses the diverse actions of the complement system in sepsis, emphasising on the defence against infection and the damage done by uncontrolled systemic activation of the complement system.

The complement system

Our first line of defence against infections is formed by the innate immune system. The innate immune system comprises a plasmatic part in which the complement system is most important, and a cellular part that recognises microbes using receptors such as the toll-like receptors (TLRs). Because components of the complement system circulate in the plasma as inactive enzymes that are ready to jump into gear upon activation, it is the only defence against infections that can be instantaneously activated when a pathogens enters the body. Thus, it is crucially important in fulminating infections since the other defence systems take more time to start up. The complement system in infectious diseases is a redundant system with diverse functions: 1. recognition of pathogens; 2. elimination of these pathogens; 3. signalling danger; 4. removal and repair of apoptotic immune cells and damaged tissue and 5. prevention of excessive activation.

Recognition of pathogens by the complement system is done by the initial pathways for complement activation: the classical, lectin and alternative pathways. Both classical pathway and lectin pathway use specific proteins designed to recognize pathogens for this aim. Classical pathway activation requires binding of antibody (IgG or IgM) to a microbial antigen, or binding of pentraxins like C-reactive protein (CRP) and pentraxin III to specific target molecules. By the mechanism of antibody- antigen mediated classical pathway activation, the complement system is linked to the adaptive immune system. The lectin pathway is activated after recognition of carbohydrates on the microbial surface by mannose binding lectin, and also L-ficolin (or ficolin 2) and H- ficolin (or ficolin 3) 1. The alternative pathway has no specific molecule that aids in activation of this route. Instead it is started up by spontaneous binding of C3 to foreign surfaces in the absence of membrane-bound complement inhibitors. All human cells posses these inhibitors while the surface of

182 most pathogens lacks these proteins. In this way, it is possible for the alternative pathway to discriminate between self and non-self. Of note, the alternative pathway is also particularly important for amplification of complement activation after deposition of C3 on a pathogen by any of the other two initial pathways of complement activation 2.

Elimination of pathogens occurs after cleavage and activation of C3 to C3b and C5 to C5b, key molecules of the complement system. Most pathogens are eliminated by opsonisation with C3b that promotes phagocytosis. C3b bound to a pathogen can be recognised by complement receptors on phagocytic cells in the blood, liver and spleen, and after recognition, phagocytosis can occur. This mechanism is especially important for Gram-positive encapsulated bacteria which have a thick cell wall. Thus, deficiency of C3 is associated with serious bacterial infections. Gram-negative bacteria, which have an outer membrane and a thin cell wall, can be killed directly by the lytic effect of the membrane attack complex (MAC). The MAC is a macromolecule composed of C5b, C6, C7, C8 and multiple C9 molecules. The MAC penetrates the bacterial cell membrane, punches a hole, and after this lysis of the bacterium will occur. This mechanism of bacterial complement lysis is principally important for killing of Neisserial species.

Cleavage and activation of C3 and C5 occurs by so-called convertases. The classical pathway C3 convertase - C4b2a - is generated when C1q is bound by antibody-antigen complexes and activates C1rs. Subsequently, C1rs can activate and cleave both C4 and C2. The lectin pathway forms the same C3 convertase, and here C4 and C2 are activated when binding of MBL to pathogens triggers the MBL-associated serine proteases (MASPs). The alternative pathway has another C3 convertase, called C3bBbP. This enzyme is formed after binding of factor B to C3b, cleavage of factor B by factor D and stabilisation of this complex by factor P. The C5 convertases (C4b2a3b for classical and lectin pathway and C3b3bBbP for the alternative pathway) are formed when a second C3b-molecule binds covalently to the C3 convertases. 12

The complement system signals danger by the actions of the anaphylatoxins: C4a, C3a and C5a. These are small peptide remnants that arise after cleavage of C4, C3 and C5. In particular C5a has been shown to be a potent pro-inflammatory mediator, whereas C3a has diverse effects also acting anti-inflammatory and possibly bactericidal 3,4. Various immune competent cells like granulocytes and mast cells possess receptors for these anaphylatoxins. The anaphylatoxins exert their actions by ligation of these receptors. In this way, the complement system is also linked to the cellular part of the innate immune system. The specific actions of

183 Classical Lectin Alternative pathway pathway pathway Antibody / CRP MBL, ficolins Surface of pathogens / artificial surfaces C1q / C1r / C1s MASP C3 C4 C3b C4 C2 B D C2 P C3 amplification C3b C5

phagocytosis C5b-9 C5a danger signal lysis of pathogens inflammation

‘DEFENCE’ ‘DAMAGE’

Figure 1. A simplified overview of the complement system.

C5a are discussed below. Unwanted or excessive activation of the complement system may cause damage to the host. Therefore, the body is equipped with complement-regulatory proteins. The most important are C1 inhibitor that prevents excessive activation of the classical and lectin pathways 5, C4-binding protein that prevents formation of C4b2a, and factor H and I that prevent alternative pathway activation. In addition, host cell-membranes are protected by membrane-bound regulators: Complement Receptor 1 (CR1), membrane cofactor protein (MCP) and decay accelerating factor (DAF). These membrane proteins prevent blood and endothelial cells against C3b formation. In addition, CD59 is present on most cells to protect against insertion of the lytic C5b-9 (MAC) complex. The anaphylatoxins are rapidly inactivated by the carboxypeptidases when they are released in the systemic circulation. Figure 1 shows a simplified overview of the complement system.

Defence

Because the complement system is crucially important for the elimination of certain pathogens, inherited or acquired deficiency for a complement protein will predispose to bacterial infection or sepsis.

In animal studies, it has been shown for nearly all complement components - i.e. C1, C2, C3, C4, C5, factor B and factor D - that deficiency leads to either increased susceptibility to infection and/or increased virulence of bacteria 6-9.

In humans, deficiency for C1q, C4 and C2, the proximal components of the classical

184 and lectin pathway, is associated with infections by encapsulated bacteria such as S. pneumonia and N. meningitidis. In addition, patients with classical pathway deficiencies are especially prone to developing auto-immune diseases such as systemic lupus erythematosus (SLE). Common pathway deficiency (C3) leads to a severe immune deficiency and predisposes to infections with a wide range of Gram-positive, Gram-negative and other infections. Patients with deficiencies for the alternative pathway and the terminal complement components C6-C9 are at risk particularly for infections with Neisseria meningitidis. Because complement deficiencies are rare, with prevalences reported to be < 0.1 % 10, they may be of minor importance in daily clinical practice. Yet, they underscore the importance of complement in defence against infections.

Deficiency for MBL, the initiating molecule of the lectin pathway of complement is much more common, with approximately 5-10 % of the population being completely deficient for this protein and over 35 % partially deficient 11. MBL deficiency is reported to be associated with pneumococcal and meningococcal infection 12,13 but overall, a only relatively small amount of these infections can be explained by MBL-deficiency. Because of the redundancy of the immune system, MBL- deficiency is probably more important in situations where the immune response is compromised by other mechanisms such as in young children 14, chemotherapy induced neutropenia 15, other immune deficiencies 16, or in critically ill patients (see below).

A number of observations have highlighted the consequences of MBL-deficiency in patients with sepsis admitted to the ICU. First, it appears that patients with sepsis admitted to the ICU have a higher incidence of MBL-deficiency than normal controls or patients with the systemic inflammatory response syndrome (SIRS) in the absence of infection 17-19. This indicates that MBL-deficiency predisposes to serious infections needing intensive care treatment. Likewise, MBL-deficiency is also linked to a higher number of infectious complications in patients with cancer following major surgery (20,21. Second, recently it was found that low MBL-plasma 12 concentrations predispose to pour outcome in sepsis and septic shock, although this effect was only evident in patients with a normal MBL-genotype 22. In addition, in pediatric patients with sepsis admitted to the ICU, low MBL plasma levels were associated with more severe disease in patients with sepsis 19. Thus, MBL-deficiency may be associated with a poor outcome in patients with sepsis admitted to the ICU. Interestingly, this may also be the case for patients without infection admitted to the ICU; as it was recently found in a trial assessing the effect of intensive insulin treatment on a surgical ICU unit, that low MBL plasma concentration in the control group, but not in the patients with intensive insulin treatment was linked to mortality

185 23. Possibly this is related to the development of more infectious complications in the patients with low MBL plasma concentrations. In sum, in critically ill patients, there is mounting evidence that MBL-deficiency predisposes to infectious complications and is associated with an increased risk for fatal outcome 24.

The role of complement in the defence against infections is indisputable; without it we would succomb to infectious diseases. The complement system is designed to function locally - on the pathogen at the site of infection - in a highly controlled way. In conditions when the control fails, systemic and insufficiently controlled activation of the complement system can be harmful and disrupt homeostasis; this is the case in sepsis and septic shock. Therefore, the complement system has been termed a ‘double-edged sword’ in the pathogenesis of sepsis. In the next paragraph, the implications of excessive systemic activation of the complement system in the pathogenesis of sepsis will be discussed.

Damage

The first study that documented increased complement activation in sepsis in humans was performed over 30 years ago 25. Two years later, the same group showed that the alternative pathway was important for this activation 26. In these studies, complement activation was seen in patients with septic shock, but not in patients with uncomplicated bacteraemia. It was concluded that complement activation in concert with activation of other mediators could play a critical role in the pathogenesis of shock. Subsequent studies showed that increased activation of complement was associated with a poor outcome 27-29, development of shock 30 and the adult respiratory distress syndrome 31.

In the above studies evidence was found for classical and alternative pathway dependent mechanisms of complement activation during sepsis. It should be noted however that activation of the lectin pathway (only discoverded in the 1990ies) of complement was indistinguishable from classical pathway activation in these studies. Recent evidence from in-vitro and animal models also points to lectin- pathway dependent complement activation during sepsis 32,33.

It is likely that the pathogen itself is responsible for the systemic activation seen during septic shock; it has been shown that in-vitro in whole blood substantial complement activation is seen after stimulation with different bacteria 32,34, at concentrations relevant for the situation during septic shock. However, several other mechanisms can also contribute to activation of complement. First, thrombin and fibrin can also activate complement 35 and both proteins are formed in

186 abundance during disseminated intravascular coagulation (DIC) seen during septic shock. Second, during septic shock, ischaemia-reperfusion (I-R) injury occurs, and activation of complement is an important pathogenic event in I-R 36.

Excessive activation of the complement system with formation of the anaphylatoxins is deleterious to the host. C5a is the best studied anaphylatoxin in sepsis, and a large body of data from animal models indicates that uncontrolled, systemic activation of C5a during sepsis is harmful. Blockade of C5a activity with monoclonal inhibitory antibodies against C5a or C5 in cecal ligation and puncture (CLP)-induced shock in rats has a protective effect 37,38, and attenuates the development of multi-organ failure 39. In addition, lung injury in mice induced by administration of cobra venom factor, an activator of complement, is mediated by C5a 40. In primates receiving with a lethal dose of E. coli, anti-C5a protects against early mortality, and inhibits the development of the adult respiratory distress syndrome and shock 41. Thus, C5a plays a critical role in the development of organ failure during sepsis.

In the human sepsis and septic shock, C5a has been studied and relates to disease severity. C5a is high in sepsis and septic shock 42,43, normalizes when the patient recovers, and remains high when patients fail to respond to treatment 43,44. In addition, soluble terminal complement complexes (sTCC or sC5b-9), that indicate C5a generation, are also increased in patients with septic shock, and correlate with mortality 28,44.

Because of the important role of C5a in the pathofysiology of sepsis, numerous studies have adressed the mechanism by which C5a can induce damage. It is now evident that during sepsis C5a exerts various separate but intertwined activities. First, C5a induces the synthesis of other mediators such as the cytokines and activates the coagulation cascade, which are both crucial factors in the development of organ damage during sepsis 45,46. Second, granulocytes and endothelial cells are activated excessively, leading to upregulation of pro-inflammatory molecules, adhesion of granulocytes to endothelium, release of oxygen radicals and activation 12 of coagulation 32,34,47,48. Third, C5a has a direct effect on vasopermeability and vasodilatation 49 and can induce cardiac dysfunction 50, events that are thought to be highly important in the development of shock. Finally, C5a impairs the bactericidal activity of neutrophils 37,51 which may counteract the host defence against infections. It is likely that these activities in concert are responsible for the harmful effect of C5a activation during sepsis (Figure 2).

The role of the other anaphylatoxins and the MAC in human sepsis is less clear. C3a has bactericidal activities 4, but in contrast to C5a it is thought to have an

187 induction of cytokines Excessive inflammation, ARDS

cellular activation

activation of coagulation Disseminated intravascular coagulation

C5a vasodilatation

vasopermeability Septic shock

cardio-depression

decreased neutrophil function Impairment of host defence

Figure 2 Actions of C5a of importance during the development of sepsis and septic shock.

anti-inflammatory role during sepsis 3. The MAC is possibly related to lytic damage during sepsis 52. In addition, the MAC and C3b aid in the induction of apaoptosis and the removal of apoptotic cells during inflammatory reactions 53,54.

Thus, uncontrolled systemic activation of the complement sytem plays an important role in the development of damage during sepsis, and it is likely that most of these effects are mediated by the anaphylatoxin C5a.

Therapy

Because of the evidence from animal models that inhibition of complement activation might be helpful and the observations in humans that excessive complement activation during sepsis is associated with severe disease and fatal outcome, modulation of complement activation during sepsis has been attempted in humans.

C1 inhibitor inhibits complement activation by classical and lectin pathway and additionally impedes activation of the contact and kinin systems 5,55 has been evaluated in a small randomized controlled trial. In this trial involving 40 patients with sepsis, treatment with C1 inhibitor had a beneficial effect on renal function and effectively inhibited activation of classical pathway, the contact system and granulocytes 56,57. However, no effect on outcome was observed. In addition, two case reports showed a possible beneficial effect of C1 inhibitor in patients with sepsis and streptococcal toxic shock syndrome 58,59. It must be noted however that the classical and lectin pathways are not the only pathways activated during sepsis, and that C1 inhibitor administration in primates suffering from lethal E. coli sepsis

188 had only a limited effect on terminal pathway activation 60.

One important problem of complement-inhibitory therapies aimed at inhibiting initial pathway (i.e. clasical, lectin or alternative) activation is that they also have a disadvantageous effect on the host defence. This might be of less importance in the early phase of the disease, when patients are treated with antibiotics and they have an excessive inflammatory response, but can be expected to be harmful later on in the course of their intensive care treatment when multiple organ failure may develop and infectious complications lie in ambush. Therefore, it would be more attractive to inhibit the effects of excessive complement activation without impairing elimination of bacteria, i.e. leaving intact the generation of C3b and the terminal C5b-9 complex. In the light of the crucial role C5a plays in the pathogenesis of sepsis and septic shock, selective inhibition of C5a or the C5a receptor is a promising option. By selective inhibition of C5a most of the damaging effects of complement activation can be prevented without significantly impairing the host defence systems 32,61. Interestingly, in a large randomised placebo controlled trial studying the effect of the C5-inhibitor pexelizumab on mortality and myocardial infarction after CABG, the incidence of septicaemia was even significantly reduced, although there were more cases of pneumonia 62.

Although inhibition of C5a may be an attractive approach to diminish the devastating effects of septic shock, it should be noted that the pathogenesis of sepsis and septic shock is complex and involves multiple mediators at multiple time points. Thus, therapies aimed at modulating the course of sepsis would most likely require a multi-target approach and should attack mediators upstream in the inflammatory reaction, of which C5a indeed is one of the candidates.

Summary and conclusions

The complement system is aimed to recognise pathogens and eliminate them. In addition, it provides dangers signals. Complement is essential in the defence 12 against certain infections, complement deficiency, of which MBL is the most frequent, predisposes to serious infections. On the other hand, during sepsis and septic shock, excessive systemic activation of complement contributes to the development of shock and organ failure. The anaphylatoxin C5a plays a key role in the pathogenesis of sepsis. Future therapies aimed at modulating the complement system should target this molecule or its receptor.

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193

Vascular endothelial growth factor is increased during the first 48 hours of human septic shock and correlates with vascular permeability. 13

Peter Pickkers, Tom Sprong, Lucas van Eijk, Hans van der Hoeven, Paul Smits and Marcel Deuren. Shock. 2005;24(6):508-512. Abstract

Meningococcal septic shock is an important cause of morbidity and mortality in children and young adults worldwide and is the prototypical gram-negative septic shock. One of the key factors in the development of shock is increased microvascular permeability. Vascular endothelial growth factor (VEGF) is a central factor in angiogenesis and is an important mediator of vascular permeability. Thirteen patients with meningococcal infection (eight presenting with shock) were investigated in the early phase of invasive meningococcal disease. Cytokines, complement activation, and VEGF plasma concentrations were measured during the first 48 h on the pediatric intensive care unit. Increased cytokine concentrations and activation of the complement system were observed. VEGF plasma concentrations were increased (median 193 pg/mL, range 71-1082) and were highest in the presence of shock (208 pg/mL, 169-1082) compared with patients presenting without shock (92 pg/ mL range 71-299). VEGF concentration at admission correlated with the severity of disease (pediatric risk of mortality score, R = 0.90 [Spearman], P = 0.0001) and the amount of fluids administered within the first 24 h (R = 0.90, P < 0.0001). In all patients, a decrease in VEGF was associated with a decrease in fluid intake during t = 24 to 48 h. The results suggest that apart from correlation with IL-1β, -10, -12, and complement activation, microvascular permeability in sepsis is also closely linked to the plasma concentration of VEGF. The role of VEGF in sepsis-associated increased microvascular permeability needs further exploration and may represent a new therapeutic target.

196 INTRODUCTION

Fulminant meningococcal sepsis is an important cause of childhood morbidity and mortality. The development of shock in meningococcal disease is caused by the activation of a range of mediators belonging to the cytokine system, and the coagulation and complement cascade. Activation of these mediators leads to increased microvascular permeability, vasoplegia, disseminated intravascular coagulation, and myocardial depression. Ultimately, this results in impaired tissue oxygenation, a critical factor in the development of multiple organ failure and limb ischemia 1. Excessive activation of the complement system in children with meningococcal shock was found to be related to outcome, severity of disease, and extent of capillary leakage 2, and the inflammation-induced loss of endothelial and basement membrane glycosaminoglycans during meningococcal sepsis may be causally correlated to the increase in permeability to proteins 3. In contrast to its clinical importance, the pathophysiology of increased microvascular permeability in human septic shock is only sparsely studied and is not a therapeutic target in today’s treatment.

Vascular endothelial growth factor (VEGF), first described in 1983 by Senger et al. as a secretory protein from hepatocarcinoma cells that increased extravasation of dye in the skin of guinea pigs, is a dimeric 46-kD endothelium-specific multifunctional cytokine that plays a pivotal role in microvascular permeability and angiogenesis 4 5. VEGF is synthesized and released by vascular smooth muscle cells, lung epithelium, platelets, leukocytes, and macrophages 6. VEGF is a key molecule in the control of vascular permeability as it is 20,000 times more potent in the induction of vascular permeability than histamine 7.

VEGF expression is influenced by cytokines and nitric oxide 8, and is found to be upregulated in a variety of pathologic conditions such as wound healing, myocardial ischemia, tumor growth, and endotoxemia 9. Recently, increased VEGF concentrations were described in patients with sepsis, but no correlation with a decline in serum albumin (as measure of vascular permeability) was found 10. Up to now, VEGF concentrations have not been measured in meningococcal sepsis, known for its profound effects on vascular permeability. 13

In view of the key role played by VEGF in regulating microvascular permeability, we measured VEGF concentrations in 13 children with invasive meningococcal infections, and correlated VEGF concentration with fluid balance as a measure of systemic vascular permeability.

197 MATERIALS AND METHODS

After approval of the local ethics committee and with informed consent of their parents, 13 previously healthy children (seven boys) with invasive meningococcal disease admitted to our pediatric intensive care unit (PICU) between September 2002 and January 2004 were included. Diagnosis was made based on positive culture of blood, cerebrospinal fluid, or skin biopsy, or typical presentation. Eight patients presented with septic shock, defined as hemodynamic instability irresponsive to fluid resuscitation and requiring sustained (>12 h) vasopressive support [dopamine and (nor)epinephrine; shock group]. One of these patients died 3.5 h after admission. Five patients presented without shock, four of them had meningitis, and one was identified as meningococcal bacteremia with purpura but without hemodynamic complications (no shock group). All five of these patients recovered completely. Time of disease onset was determined by taking a detailed disease history of the parents and noting the first symptom that appeared, e.g., malaise or fever. Disease severity was determined by the pediatric risk of mortality score (PRISM) based on the worst value of parameters during the first 24 h 11. Hemodynamic parameters, net fluid amount (the amount of fluids administrated within the first 24 h minus diuresis), and tumor necrosis factor-α (TNFa), and interleukin 1β (IL-1β), -10, and -12 were determined using a commercially available Bio-Plex protein array system (Bio-Rad Laboratories, Hercules, CA) with lower limits of detection of 8 to 16 pg/ mL. Complement activation was measured by an enzyme-linked immunoabsorbant assay (ELISA) specific for soluble C5b-9 (soluble terminal complement complex, sTCC) as described previously 12. VEGF was measured in EDTA plasma 0, 2, 8, 12, 24, and 48 h after admission to PICU by a commercially available ELISA (DuoSet ELISA development system; R&D Systems, Minneapolis, MN) with a lower limit of detection of 16 pg/mL. Also, VEGF levels were measured in 12 healthy controls (aged 23.1 ± 0.9 years).

Statistics Differences between groups were analyzed using a two-sided nonparametric Mann-Whitney test. Linear regression was calculated using Spearman’s correlation coefficient. Time-dependent changes were analyzed by analysis of variance (ANOVA) with repeated measures. A P value <0.05 was considered significant. Statistical analyses were performed with GraphPad Prism version 3.00 software (GraphPad, San Diego, CA).

RESULTS

Baseline characteristics of the patients are illustrated in Table 1. The shock patients

198 Table 1. Patient characteristics at admission ‘No Shock’ group ‘Shock’group P N 5 8 n.d. Age (years) 4.7 (0.6 - 5.6) 2.7 (0.6 - 5.5) 0.354 Time from disease 28 (19 - 100) 16.5 (13 - 21.5) 0.003 onset to ICU admission PRISM II 5 (0 - 10) 14 (6 - 35) 0.011 RR (MAP) 80 ( 40 - 88) 73 (54 - 87) 0.724 Heart rate (bpm) 145 (92 - 163) 162 (147 - 210) 0.065 Net Fluid amount 32 (0 - 80) 117 (55 - 484) 0.006 (ml/kg) CRP (mg/L) 147 (99 - 307) 82 (37 - 118) 0.008 Inotrope/ 2/5 (Dobu) 8/8 n.d. vasopressor therapy Mortality 0/5 1/8 n.d. Sequelae 0/5 1/7 (amputation) n.d. PRISM = pediatric risk of mortality score, MAP = mean arterial pressure, bpm = beats per minute, Net fluid amount is the amount of fluids administrated within the first 24 hours minus diuresis (in mL/kg body weight), CRP = C-reactive protein. Statistics by Mann-Whitney test, P < 0.05 is considered significant. n.d. means not determined. tended to be younger, but this difference was not significant. Time from onset of disease to admission was significantly shorter in the shock group, demonstrating a more fulminant course of disease. The patients in shock had a significantly higher disease severity score and a higher net fluid intake, indicating that a higher volume of fluids was necessary for resuscitation. MAP and heart rate did not differ significantly between the shock and no shock groups, reflecting adequate resuscitation of the shock patients before PICU admission. The inflammatory parameter CRP was lower in the shock group, whereas all proinflammatory cytokines were significantly higher. Taken together, these data show that our patients with meningococcal septic shock had a more rapidly progressing and severe systemic disease than patients without shock.

13 Figure 1. VEGF concentration (pg/mL, ELISA) in the plasma of eight patients presenting with meningococcal septic shock (shock), five patients with meningococcal meningitis (n = 4) or bacteremia (n = 1) without shock (no shock), and 12 healthy control subjects (controls). The horizontal line represents the median value of the group. †Patient who died. Significance was tested by two-sided Mann-Whitney test.

199 Figure 2. Plasma concentration of VEGF (pg/mL, ELISA) in patients with meningococcal disease presenting with shock or without shock from admission at PICU to 48 h after admission. Median and upper quartile are presented, n = 8 for the shock group at T = 0 and T = 2, and n = 6 for the shock group at subsequent time points. n = 5 for the patients without shock. The dotted line represents the upper limit of VEGF concentration of the control subjects. During the first 48 h, there was a significant decrease of VEGF in the shock patients (ANOVA repeated measures, P = 0.03), whereas the VEGF concentration in the no shock group remained unchanged.

Figure 3. Plasma concentration of VEGF (pg/mL, ELISA) in patients with meningococcal disease presenting with shock (•) or without shock ([white circle]) correlated with net fluid balance (R = 0.90, P < 0.0001). Data was analyzed without one outlier in the shock group, indicated by the arrow. Net fluid amount is the amount of fluids administrated within the first 24 h minus diuresis (in mL/kg body weight).

At presentation, patients with meningococcal septic shock had significantly higher VEGF plasma concentrations (median 208 pg/mL, range 169-1082) than the no shock patients (median 92 pg/mL, range 71-299, P = 0.03) or controls (median 39 pg/mL, range 25-55, P < 0.001; Figure 1). The patient with the highest VEGF concentration in the no shock group was the bacteremic patient who required no vasopressor support. During the first 48 h, VEGF levels decreased in the patients

Table 2. Spearman correlation coefficient for VEGF (for all patients). R (Spearman) P-value PRISM 0.90 0.0001 Net fluid amount 0.90 0.0001 Heart rate 0.75 0.0031 Mean arterial pressure (MAP) -0.33 0.27 CRP -0.57 0.05 IL-1β 0.82 0.0017 Leucocytes -0.49 0.1 Platelets -0.90 <0.0001 PRISM = pediatric risk of mortality score, MAP = mean arterial pressure, Net fluid amount is the amount of fluids administrated within the first 24 hours minus diuresis (in mL/kg body weight), CRP = C-reactive protein, IL-1β = interleukin 1β.

200 Table 3. Spearman correlation coefficient of cytokines and complement activation products with net fluid amount (for all patients). Admission Value R (Spearman) P-value median, [range] (pg/mL) TNFα 39 [8-726] 0.56 0.06 IL-1β 58 [8-304] 0.81 0.001 IL-10 5710 [14-13571] 0.91 <0.0001 IL-12 71 [8-3771] 0.91 <0.0001 TCC 2.8 [0.9-8.0] 0.71 0.01 Net fluid amount is the amount of fluids administrated within the first 24 hours minus diuresis (in mL/kg body weight). with shock (P = 0.03 with repeated-measures ANOVA), and remained unchanged in the patients without shock (Figure 2). As shown in Table 2, VEGF correlated well with the severity of disease expressed by the PRISM score, the activation of the cytokine network expressed by the IL-1β concentration, and the net amount of fluid administered within the first 24 h (Figure 3). As indicated in Figure 3, one outlier was excluded from the correlation analysis; however, with inclusion of this patient, the correlation remains significant (R = 0.79, P = 0.003). Table 3 illustrates that IL-1β, -10, and -12 and complement activation also correlated with fluid balance. The time-dependent decrease in VEGF in shock patients was associated with a significant decrease in fluid intake in all patients during the first 48 h (P = 0.03), however, the number of patients was too small to find a significant correlation between the decrease in VEGF and the decrease in positive fluid balance.

Finally, we found that leukocyte count was not correlated with VEGF concentrations (R = -0.49, P = 0.1), but a significant negative correlation was found for platelets (R = -0.90, P < 0.0001; see Table 2).

DISCUSSION

In vitro as well as in vivo in animals, VEGF is shown to be an important mediator of vascular permeability. Serum VEGF levels were recently found elevated in human sepsis, but no correlation with capillary leakage was found 10. In the present study, we confirmed previous results concerning the activation of cytokine network and the complement system during meningococcal sepsis, but more importantly, we 13 observed that the early phase of meningococcal septic shock is associated with increased concentrations of VEGF. ILs, complement activation, and VEGF levels all correlated with the net amount of fluid intake in these patients, indicating that in concert with various cytokines and complement, VEGF may mediate vascular permeability during sepsis. Moreover, cytokines, complement, and VEGF interact with each other in their permeability increasing effect 13,14.

201 The high VEGF concentrations at presentation, its decline during the first 48 h in shock patients, and the correlation with the proinflammatory cytokine IL-1β suggest that the early VEGF peak is induced by cytokines or directly by the meningococcal components itself. In accordance, it has been demonstrated that proinflammatory cytokines account for VEGF release from various tissues 8, whereas the effect on VEGF receptor density is unclear as up- 15 and down-regulation 16 have been reported. The source of VEGF is not clear. It is likely that activated leukocytes and thrombocytes are sequestered, and that these are the ones that release VEGF. In accordance, we found a negative correlation of VEGF concentrations with the platelet count. This can indicate that activated platelets are the source of VEGF. However, another possibility is that activated endothelial cells are responsible for the release of VEGF, as well as for the binding of the platelets.

In patients with bacterial meningitis, circulating VEGF concentrations were normal, but the VEGF concentrations were elevated in cerebrospinal fluid, indicating intrathecal production 17. Cryptococcal meningitis induced elevated VEGF concentrations in cerebrospinal fluid and plasma 18. In acute respiratory distress syndrome (ARDS), another form of inflammatory-mediated tissue damage, it was found that plasma VEGF concentrations were elevated and that peripheral blood mononuclear cells of these patients produced more VEGF in vitro than cells from the control group 19. Also, plasma of these patients increased endothelial permeability in vitro, an effect that could be inhibited by the addition of a soluble VEGF inhibitor. These studies indicate that VEGF is a potent inducer of inflammation-associated capillary leakage in critically ill patients. In accordance with our study, Van der Flier et al. 10 recently reported elevated circulating VEGF concentrations in severe septic patients. Because of the clinical entity of meningococcal disease, our patients represent a more homogeneous group of patients with sepsis, and the time from disease onset of meningococcal sepsis is more precise compared with sepsis from other causes. Also, meningococcal sepsis is especially known for its severe effect on capillary leakage. These factors may explain that a correlation of elevated VEGF levels with vascular permeability was found in our study, in contrast to the study of Van der Flier et al. 10.

Angiogenesis is a key component of the repair mechanisms triggered by tissue injury. The cellular expression of VEGF may be important for tissue repair during hypoxia, trauma, burns, and inflammatory diseases such as ARDS. In this context, VEGF may in fact be induced by the disease to promote tissue repair. In burn and trauma patients, markedly enhanced levels of serum VEGF are present 2 to 3 weeks after trauma or burn injury 20,21. Furthermore, the subgroup of patients with uncomplicated healing showed significantly higher increases of serum VEGF than

202 the subgroup who developed severe complications during the post-traumatic course, such as sepsis, ARDS, or multiple organ failure 20. Because occurrence of severe complications during the post-traumatic period was associated with lower levels of serum VEGF, one might argue that VEGF is necessary rather than deleterious. It is of evident importance to notice that the VEGF-mediated repair mechanisms (angiogenesis) appear to coincide with the VEGF peak 2 to 3 weeks after the insult. In contrast, it is possible that early during sepsis, VEGF mediates an increase in vascular permeability, explaining the correlation with a positive fluid balance we found in this study. These apparent contradictory effects may be explained by the absence or presence of angiopoietin-1. Like VEGF, angiopoietin-1 is important during vascular development, but acts reciprocally as an antipermeability factor. VEGF alone causes an increase in vascular permeability, whereas simultaneous transgenic overexpression or administration of angiopoietin-1 22,23 normalized this effect. Combining these results, it is tempting to speculate that the early VEGF- induced increase in vascular permeability may be deleterious, whereas the later peak is favorable in the healing process.

Therapeutic opportunities During gram-negative septic shock, at least three pathophysiologic processes occur: increased capillary leakage, disseminated intravascular coagulation, and myocardial depression contribute to the development of shock and multiple organ failure. Although an increased microvascular permeability is frequently encountered and is an important cause of multiple organ failure in patients with sepsis, it is only sparsely studied in humans. At present, there is no therapy available directed specifically against the sepsis-induced increase in microvascular permeability. Agents successfully used in animal models are not effective or are not applicable in humans 24-27.

Recently, an anti-VEGF antibody became available for human use 28. Administration of the anti-VEGF antibody bevacizumab in patients with metastatic renal cancer was associated with only mild asymptomatic side effects. The recent findings that inflammation-mediated capillary leakage is associated with elevated VEGF concentrations is of particular interest as VEGF may be considered as a new therapeutic target for the treatment with anti-VEGF antibodies. However, blocking 13 VEGF during critical illness might devoid patients from the positive effects of VEGF during the chronic phase. Because mainly the later peak of VEGF seems to be beneficial, blocking VEGF in the early phase of sepsis could be considered. However, before this hypothesis can be tested, more research to obtain Koch’s postulates regarding disease causation is needed.

203 In conclusion, we found that the positive fluid balance of patients in meningococcal septic shock correlates with increased plasma concentrations of VEGF. Further efforts should be made to confirm that VEGF mediates vascular permeability in meningococcal septic shock and other types of human sepsis and to establish whether modulation of VEGF release may be beneficial in the treatment of inflammation-induced capillary leakage.

204 REFERENCES 1. Van Deuren M, Brandtzaeg P, Van der Meer JWM. Update on meningococcal disease with emphasis on pathogenesis and clinical management. Clin Microbiol Rev. 2000;13:144- 166 2. Hazelzet JA, de Groot R, van Mierlo G, Joosten KFM, van der Voort E, Eerenberg A, Suur MH, Hop WCJ, Hack CE. Complement activation in relation to capillary leakage in children with septic shock and purpura. Infect. Immun. 1998;66:5350-5356 3. Oragui EE, Nadel S, Kyd P, Levin M. Increased excretion of urinary glycosaminoglycans in meningococcal septicemia and their relationship to proteinuria. Crit Care Med. 2000;28:3002-3008 4. Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science. 1983;219:983-985 5. Ferrara N. Molecular and biological properties of vascular endothelial growth factor. J Mol Med. 1999;77:527-543 6. Berse B, Brown LF, Van de Water L, Dvorak HF, Senger DR. Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors. Mol Biol Cell. 1992;3:211-220 7. Dvorak HF, Brown LF, Detmar M, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol. 1995;146:1029-1039 8. Dulak J, Jozkowicz A, Dembinska-Kiec A, Guevara I, Zdzienicka A, Zmudzinska- Grochot D, Florek I, Wojtowicz A, Szuba A, Cooke JP. Nitric oxide induces the synthesis of vascular endothelial growth factor by rat vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2000;20:659-666 9. Mittermayer F, Pleiner J, Schaller G, Weltermann A, Kapiotis S, Jilma B, Wolzt M. Marked increase in vascular endothelial growth factor concentrations during Escherichia coli endotoxin-induced acute inflammation in humans. Eur J Clin Invest. 2003;33:758-761 10. van der Flier M, van Leeuwen HJ, van Kessel KP, Kimpen JL, Hoepelman AI, Geelen SP. Plasma vascular endothelial growth factor in severe sepsis. Shock. 2005;23:35-38 11. van Brakel MJ, van Vught AJ, Gemke RJ. Pediatric risk of mortality (PRISM) score in meningococcal disease. Eur J Pediatr. 2000;159:232-236. 12. Mollnes TE, Lea T, Froland SS, Harboe M. Quantification of the terminal complement complex in human plasma by an enzyme-linked immunosorbent assay based on monoclonal antibodies against a neoantigen of the complex. Scand J Immunol. 1985:197- 202 13. Clauss M, Sunderkotter C, Sveinbjornsson B, Hippenstiel S, Willuweit A, Marino M, Haas E, Seljelid R, Scheurich P, Suttorp N, Grell M, Risau W. A permissive role for tumor necrosis factor in vascular endothelial growth factor-induced vascular permeability. Blood. 2001;97:1321-1329 14. Barton PA, Warren JS. Complement component C5 modulates the systemic tumor necrosis factor response in murine endotoxic shock. Infect Immun. 1993;61:1474-1481 15. Girardin E, Grau GE, Dayer J-M, Roux-Lombard P, The, J5, Study, Group, Lambert PH. Tumor necrosis factor and interleukin-1 in the serum of children with severe infectious purpura. N Engl J Med. 1988;319:397-400 16. Patterson C, Perrella MA, Endege WO, Yoshizumi M, Lee ME, Haber E. Downregulation of vascular endothelial growth factor receptors by tumor necrosis factor-alpha in cultured 13 human vascular endothelial cells. J Clin Invest. 1996;98:490-496 17. van der Flier M, Stockhammer G, Vonk GJ, Nikkels PG, van Diemen-Steenvoorde RA, van der Vlist GJ, Rupert SW, Schmutzhard E, Gunsilius E, Gastl G, Hoepelman AI, Kimpen JL, Geelen SP. Vascular endothelial growth factor in bacterial meningitis: detection in cerebrospinal fluid and localization in postmortem brain. J Infect Dis. 2001;183:149-153 18. Coenjaerts FE, van der Flier M, Mwinzi PN, Brouwer AE, Scharringa J, Chaka WS, Aarts M, Rajanuwong A, van de Vijver DA, Harrison TS, Hoepelman AI. Intrathecal production and secretion of vascular endothelial growth factor during Cryptococcal Meningitis. J

205 Infect Dis. 2004;190:1310-1317 19. Thickett DR, Armstrong L, Christie SJ, Millar AB. Vascular endothelial growth factor may contribute to increased vascular permeability in acute respiratory distress syndrome. Am J Respir Crit Care Med. 2001;164:1601-1605 20. Grad S, Ertel W, Keel M, Infanger M, Vonderschmitt DJ, Maly FE. Strongly enhanced serum levels of vascular endothelial growth factor (VEGF) after polytrauma and burn. Clin Chem Lab Med. 1998;36:379-383 21. Infanger M, Schmidt O, Kossmehl P, Grad S, Ertel W, Grimm D. Vascular endothelial growth factor serum level is strongly enhanced after burn injury and correlated with local and general tissue edema. Burns. 2004;30:305-311 22. Thurston G, Suri C, Smith K, McClain J, Sato TN, Yancopoulos GD, McDonald DM. Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science. 1999;286:2511-2514 23. Thurston G, Rudge JS, Ioffe E, Zhou H, Ross L, Croll SD, Glazer N, Holash J, McDonald DM, Yancopoulos GD. Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med. 2000;6:460-463 24. Ravage ZB, Gomez HF, Czermak BJ, Watkins SA, Till GO. Mediators of microvascular injury in dermal burn wounds. Inflammation. 1998;22:619-629 25. Eibl G, Forgacs B, Hotz HG, Buhr HJ, Foitzik T. Endothelin A but not endothelin B receptor blockade reduces capillary permeability in severe experimental pancreatitis. Pancreas. 2002;25:e15-20 26. Nunes FB, Simoes Pires MG, Alves Filho JC, Wachter PH, Rodrigues De Oliveira J. Physiopathological studies in septic rats and the use of fructose 1,6-bisphosphate as cellular protection. Crit Care Med. 2002;30:2069-2074 27. Gautam N, Olofsson AM, Herwald H, Iversen LF, Lundgren-Akerlund E, Hedqvist P, Arfors KE, Flodgaard H, Lindbom L. Heparin-binding protein (HBP/CAP37): a missing link in neutrophil-evoked alteration of vascular permeability. Nat Med. 2001;7:1123-1127 28. Yang JC, Haworth L, Sherry RM, Hwu P, Schwartzentruber DJ, Topalian SL, Steinberg SM, Chen HX, Rosenberg SA. A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med. 2003;349:427-434

206 13

207

Tom Sprong, Peter Pickkers, Anneke Geurts-Moespot, Johanna van der Ven-Jongekrijg, Chris Neeleman, Marlies Knaup, Didier Leroy, Thierry Calandra, Jos W.M. van der Meer, Fred Sweep and Marcel van Deuren. Shock. 2007;27(5):482-487.

Macrophage migration inhibitory factor (MIF) in meningococcal septic shock and experimental human endotoxemia. 14 ABSTRACT

Macrophage migration inhibitory factor (MIF) is a mediator of innate immunity and important in the pathogenesis of septic shock. Lipopolysaccharide (LPS) and tumor necrosis factor (TNF)α are reported to be inducers of MIF. We studied MIF and cytokines in vivo in patients with meningococcal disease, in human experimental endotoxemia, and in whole blood cultures using a newly developed sensitive and specific enzyme-linked immunosorbent assay. Twenty patients with meningococcal disease were investigated. For the human endotoxemia model, 8 healthy volunteers were intravenously injected with 2 ng/kg Escherichia coli LPS. Whole blood from healthy volunteers was incubated with LPS or heat-killed meningococci. Macrophage migration inhibitory factor concentration in blood was increased during meningococcal disease and highest in the patients presenting with shock compared with patients without shock. Plasma concentration of MIF correlated with disease severity, the presence of shock and with the cytokines interleukin (IL) 1β, IL-10, IL-12, and vascular endothelial growth factor, but not with TNF-α. MIF was not detected in blood in experimental endotoxemia, nor after stimulation of whole blood with LPS or meningococci, although high levels of TNF-α were seen in both models. In conclusion, MIF is increased in patients with meningococcal disease and highest in the presence of shock. Macrophage migration inhibitory factor cannot be detected in a human endotoxemia model and is not produced by whole blood cells incubated with LPS or meningococci.

210 INTRODUCTION

Macrophage migration inhibitory factor (MIF), discovered more than 40 years ago 1,2, has recently emerged as a major mediator of the inflammatory response. In in vitro and animal models, MIF is induced by bacterial components such as endotoxin [lipopolysaccharide (LPS)] 3,4 and proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) 3, and is expressed in macrophages, T cells, cells in the anterior pituitary gland, and other tissues throughout the body 5. Once released, MIF regulates the inflammatory response by modulating apoptosis 6, influencing the carbohydrate metabolism 7, activating macrophages and T cells 8,9, and by counteracting the anti-inflammatory actions of glucocorticoids on immune cells 8. In addition, MIF has recently been shown to modulate the expression of Toll- like receptor 4, an integral part of the LPS receptor complex 10.

Because of its important immunoregulatory function, MIF has been extensively studied in murine models of sepsis, and it was shown that targeted disruption of the MIF gene or neutralization of MIF by anti-MIF antibodies (Abs) results in increased survival in these models 11-13. This suggests that MIF is a player in the pathogenesis of septic shock, together with the cytokine network and the complement, coagulation, fibrinolytic and kinin/kallikrein systems.

Meningococcal disease is caused by bloodstream invasion of the Gram-negative pathogen Neisseria meningitidis and presents as meningococcal meningitis or bacteremia, with a relatively benign clinical picture and low mortality, or as meningococcal septic shock, an overwhelming septic shock syndrome with a high morbidity and mortality 14. Because it strikes mostly previously healthy children and the time from start of symptoms to clinically overt disease is usually short, it has been termed the prototypical Gram-negative sepsis, and functions as an excellent model for studying sepsis. In the human situation, increased plasma concentrations of MIF have been found in intensive care unit (ICU) patients with diverse types of Gram-negative, Gram-positive, or polymicrobial sepsis 13,15-18. However, MIF in meningococcal disease and the interplay between endotoxin (LPS), cytokines, and MIF in the human situation has not been studied. Therefore, we used a newly developed specific and sensitive enzyme-linked immunosorbent assay (ELISA) method to determine the plasma concentrations of MIF and the relation of MIF to cytokine production in meningococcal disease, in experimental human endotoxemia, 14 and ex vivo in whole blood incubated with LPS or meningococci.

211 PATIENTS AND METHODS Ethics All studies were performed with approval of the local ethics committee, and informed consent was obtained for all patients and volunteers.

Meningococcal disease Patients admitted to our pediatric intensive care unit (PICU) between September 2002 and January 2006 with invasive meningococcal disease were included; diagnosis was made based on positive culture of blood, cerebrospinal fluid or skin biopsy, or typical presentation. Patients were admitted after first being treated in a secondary care hospital (n = 17; delay between presentation on PICU and first admission, 3.4 h; range, 1.8-6 h) or referred directly to our hospital (n = 3). Septic shock was defined as hemodynamic instability irresponsive to fluid resuscitation and requiring sustained (>12 h) vasopressive support [dopamine or (nor-)epinephrine]. Two patients died, both within 8 h after admission. All patients received antibiotic therapy (ceftriaxone, i.v.) and i.v. fluids at first admission and 4 × 0.15 mg/kg dexamethasone during the first 3 days of admission. EDTA-anticoagulated blood was sampled serially at admission to PICU (t = 0) and at t = 2, t = 8, t = 24 and t = 48 h after admission when possible. Heparin-anticoagulated blood was obtained at admission (t = 0) for determination of cytokines in all 20 patients. Data on vascular endothelial growth factor (VEGF) were obtained for 13 patients.

Human endotoxemia Fourteen healthy adult human volunteers aged 18 to 25 years (7 men) and checked by routine medical examination, laboratory testing, and electrocardiography participated in the study. During the experiment, vital signs were continuously monitored. All subjects received i.v. infusion of glucose/saline solution (2.5% glucose, 0.45% NaCl, 75 mL/h). At t = 0 h, a bolus of 2 ng/kg body weight Escherichia coli 0113 LPS (batch O:113, United States Pharmacopia Convention, Rockville, Md) was injected intravenously in 1 to 2 min in 8 of the subjects (endotoxin group); 6 subjects received an infusion of 10 mL of physiological saline (0.9% NaCl, control group). EDTA-anticoagulated blood for determination of MIF and VEGF concentration and heparin anticoagulated blood for cytokines were obtained just before infusion and serially thereafter at regular time intervals up to 22 h after infusion of LPS. Blood was centrifuged at 2800g for 15 min and stored at -80°C until analysis of the parameters in 1 batch. Baseline MIF values of these subjects were used as control for comparison with the MIF concentration in patients with meningococcal disease.

212 Whole blood model of sepsis Heparin-anticoagulated whole blood was collected by venapuncture from 8 healthy adult volunteers and incubated for 20 h at 37°C in sterile, endotoxin-free cryotubes (NUNC, Roskilde, Denmark) with one of the following substances: E. coli B55:05 LPS (Sigma Chemical Co, St Louis, Mo), N. meningitidis H44/76 [an international reference strain isolated from a case of meningococcal disease 19 heat-killed (HK) for 1 h at 56°C before use], and N. meningitidis H44/76 LPS (isolated and purified from strain H44/76 by phenol extraction 20. Both N. meningitidis H44/76 and the neisserial LPS were a kind gift from Dr. Peter van der Ley (Netherlands Vaccine Institute, Bilthoven, the Netherlands).

Measurements An ELISA for human MIF (hMIF) was developed by our group according to the 4-span approach, described in detail by Grebenschikov et al. 21,22. Antibodies were raised in chicken and rabbits using rhMIF as an immunogen. The sandwich structure used includes 4 different Abs, a coating Ab (duck anti-chicken) a capture Ab (chicken anti-hMIF), a trapping Ab (rabbit anti-hMIF), and finally, a detection Ab (HRP-labeled goat antirabbit). The procedure started with treating the microtiter plates with coating Ab overnight at 4°C. The next step was the incubation with capture Ab for 2 h at 37°C. Incubation with patient samples, references, and standards was performed at 4°C overnight. Hereafter, trapping and detection Abs were sequentially incubated for 2 h at room temperature. Plates were developed with substrate solution in the dark for 30 min at room temperature, color reaction was stopped with H2SO4, and optical density was measured at 492 nm within 30 min. In each run, interassay variability and performance were checked using a reference preparation (23). The analytical sensitivity for hMIF is 39 pg/mL. Precision profiling showed a coefficient of variation (CV) of 20% at 45 pg/mL (i.e., functional level) decreasing to 7% at higher concentrations. For estimation of the accuracy of the method, a lyophilized reference preparation (marked 140799) is used. The mean hMIF concentration in this preparation was 20.7 ng/mL, whereas the within- run CV and between-run CV amounted to 6% (n = 8) and 12% (n = 11, over a period of 13 months), respectively.

Interleukin (IL) 1β, IL-8, IL-12, IL-10, TNF-α, and interferon-γ (IFN-γ) were measured using commercially available Bio-Plex protein array system (Bio-Rad Laboratories, Inc, Hercules, Calif) 23. Lower limits of detection were 8 pg/mL for IL-1β, IL-8, IL- 14 12, IL-10, and TNF-α, and 32 pg/mL for IFN-γ. Vascular endothelial growth factor was measured using a commercially available ELISA (DuoSet ELISA development system; R&D Systems, Minneapolis, Minn) with a lower limit of detection of 16 pg/ mL.

213 Statistics Differences were tested for statistical significance by 2-sided Mann-Whitney test. Correlation was calculated by Spearman rank test. GraphPad PRISM version 3.00 software was used for the calculations. P < 0.05 was considered significant.

RESULTS MIF and cytokines in patients with meningococcal disease Patients were divided in 2 groups: patients with meningococcal septic shock (n = 12) and patients without shock (in this group, 6 patients presented with meningitis and 2 patients with meningococcal bacteremia), illustrated in Table 1. The shock group experienced a more fulminant and severe disease, reflected by a higher mortality and more sequelae, a shorter time from disease onset to ICU admission, and a higher PRISM score 24,25. The presence of shock is demonstrated by the sustained requirement for vasopressors, the higher heart rate, and the higher amount of i.v. fluids needed during resuscitation in the first 24 h. Plasma concentrations of the cytokines IL-1β, IL-12, IL-10, VEGF, and TNF-α were significantly higher in the shock group compared with the patients without shock. There was no significant difference for IL-8 and IFN-γ.

Table 1. Characteristics of patients presenting with or without shock during meningococcal disease. no shock shock P N 8 12 Male/female 4/4 6/6 Age (years) 3.1 (0.6 - 5.6) 2.6 (0.6 - 5.5) .671 Mortality 0/8 2/8 Sequelae 0/8 2/8 Culture positive 5 (62.5%) 10 (83%) Time from disease onset 26.5 (16 - 100) 16.5 (12.5 - 21.5) .028 to ICU admission (h) PRISM 5 (0 - 13) 16.5 (6 - 35) .003 MAP (mmHg) 78 ( 40 - 93) 68 (30 - 93) .724 HR (bpm) 142 (92 - 188) 162 (147 - 226) .031 Net Fluid Therapy (ml/kg) 57.5 (0 - 212) 131 (55 - 484) .01 IL-1β (ng/mL) 0.017 (0.008 – 0.175) 0.04 (0.008 – 0.125) .017 IL-12 (ng/mL) 0.008 (0.008 – 0.087) 0.112 (0.008 – 0.365) .009 IL-10 (ng/mL) 0.634 (0.014 – 21.97) 4.109 (0.89 – 30.73) .010 VEGF (ng/mL) 0.091 (0.071 – 0.299) 0.208 (0.169 – 1.082) .03 TNFα (ng/mL) 0.008 (0.008 – 0.093) 0.040 (0.008 – 0.125) .012 IL-8 (ng/mL) 0.017 (0.008 – 0.175) 4.23 (0.008 – 3.20) .080 IFNγ (ng/mL) 0.100 (0.032 – 2.684) 0.270 (0.106 – 1.423) .211 Where appropriate, median and range is presented. Differences were tested for significance using Mann-Whithney test. culture positive = culture of blood, cerebrospinal fluid or skin biopsy positive for N. meningitidis PRISM = Pediatric Risk of Mortality, MAP = mean arterial pressure, HR = heart rate, bpm = beats per minute. Net fluid therapy indicates the amount of fluids infused during resuscitation and in the first 24 hours subtracted with diuresis. Cytokines were measured at admission.

214 FIGURE 1

Patients with meningococcal disease had increased plasma concentrations of MIF at admission to the PICU (median, 70.2 ng/mL; range, 6.2-347 ng/mL) in comparison to healthy adult controls. Macrophage migration inhibitory factor plasma concentration was higher at admission (P = 0.02) and 2 h after admission (P = 0.08) in the patients with shock as compared with the patients without shock, but there was a very large variation in MIF concentration in this group (Figure 1). MIF plasma 400 P < .0001 concentration in theP = shock.02 group decreased within 8 h to concentrations similar to

those300 seen in the patients without shock. In the ensuing 24 to 48 h, MIF plasma concentration remained higher than seen in normal controls for both the shock and P = .007 no-shock200 groups (Figure 2). MIF ng/mL Macrophage100 migration inhibitory factor at admission significantly correlated with disease severity in all patients with meningococcal disease (as defined by FIGURE 1 PRISM0 score) and the presence of shock (heart rate and the amount of fluids shock no control needed for resuscitation)shock (Table 2). Macrophage migration inhibitory factor plasma concentration at admission significantly correlated with plasma concentrations of IL-1β, IL-10, IL-12, and VEGF at admission. No significant correlation was observed between MIF and IL-8, TNF-α, or IFN-γ (Table 3).

400 P < .0001 P = .02

1 300 P = .007 FIGURE 2 200 Figure 1.

MIF ng/mL MIF at admission in meningococcal disease. 100 Macrophage migration inhibitory factor plasma concentration at admission in patients with meningococcal disease presenting with shock (shock, n =12), without shock (no shock, n = 8), 0 and healthy control subjects (n = 14). Differences shock no control were tested for significance using Mann-Whitney shock test. † indicates the patients that died.

400 P = .02

P = .08

300 )

L P = .33

/ g

n 200 (

P = 1.0

F P = .76 I

M Figure 2. 1 100 Time-course of MIF in meningococcal disease. 14 Macrophage migration inhibitory factor plasma concentration from admission to 48 h after FIGURE 2 0 admission in patients with meningococcal disease 0 2 8 24 48 presenting with shock (dashed boxes) or without shock (white boxes). Box-whisker plots indicate Time after admission (h) medians, 25 and 75 percentiles, and ranges.

215

400 P = .02

P = .08

2 300 )

L P = .33

/ g

n 200 (

P = 1.0

F P = .76

I M 100

0 0 2 8 24 48 Time after admission (h)

2 Table 2. Correlation of MIF plasma concentration at admission with disease severity and markers of shock R (Spearman) P PRISM 0.62 .004 MAP - 0.22 .34 HR (bpm) 0.74 < .001 Net Fluid therapy 0.69 < .001 Correlation of MIF plasma concentration at admission with disease severity and markers of shock. PRISM = Pediatric Risk of Mortality, MAP = mean arterial pressure, HR = heart rate, bpm = beats per minute. Net fluid therapy indicates the amount of fluids infused during resuscitation and in the first 24 hours subtracted with diuresis.

Table 3. Correlation of MIF plasma concentration at admission with plasma concentration of cytokines and VEGF at admission R (Spearman) P IL-1β 0.53 .016 IL-12 0.56 .010 IL-10 0.59 .006 VEGF 0.73 .005 TNFα 0.375 .103 IL-8 0.21 .370 IFNγ 0.17 .482

Cytokines and MIF in experimental human endotoxemia and in a whole blood in vitro model of sepsis To define more closely the pathophysiological mechanism leading to production of MIF during meningococcal sepsis, we studied MIF in experimental human endotoxemia and in an in vitro whole blood model of sepsis.

Infusion of 2 ng/kg E. coli LPS in healthy volunteers induced significant clinical symptoms such as nausea, pyrexia, increased heart rate, and a drop in blood pressure. LPS infusion did not affect MIF plasma concentrations. In contrast, TNF-α peaked 1.5 h after LPS infusion, and a small increase in plasma concentrations of IL-1β and IL-10 was seen 2 to 3 h after infusion (Figure 3).

Incubation of human whole blood with E. coli LPS, LPS isolated from N. meningitidis, or N. meningitidis HK bacteria did not induce MIF production, whereas increased concentrations of TNF-α were found (Figure 4). Lower concentrations of LPS or bacteria (up to 1 ng/mL or 105 bacteria) did also not induce MIF production (data not shown). MIF in control samples of unstimulated heparinized plasma differed from that in EDTA plasma (62 vs. 10 ng/mL, P < 0.001).

216 FIGURE 3

25 1.2 MIF TNFααα 20 1.0

0.8

) )

L 15

m /

/ 0.6

n n

( 10 ( 0.4 5 0.2

FIGURE 3 FIGURE 3 0 0 0 6 12 18 24 30 25 25 1.2 0 6 121.2 18 24 30 MIF Time afterMIF endotoxin infusionTNF (h)ααα TimeTNF afterααα endotoxin infusion (h) 1.0 20 20 1.0 0.1 0.8 0.1

) 0.8 βββ )

) IL-1 IL-10 )

L 15 L

L 15

/ m

/ 0.08 0.6 /

g 0.08

0.6 g

n (

n 10

( n

( 10 (

) 0.4 )

L 0.060.4

L 0.06 m

5 m /

5 0.2 / g

0.2 g

n n

( 0.04 0.04 ( 0 0 0 0 0 60.02 12 18 24 30 0 6 120.02 18 24 30 0 6 12 18 24 30 0 6 12 18 24 30 Time after endotoxin infusion (h) Time after endotoxin infusion (h) Time after endotoxin infusion (h) Time after endotoxin infusion (h) 0 0 0.1 0 6 120.1 18 24 30 0 6 12 18 24 30 0.1 0.1 IL-1βββ IL-10 IL-1βββ Time after endotoxin infusionIL-10 (h) Time after endotoxin infusion (h) 0.08 0.08

0.08 0.08

) )

L 0.06

)

/ L

0.06 / L 0.06

g 1

n /

( 0.04 0.04

(

n n

( 0.04 0.04 ( 0.02 FIGURE 4 0.02 0.02 0.02 0 0 0 0 6 120 18 24 30 0 6 12 18 24 30 0 6 12 18Time 24 after 30 endotoxin infusion0 (h) 6 12200 18Time 24 after 30 endotoxin infusion (h) Time after endotoxin infusion (h) Time after endotoxin infusionMIF (h) 150 Figure 3. 1 MIF and cytokines in experimental human endotoxaemia. 1 Macrophage migration inhibitory factor, TNF-α, IL-1β, and100 IL-10 plasma concentration at baseline (T = 0)

and at subsequent time points up to 22 h after i.v. injectionng/mL of a bolus of 2 ng/kg E. coli LPS (endotoxin FIGUREgroup, 4 n = 8, open squares, pyramids or circles) or healthy volunteers with saline (control group, n = 6, closed circles, only shown for MIF, TNF-α, IL-1β, and IL-1050 were below the lower limit of detection in the FIGURE 4 control group). Median values ± interquartile range are presented. 0 E. coli N. men N. men 200 control MIF LPS LPS 200 150 40 *** MIF TNFααα 150 100 30 ng/mL *** 100 50 20 ng/mL ng/mL *** 50 0 10 control E. coli N. men N. men LPS LPS 0 0 control E. coli N. men40 N. men control***E. coli N. men N. men TNFααα LPS LPS LPS LPS 30 40 *** TNFααα 14 *** Figure30 4. 20

MIF and TNF-α in an in vitro wholeng/mL blood model of sepsis. Macrophage migration inhibitory factor and TNF-α concentration*** after incubation of whole blood with 1 µg/ mL of20 E2. coli LPS, 1 µg/mL or N. meningitidis***10 (N. men) LPS, N. meningitidis bacteria (1 × 109/mL), or PBS

(control)ng/mL for 20 h. n = 8 for hMIF, n = 7 for TNF-α. Medians and upper quartiles are presented. ***P < 0.001 for the comparison between*** sample and control. 10 0 control E. coli N. men N. men LPS LPS 0 2 E. coli N. men N. men control 217 LPS LPS

2 2

2 DISCUSSION

In the present study, we showed that MIF was increased in patients with meningococcal disease. The highest plasma concentrations of MIF were seen in the patients with shock. Not withstanding the very high cytokine levels reached, MIF was not detected in blood after infusion of endotoxin in humans-neither is MIF induced by incubation of whole blood with LPS or N. meningitidis HK bacteria.

In the patients with meningococcal disease described in this study, plasma concentrations of MIF at admission to PICU were increased up to 20-fold in comparison with the control subjects, which is in line with earlier studies in adults with mixed types of sepsis or SIRS 13,15-17. Macrophage migration inhibitory factor remained increased in the patients with meningococcal disease up to 48 h after admission, which corresponds to the results obtained by Gando et al. 16. Although the control group was older than the patients studied, we believe these conclusions can be drawn because in a previous study, no effect of age on MIF plasma concentration was found, and MIF levels in our control group are even higher as reported in a control group for a study of MIF in children with malaria 26,27.

In patients with meningococcal septic shock, MIF plasma concentration was higher during the first 2 h after admission compared with the patients without shock and was correlated with several parameters of shock. This observation suggests that MIF plays a role in the development of shock in patients with sepsis, which must be mediated in concert with other pathological events because infusion of recombinant MIF into mice alone did not induce lethality by itself but rather potentiates the effect of endotoxemia or sepsis 11. A well-known mechanism that contributes to fluid-refractory septic shock is relative adrenal insufficiency, a situation wherein the adrenal glands synthesize less endogenous glucocorticoids than is needed in the stress situation of sepsis 17,28. This relative deficiency of glucocorticoids can lead to vasopressor hyporesponsiveness 29. Because MIF antagonizes the effects of endogenous glucocorticoids, it seems possible that this overriding effect of MIF on glucocorticoid action 5 exacerbates the relative adrenal insufficiency observed in patients with septic shock. Indeed, in a previous study, the increase in cortisol production is matched by the increase in MIF production in patients with sepsis. In this way, MIF is able to antagonize the effect of endogenous glucocorticoids 17. Another mechanism by which MIF can contribute to the development of shock is by modulation of VEGF production. We recently reported that VEGF is a mediator of increased vascular permeability in meningococcal disease 30. In the present study, MIF plasma concentration at admission was strongly correlated with VEGF, suggesting a link between VEGF and MIF in the development of shock. Indeed,

218 preclinical studies have shown that MIF induces VEGF production in tumor cell lines, and that MIF and VEGF plasma concentrations are correlated in patients with cancer 31,32.

After admission, the increased concentration of MIF in the shock patients rapidly declines to concentrations that are also seen in less severely ill patients. In one of the fatalities, MIF declined between 0 and 2 h after admission (164 -> 115 ng/mL). In the other patient that died, MIF increased in the first 2 h after admission (172 -> 302 ng/mL). The quick decline in MIF plasma concentrations in the patients with shock can reflect the normal clearance of MIF from the circulation after successful antimicrobial therapy. Alternatively, this decline can possibly also be accounted for by the administration of corticosteroids in our patients because it was recently discovered that corticosteroids normalize leukocyte production of MIF during human sepsis 33, and in a murine cecal ligation and puncture model of sepsis, administration of dexamethasone led to a decrease in MIF plasma concentration 34. After 8 h, MIF plasma concentration in the patients with septic shock is comparable with that in the patients without shock. Because none of the patients without shock died or had severe sequelae, it can be questioned whether this relatively small increase of MIF found more than 8 h after admission in the shock group is of importance for the clinical outcome in meningococcal disease.

To define more closely the pathophysiological mechanism leading to production of MIF during sepsis, we studied MIF during experimental human endotoxemia and in whole blood. In the first in vivo model, E. coli LPS, a component of Gram-negative bacteria and important in the pathogenesis of septic shock, is infused as a bolus. It is a widely accepted model to study acute inflammatory responses during Gram- negative sepsis 35-38. Reportedly, MIF is induced by LPS and the proinflammatory cytokines TNF-α and IFN-γ 3,4,8. In the human endotoxemia model, however, we found no MIF induction up to 22 h after LPS infusion, although high peak plasma concentrations of TNF-α did occur 1.5 h after infusion. Correlation of MIF to cytokines found in meningococcal disease showed that MIF did not correlate with TNF-α, whereas MIF positively correlated with IL-1β, IL-10, and IL-12. These data suggest that LPS or TNF-α are not main driving forces for MIF induction during Gram-negative sepsis. Support for this is found in the observation that MIF is also increased in nonseptic inflammatory diseases and after stimulation with Gram- positive bacteria 39-41. Possibly, other cytokines such as IL-1β or other inflammatory 14 compounds such as C5a are responsible for the induction of MIF 42 or may be necessary in concert with LPS or TNF-α to induce MIF. In addition, components of Gram-negative bacteria other than LPS can be responsible for the induction of MIF because it has been shown that an inflammatory cytokine response and

219 complement activation by meningococci can occur also in the absence of LPS 20,43.

In human whole blood cultures, we attempted to induce MIF by incubation with LPS or HK meningococci, as was recently published for isolated mononuclear cells 33. At concentrations of LPS or meningococci ranging from 1 ng LPS/mL to 1 µg LPS/ mL or 106 meningococci per milliliter to 109 meningococci per milliliter, and in the presence of high levels of TNF-α, no MIF production was seen in whole blood. The concentrations of LPS and meningococci in the whole blood experiments were chosen to match the situation during meningococcal sepsis. Previously, it has been found that during meningococcal septic shock, plasma concentrations of bacteria are median 2 × 107/mL. Highest concentrations of bacteria found were 5.4 × 108, with maximal concentrations of LPS up to 215 ng/mL 44-46. Thus, the concentrations we used in the experiments are clinically relevant. In heparinized human whole blood, high background levels of MIF were observed, higher than in EDTA-anticoagulated samples. It is uncertain whether this is an effect of the ELISA method used or that it signifies MIF release by human blood cells independently of a stimulus. It is likely that MIF is produced in other tissues such as the liver, the spleen, and the kidney by resident macrophages and by other tissues such as the heart 47 and, more specifically for MIF, the anterior pituitary gland 5.

In conclusion, MIF concentration in blood was increased in patients with meningococcal disease, where it may play a role in the development of shock, acting in concert with other cytokines. Macrophage migration inhibitory factor plasma concentration did not correlate with TNF-α. No MIF production was observed in experimental human endotoxemia. In addition, MIF was not produced by human whole blood cultures after challenge with LPS or meningococci. Therefore, it can be questioned whether MIF in human sepsis is induced by LPS or TNF-α in the blood compartment.

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221 LPS to proinflammatory cytokine response. J Leukoc Biol. 2001;70:283-288. 21. Grebenschikov N, Geurts-Moespot A, De Witte H, Heuvel J, Leake R, Sweep F, Benraad T. A sensitive and robust assay for urokinase and and tissue-type plasminogen activators (uPA and tPA) and their inhibitor type 1 (PAI-1) in breast tumor cytosols. Int J Biol Markers. 1997;12:6-14 22. Grebenschikov N, Sweep F, Geurts-Moespot A, Piffanelli A, Foekens JA, Benraad T. An ELISA avoiding interference by heterophilic antibodies in the measurement of components of the plasminogen activation system in blood. J Immunol Methods. 2002;268:219-231 23. de Jager W, te Velthuis H, Prakken BJ, Kuis W, Rijkers GT. Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells. Clin Diagn Lab Immunol. 2003;10:133-139 24. Pollack MM, Ruttimann UE, Getson PR. Pediatric risk of mortality (PRISM) score. Crit Care Med. 1988;16:1110-1116. 25. van Brakel MJ, van Vught AJ, Gemke RJ. Pediatric risk of mortality (PRISM) score in meningococcal disease. Eur J Pediatr. 2000;159:232-236. 26. Mizue Y, Nishihira J, Miyazaki T, Fujiwara S, Chida M, Nakamura K, Kikuchi K, Mukai M. Quantitation of macrophage migration inhibitory factor (MIF) using the one-step sandwich enzyme immunosorbent assay: elevated serum MIF concentrations in patients with autoimmune diseases and identification of MIF in erythrocytes. Int J Mol Med. 2000;5:397-403 27. Awandare GA, Hittner JB, Kremsner PG, Ochiel DO, Keller CC, Weinberg JB, Clark IA, Perkins DJ. Decreased circulating macrophage migration inhibitory factor (MIF) protein and blood mononuclear cell MIF transcripts in children with Plasmodium falciparum malaria. Clin Immunol. 2006;119:219-225 28. Bollaert PE, Fieux F, Charpentier C, Levy B. Baseline cortisol levels, cortisol response to corticotropin, and prognosis in late septic shock. Shock. 2003;19:13-15 29. Rothwell PM, Udwadia ZF, Lawler PG. Cortisol response to corticotropin and survival in septic shock. Lancet. 1991;337:582-583 30. Pickkers P, Sprong T, Eijk L, Hoeven H, Smits P, Deuren M. Vascular endothelial growth factor is increased during the first 48 hours of human septic shock and correlates with vascular permeability. Shock. 2005;24:508-512 31. Ren Y, Law S, Huang X, Lee PY, Bacher M, Srivastava G, Wong J. Macrophage migration inhibitory factor stimulates angiogenic factor expression and correlates with differentiation and lymph node status in patients with esophageal squamous cell carcinoma. Ann Surg. 2005;242:55-63 32. Ren Y, Tsui HT, Poon RT, Ng IO, Li Z, Chen Y, Jiang G, Lau C, Yu WC, Bacher M, Fan ST. Macrophage migration inhibitory factor: roles in regulating tumor cell migration and expression of angiogenic factors in hepatocellular carcinoma. Int J Cancer. 2003;107:22- 29 33. Maxime V, Fitting C, Annane D, Cavaillon JM. Corticoids normalize leukocyte production of macrophage migration inhibitory factor in septic shock. J Infect Dis. 2005;191:138- 144 34. Bruhn A, Verdant C, Vercruysse V, Su F, Vray B, Vincent JL. Effects of dexamethasone on macrophage migration inhibitory factor production in sepsis. Shock. 2006;26:169-173 35. Spinas GA, Keller U, Brockhaus M. Release of soluble receptors for tumor necrosis factor (TNF) in relation to circulating TNF during experimental endotoxinemia. J. Clin. Invest. 1992;90:533-536 36. Van der Poll T, de Waal Malefyt R, Coyle SM, Lowry SF. Antiinflammatory cytokine responses during clinical sepsis and experimental endotoxemia: sequential measurements of plasma soluble interleukin (IL)-1 receptor type II, IL-10, and IL-13. J. Infect. Dis. 1997;175:118-122 37. Pajkrt D, Doran JE, Koster F, Lerch PG, Arnet B, van der Poll T, ten Cate JW, van Deventer SJH. Antiinflammatory effects of reconstituted high-density lipoprotein during human endotoxemia. J. Exp. Med. 1996;184:1601-1608 38. Lowry SF. Human endotoxemia: a model for mechanistic insight and therapeutic targeting. Shock. 2005;24 Suppl 1:94-100

222 39. Lehmann AK, Halstensen A, Sørnes S, Røkke O, Waage A. High levels of interleukin 10 in serum are associated with fatality in meningococcal disease. Infect Immun. 1995;63:2109-2112 40. Radstake TR, Sweep FC, Welsing P, Franke B, Vermeulen SH, Geurts-Moespot A, Calandra T, Donn R, van Riel PL. Correlation of rheumatoid arthritis severity with the genetic functional variants and circulating levels of macrophage migration inhibitory factor. Arthritis Rheum. 2005;52:3020-3029 41. Calandra T, Spiegel LA, Metz CN, Bucala R. Macrophage migration inhibitory factor is a critical mediator of the activation of immune cells by exotoxins of Gram-positive bacteria. Proc Natl Acad Sci U S A. 1998;95:11383-11388 42. Riedemann NC, Guo RF, Gao H, Sun L, Hoesel M, Hollmann TJ, Wetsel RA, Zetoune FS, Ward PA. Regulatory role of C5a on macrophage migration inhibitory factor release from neutrophils. J Immunol. 2004;173:1355-1359 43. Sprong T, Moller ASWM, Bjerre A, Wedege E, Kierulf P, van Der Meer JW, Brandtzaeg P, van Deuren M, Mollnes TE. Complement activation and complement-dependent inflammation by Neisseria meningitidis occurs independent of lipopolysaccharide. Infect Immun. 2004;72:3344-3349 44. Brandtzaeg P, Bryn K, Kierulf P, Øvstebø R, Namork E, Aase B, Jantzen E. Meningococcal endotoxin in lethal septic shock plasma studied by gas chromatography, mass- spectrometry, ultracentrifugation, and electron microscopy. J Clin Invest. 1992;89:816- 823 45. Ovstebo R, Brandtzaeg P, Brusletto B, Haug KB, Lande K, Hoiby EA, Kierulf P. Use of robotized DNA isolation and real-time PCR to quantify and identify close correlation between levels of Neisseria meningitidis DNA and lipopolysaccharides in plasma and cerebrospinal fluid from patients with systemic meningococcal disease. J Clin Microbiol. 2004;42:2980-2987 46. Hackett SJ, Guiver M, Marsh J, Sills JA, Thomson APJ, Kaczmarski EB, Hart CA. Meningococcal bacterial DNA load at presentation correlates with disease severity. Arch Dis Child. 2002;86:44-46 47. Garner LB, Willis MS, Carlson DL, DiMaio JM, White MD, White DJ, Adams GAt, Horton JW, Giroir BP. Macrophage migration inhibitory factor is a cardiac-derived myocardial depressant factor. Am J Physiol Heart Circ Physiol. 2003;285:H2500-2509

223

Tom Sprong, Giuseppe Peri, Chris Neeleman, Alberto Mantovani, Stefano Signorini, Jos W.M. van der Meer, Marcel van Deuren Accepted: Shock

PTX3 and C-reactive protein in severe meningococcal disease 15 Abstract

The long pentraxin PTX3 is an important element of the innate immune system and has potential as a diagnostic tool in inflammatory conditions. We studied PTX3 in patients admitted to an ICU with severe meningococcal disease and compared it with the short pentraxin CRP. 26 patients with meningococcal disease were studied, 17 patients presented with meningococcal septic shock (shock group) and 9 patients with meningococcal meningitis or bacteraemia (no shock group). PTX3 and CRP were measured by ELISA. High plasma concentrations of PTX3 (median 579 µg/L) were seen at admission in patients with meningococcal disease. Concentrations were significantly higher in patients with shock compared to patients without shock (median 801 µg/L and median 256 µg/L, P = .006, respectively). In contrast, CRP at admission was lower in the shock group as compared to the no shock group (median 58 mg/L and median 165 mg/L, P = .008, respectively). High PTX3 and low CRP concentration at admission discriminated between presence and absence of shock (area under the ROC curve 0.85, P = .007 for PTX3 and 0.84 P = 0.01 for CRP). PTX3 did not correlate with disease severity (PRISM) and days spent in ICU. PTX3 at admission and PTX3 peak concentration both showed a negative correlation with plasma fibrinogen concentrations. CRP concentration at admission correlated negatively with disease severity. In conclusion, PTX3 was an early indicator of shock in patients with severe meningococcal disease that followed a pattern of induction distinct from CRP.

226 Introduction

Meningococcal disease is an important cause of morbidity and mortality world-wide. Invasive meningococcal disease occurs when a pathogenic strain of Neisseria meningitidis penetrates the nasopharyngeal mucosa and invades the bloodstream. When outgrowth in the bloodstream is restricted, meningococci seeded into the subarachnoideal space will proliferate within that compartment and meningococcal meningitis develops, with a localized inflammatory response and a mild clinical course. In contrast, meningococcal septic shock has a fulminant course and is characterized by a seemingly unimpeded outgrowth of meningococci in the bloodstream causing a massive systemic activation of a range of inflammatory systems, such as the cytokine network and the coagulation and complement cascades 1-3.

Pentraxins such as C-reactive protein (CRP), serum amyloid P (SAP) and pentraxin 3 (PTX3) are acute-phase reactants which play major roles as soluble innate immune pattern recognition receptors in defence against microbes and clearance of apoptotic or necrotic cells 4. The long pentraxin PTX3 is structurally related to CRP and SAP. Whereas CRP and SAP are made by the liver in response to inflammatory mediators such as interleukin-6 (IL-6) 5-8, PTX3 is induced by microbial components and by TNF and IL-1 in a variety of cell types, most prominently endothelial cells and mononuclear phagocytes 9-13. PTX3 is elevated in a variety of viral, bacterial and inflammatory diseases 14-16.

In daily practice, plasma concentrations of the short pentraxin CRP are often used to discern severe bacterial infections from diseases with a milder course. In addition, multiple other prognostic markers have been studied in the past to aid diagnosis and treatment of severe meningococcal disease, including leukocyte count, endotoxin (LPS), TNF-alpha and pro-calcitonin. Although some of these markers may have value in assessing disease severity, most have low sensitivity or specificity 17-20.

Based on the observations that PTX3 is essential in defence against certain pathogens and prompted by reported correlations of PTX3 plasma concentration with severity of disease 21, we evaluated PTX3 in comparison with the short pentraxin CRP in patients admitted to an ICU with severe meningococcal disease.

227 Materials and Methods Patient characteristics Twenty six patients with meningococcal disease admitted between 1994 and 2004 to the intensive care unit of the Radboud University Nijmegen Medical Centre were studied : 24 children (median age 3.1y, range 0.6-14y, 13 boys, 11 girls) and 2 adult patients (1 male, 25.8y and 1 female, 30.6y). Informed consent was obtained for all patients, and the studies were conducted according to the guidelines of the local ethical committee.

Diagnosis of meningococcal disease was based on typical clinical picture, in 88 % of the cases this was microbiologically confirmed. Sixteen children presented with meningococcal septic shock, defined as haemodynamic instability requiring sustained (> 12 hours) vasopressive support in spite of adequate fluid resuscitation. Four shock patients also had signs of meningitis (defined as > 100 x 103 leukocytes / L in the CSF or clear nuchal rigidity on physical examination). Eight presented without shock, six children had meningitis and two children presented with neither shock nor meningitis (no-shock group). The one female adult patient presented with shock and died early in the course of disease, the male adult patient presented with meningitis and survived. Patients received standardised treatment consisting of early appropriate antibiotics (mostly penicillin or ceftriaxone), fluid resuscitation with if necessary inotropes or vasopressors, and mechanical ventilation when indicated. In addition, corticosteroids were given to all but 1 patient and - as part of a clinical trial - 8 of the patients received whole blood exchange transfusion or plasmaferesis.

The time from onset of disease to admission was defined as the time between the first symptom noted (for example fever, malaise or vomiting) and admission to the hospital. Patient data were collected prospectively.

PTX3 and CRP analysis EDTA-anticoagulated samples stored at -80°C were used for the analysis. Admission samples were obtained within 4 hours after admission to the ICU and were available for 20 children and both adults. Peak concentrations were the highest concentrations found during admission. Time course was made by grouping the data in different categories (t = 8 were samples obtained between 4 and 12 hours after admission, t = 24 were samples obtained between 12 and 36 hours after admission, t = 48 were samples obtained between 36 and 60 hours after admission). In the time-course analysis, only patients of which sequential samples were available were included (n=20).

228 PTX3 was measured using an in-house enzyme-linked immunosorbent assay (ELISA) 14,22. PTX3 concentration is expressed as µg/L and plasma concentrations in healthy controls are < 2 µg/L. CRP was measured using a commercially available ELISA purchased from DAKO, Glastrup, Denmark. .

Statistics

Differences between medians were tested using Mann-Whitney test. Fishers exact test and Spearman correlation test were used as indicated in text or legends. Graph pad PRISM 4.0 software was used to perform the analyses and to create the ROC curves.

Results Patient characteristics The 26 patients presenting with meningococcal disease were divided into two groups: patients that developed shock and patients without shock (Table 1). The patients with shock had an unfavourable prognosis: they had higher disease severity scores (PRISM, 23,24), spent more days in ICU, had more sequelae and higher mortality than patients without shock. Importantly, patients with shock had also more fulminant disease, as reflected by the shorter time from disease onset to

Table 1. Patient characteristics shock no shock P n 17 9 n.s. male / female 9/8 5/4 n.s. age (y) 3.5 (0.6-30.6) 2.7 (0.6-25.8) n.s. patients included in 1994 (1), 1995 (2), 1994 (1), 1995 (2), 1996 (3), 1997 (2), 1996 (1), 2002 (4), 1998 (1), 2002 (3), 2003 (1) 2003 (4), 2004 (1) time to admission1 (h) 13 (9.3-23.5) 23.3 (16-96) .0004 time to admission2 (h) 16.5 (12-27) 25 (18.5-100) .0004 PRISM* 17 (6-35) 3 (0-10) .0004 days in ICU (days)** 8 (1-23) 1 (1-4) .001 MAP (mmHg) 68.5 (35-100) 82.5 (40-90) n.s. fibrinogen (mg/L) 1.6 (0.1-2.8) 4.4 (3.4-6.4) <.0001 sequelae** 10/15 (66%) 0/8 .0027 mortality 2/17 (12%) 0/8 n.s. Characteristics of patients presenting with or without shock during meningococcal disease. Where appropriate, median and range is presented. Differences were tested for significance using Mann-Whithney test and Fishers exact test when appropriate. PRISM = Pediatric Risk of Mortality, MAP = mean arterial pressure. * PRISM score and MAP value are presented only for the children. ** days in ICU and sequelae 15 were calculated only for survivors. 1 time from first symptom to admission to referring hospital, 2 time from first symptom to admission to the ICU of the Radboud University Nijmegen Medical Centre. Sequelae observed were amputations, severe scars, prolonged hypocortisism, and concentration disturbances / learning disability.

229 admission. In addition, time to admission correlated negatively with PRISM scores and length of ICU stay (Spearman R = -0.55, P = .007 and R = -0.51, P = .01, respectively).

PTX3 and CRP plasma concentrations in severe meningococcal disease High plasma concentrations of PTX3 were seen at admission: median 579 µg/L (range 21 - 2335 µg/L). Concentrations were significantly higher in patients with shock than in patients without shock (median 801 µg/L, range 95 - 2335 µg/L and median 256 µg/L, range 21 - 702 µg/L, P = .006, respectively). Peak concentration of PTX3 also showed a clear-cut difference between patients with and without shock (median 1036 µg/L, range 139-3480 µg/L and median 473 µg/L, range 37 - 1310 µg/L, P = .002, respectively). In contrast, CRP at admission was lower in the patients with meningococcal septic shock as compared to the patients without shock (median 58 mg/L, range 19 - 197 mg/L and median 165 mg/L, range 63 - 327 mg/L, P = .008, respectively). Peak concentration of CRP in patients with shock was equal to that in patients without shock (median 244 mg/L, range 66 - 391 mg/L and median 265 mg/L, range 151 - 327 mg/L, P = .945, respectively).

Time course of PTX3 and CRP showed a divergent pattern of induction: in patients with shock, median PTX3 concentration was highest at admission, whereas CRP peaked 48 hours after admission. In patients with meningitis CRP peaked within the first 24 hours after admission, reflecting a longer pre-clinical disease period in the meningitis patients (Figure 1).

PTX3, CRP and severity of disease High PTX3 and low CRP concentration at admission discriminated between presence and absence of shock: area under the ROC curve 0.85, P = .007 for PTX3 and 0.84 P = 0.01 for CRP (Figure 2). PTX3 and CRP did not correlate with each other. PTX3 at admission and PTX3 peak concentration did not correlate with disease severity (PRISM) and days spent in ICU, and correlated negatively with time to admission. CRP concentrations at admission however, showed a strong negative correlation with parameters of disease severity, and correlated positively with time to admission. The CRP peak concentration showed no relation to parameters of disease severity or time to admission. Interestingly, PTX3 at admission and PTX3 peak concentration both correlated negatively with plasma fibrinogen concentrations (Table 2).

230 2000 PTX3 SHOCK

1500 NO SHOCK

g/L 1000 µ

500

0 0 8 24 48 72 400 CRP

300

200 mg/L Figure 1. 100 Time course of PTX3 and CRP in meningococcal disease. Dashed bars indicate patients with 0 shock, white bars patients without 0 8 24 48 72 shock. Median and upper quartile Time after admission to ICU (h) is shown.

ROC of PTX3 ROC of CRP 100 100

80 80

60 60

40 40

Sensitivity (%) 20 20

0 0 0 20 40 60 80 100 0 20 40 60 80 100 Figure 2. Receiver operating charac-teristic 100 - specificity (%) 100 - specificity (%) curve for PTX3 and CRP.

Table 2. Correlation of PTX3 and CRP PTX3 PTX3 CRP CRP admission peak admission peak PTX3 peak 0.74 (<.01) CRP admission -0.24 (.28) -0.31 (.20) CRP peak 0.33 (.18) 0.33 (.14) 0.42 (.08) time to admission 1 -0.47 (.03) -0.55 (<.01) 0.58 (<.01) 0.08 (.71) PRISM 0.30 (.20) 0.40 (.08) -0.73 (<.01) -0.25 (.28) days in ICU 0.27(.27) 0.42 (.05) -0.76 (<.01) -0.08 (.72) MAP 0.12 (.63) -0.08 (0.74) .24 (0.32) -0.07 (.76) fibrinogen -0.55 (<.01) -0.66 (<.01) 0.55 (<.01) 0.06 (.58) Spearman correlation of PTX3 and CRP with different disease characteristics. P-values are shown betwen brachets. PRISM = Pediatric Risk of Mortality, MAP = mean arterial pressure. Correlation with PRISM 15 scores and MAP were only calculated for the children. 1 time from first symptom to admission to referring hospital

231 Discussion

This is the first study that has analyzed the pentraxins PTX3 and CRP in the various manifestations of meningococcal disease and that has taken into account their time course. We found that PTX3 peaks in the first hours after admission to the ICU and thereby was an early indicator of shock. This pattern greatly differs from that of CRP which is low at admission and peaks 48 hours after admission in the shock patients. Thus, a high PTX3 and a low CRP at admission discriminates between the presence or absence of shock. CRP at admission showed a strong negative correlation with disease severity, and was positively correlated with time to admission. In contrast, PTX3 at admission did not correlate with parameters of disease severity other than shock.

The difference in the time course of PTX3 and CRP is due to differences in their regulation: PTX3 is induced by microbial components and early pro-inflammatory cytokines such as TNF and IL-1 in endothelial cells and macrophages 9-12,25,26. Therefore, PTX3 is expected to appear early in the course of diseases that are characterized by activation of these cells. Reportedly, in patients with myocardial infarction PTX3 peaked only 7.5 hours after the primary event 22. CRP, in contrast, is induced by the secondary cytokine IL-6 and produced in the liver, and has a slower time course 27-30. Maximal CRP concentrations in humans experimentally challenged with endotoxin, peaked 24 hours after the primary event 31.

In meningococcal disease, a short time from disease onset to admission predicts a fulminant course and severe disease 1. In the present study, time to admission was lower in patients with shock (13 hours compared to 23.3 hours in patients without shock) and correlated negatively with PRISM scores and length of ICU stay. In addition, CRP at admission was positively correlated with time to admission, indicating that patients with a shorter time to admission had lower CRP values. Therefore, the correlation we found of low CRP with shock and parameters of disease severity is most likely caused by the slow kinetic of CRP, indicating a more fulminant course in patients with low CRP plasma concentration at admission 17,18,32,33. High PTX3 plasma concentrations discriminate well between the presence or absence of shock. The high plasma concentrations of PTX3 in the shock patients at admission possibly reflect higher bacterial loads and the higher concentrations of TNF and IL-1 found in the shock patients 34-36. PTX3 did not significantly correlate with parameters of disease severity other than shock such as PRISM score and days spent in ICU. This contrasts with earlier findings in patients with sepsis and

232 septic shock of diverse origin 14. However, the correlation coefficient found in that study was moderate and comparable to that found in the present study. It may be that the relatively small number of patients in our study or the more homogenous study population explains the discrepancy between both studies. Previous studies in vitro have found that PTX3 is able to up-regulate tissue factor, a crucial factor in the initiation of disseminated intravascular coagulation (DIC) 37,38. Our finding that PTX3 correlated negatively with fibrinogen (low fibrinogen reflects severe DIC) suggests that this link between PTX3 and activation of coagulation is also present in vivo.

The biological roles of PTX3 and CRP are intriguing. Both pentraxins are acute phase proteins involved in the direct and complement-mediated clearance of bacteria, debris and apoptic cell remnants, created during the primary infectious insult or set free throughout the subsequent inflammatory response 4. Whereas CRP is known to bind the capsule of pneumococci, preliminary data indicates that PTX3 binds to meningococci (personal communication, B. Botazzi, unpublished data) and the Gram-negative outer membrane protein OmpA 39. Whether this implies a protective role for PTX3 in meningococcal disease should be the subject of further research. On the other hand, PTX3 could also be a factor that aggravates DIC or have a role as a secondary mediator involved in the direct or complement-mediated clearance of damaged cells.

In conclusion, PTX3 is an early indicator of shock in patients with severe meningococcal disease that follows a pattern of induction distinct from CRP. High PTX3 and low CRP plasma concentrations discriminate between the presence or absence of shock. Thus, a high PTX3 level at admission should alert the clinician for imminent deterioration and shock.

233 References 1. Van Deuren M, Brandtzaeg P, Van der Meer JWM. Update on meningococcal disease with emphasis on pathogenesis and clinical management. Clin Microbiol Rev. 2000;13:144- 166 2. Sprong T, Pickkers P, Geurts-Moespot A, van der Ven-Jongekrijg J, Neeleman C, Knaup M, Leroy D, Calandra T, van der Meer JW, Sweep F, van Deuren M. Macrophage migration inhibitory factor (MIF) in meningococcal septic shock and experimental human endotoxemia. Shock. 2007;27:482-487 3. Pickkers P, Sprong T, Eijk L, Hoeven H, Smits P, Deuren M. Vascular endothelial growth factor is increased during the first 48 hours of human septic shock and correlates with vascular permeability. Shock. 2005;24:508-512 4. Garlanda C, Bottazzi B, Bastone A, Mantovani A. Pentraxins at the crossroads between innate immunity, inflammation, matrix deposition, and female fertility. Annu Rev Immunol. 2005;23:337-366 5. Baumann H, Gauldie J. The acute phase response. Immunol Today. 1994;15:74-80 6. Steel DM, Whitehead AS. The major acute phase reactants: C-reactive protein, serum amyloid P component and serum amyloid A protein. Immunol Today. 1994;15:81-88 7. Volanakis JE. Human C-reactive protein: expression, structure, and function. Mol Immunol. 2001;38:189-197 8. Agrawal A. CRP after 2004. Mol Immunol. 2005;42:927-930 9. Alles VV, Bottazzi B, Peri G, Golay J, Introna M, Mantovani A. Inducible expression of PTX3, a new member of the pentraxin family, in human mononuclear phagocytes. Blood. 1994;84:3483-3493 10. Bottazzi B, Vouret-Craviari V, Bastone A, De Gioia L, Matteucci C, Peri G, Spreafico F, Pausa M, D’Ettorre C, Gianazza E, Tagliabue A, Salmona M, Tedesco F, Introna M, Mantovani A. Multimer formation and ligand recognition by the long pentraxin PTX3. Similarities and differences with the short pentraxins C-reactive protein and serum amyloid P component. J Biol Chem. 1997;272:32817-32823 11. Basile A, Sica A, d’Aniello E, Breviario F, Garrido G, Castellano M, Mantovani A, Introna M. Characterization of the promoter for the human long pentraxin PTX3. Role of NF- kappaB in tumor necrosis factor-alpha and interleukin-1beta regulation. J Biol Chem. 1997;272:8172-8178 12. Doni A, Michela M, Bottazzi B, Peri G, Valentino S, Polentarutti N, Garlanda C, Mantovani A. Regulation of PTX3, a key component of humoral innate immunity in human dendritic cells: stimulation by IL-10 and inhibition by IFN-gamma. J Leukoc Biol. 2006;79:797-802 13. He X, Han B, Liu M. Long pentraxin 3 in pulmonary infection and acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2007;292:L1039-1049 14. Muller B, Peri G, Doni A, Torri V, Landmann R, Bottazzi B, Mantovani A. Circulating levels of the long pentraxin PTX3 correlate with severity of infection in critically ill patients. Crit Care Med. 2001;29:1404-1407 15. Mairuhu AT, Peri G, Setiati TE, Hack CE, Koraka P, Soemantri A, Osterhaus AD, Brandjes DP, van der Meer JW, Mantovani A, van Gorp EC. Elevated plasma levels of the long pentraxin, pentraxin 3, in severe dengue virus infections. J Med Virol. 2005;76:547-552 16. Fazzini F, Peri G, Doni A, Dell’Antonio G, Dal Cin E, Bozzolo E, D’Auria F, Praderio L, Ciboddo G, Sabbadini MG, Manfredi AA, Mantovani A, Querini PR. PTX3 in small- vessel vasculitides: an independent indicator of disease activity produced at sites of inflammation. Arthritis Rheum. 2001;44:2841-2850 17. Casado-Flores J, Blanco-Quiros A, Nieto M, Asensio J, Fernandez C. Prognostic utility of the semi-quantitative procalcitonin test, neutrophil count and C-reactive protein in meningococcal infection in children. Eur J Pediatr. 2006;165:26-29 18. Van der Kaay DC, De Kleijn ED, De Rijke YB, Hop WC, De Groot R, Hazelzet JA. Procalcitonin as a prognostic marker in meningococcal disease. Intensive Care Med. 2002;28:1606-1612

234 19. Kuppermann N, Malley R, Inkelis SH, Fleisher GR. Clinical and hematologic features do not reliably identify children with unsuspected meningococcal disease. Pediatrics. 1999;103:E20 20. Hsiao AL, Baker MD. Fever in the new millennium: a review of recent studies of markers of serious bacterial infection in febrile children. Curr Opin Pediatr. 2005;17:56-61 21. Mantovani A GC, Doni A, Bottazzi B. Pentraxins in Innate Immunity: From C-Reactive Protein to the Long Pentraxin PTX3. J clin Immunol. 2007 22. Peri G, Introna M, Corradi D, Iacuitti G, Signorini S, Avanzini F, Pizzetti F, Maggioni AP, Moccetti T, Metra M, Cas LD, Ghezzi P, Sipe JD, Re G, Olivetti G, Mantovani A, Latini R. PTX3, A prototypical long pentraxin, is an early indicator of acute myocardial infarction in humans. Circulation. 2000;102:636-641 23. Pollack MM, Ruttimann UE, Getson PR. Pediatric risk of mortality (PRISM) score. Crit Care Med. 1988;16:1110-1116. 24. van Brakel MJ, van Vught AJ, Gemke RJ. Pediatric risk of mortality (PRISM) score in meningococcal disease. Eur J Pediatr. 2000;159:232-236. 25. Lee GW, Goodman AR, Lee TH, Vilcek J. Relationship of TSG-14 protein to the pentraxin family of major acute phase proteins. J Immunol. 1994;153:3700-3707 26. Introna M, Alles VV, Castellano M, Picardi G, De Gioia L, Bottazzai B, Peri G, Breviario F, Salmona M, De Gregorio L, Dragani TA, Srinivasan N, Blundell TL, Hamilton TA, Mantovani A. Cloning of mouse ptx3, a new member of the pentraxin gene family expressed at extrahepatic sites. Blood. 1996;87:1862-1872 27. Kalil AC, Coyle SM, Um JY, LaRosa SP, Turlo MA, Calvano SE, Sundin DP, Nelson DR, Lowry SF. Effects of drotrecogin alfa (activated) in human endotoxemia. Shock. 2004;21:222-229 28. Derhaschnig U, Bergmair D, Marsik C, Schlifke I, Wijdenes J, Jilma B. Effect of interleukin-6 blockade on tissue factor-induced coagulation in human endotoxemia. Crit Care Med. 2004;32:1136-1140 29. Kvalsvig AJ, Unsworth DJ. The immunopathogenesis of meningococcal disease. J Clin Pathol. 2003;56:417-422 30. Moller AS, Bjerre A, Brusletto B, Joo GB, Brandtzaeg P, Kierulf P. Chemokine patterns in meningococcal disease. J Infect Dis. 2005;191:768-775 31. van Eijk LT, Pickkers P, Smits P, van den Broek W, Bouw MP, van der Hoeven JG. Microvascular permeability during experimental human endotoxemia: an open intervention study. Crit Care. 2005;9:R157-164 32. Neeleman C, van Deuren M, van der Meer JWM. Acute meningokokkeninfecties: lage C-reactief proteïne (CRP)-concentratie in het serum bij opname wijst op een fulminant beloop. Tijdschr Kindergeneeskd. 1998;66:107-110 33. Marzouk O, Bestwick K, Thomson AP, Sills JA, Hart CA. Variation in serum C-reactive protein across the clinical spectrum of meningococcal disease. Acta Paediatr. 1993;82:729-733 34. Van Deuren M, van der Ven Jongekrijg J, Bartelink AKM, van Dalen R, Sauerwein RW, van der Meer JWM. Correlation between proinflammatory cytokines and antiinflammatory mediators and the severity of disease in meningococcal infections. J Infect Dis. 1995;172:433-439 35. Brandtzaeg P, Kierulf P, Gaustad P, Skulberg A, Bruun JN, Halvorsen S, Sørensen E. Plasma endotoxin as a predictor of multiple organ failure and death in systemic meningococcal disease. J Infect Dis. 1989;159:195-204 36. Waage A, Halstensen A, Espevik T. Association between tumour necrosis factor in serum and fatal outcome in patients with meningococcal disease. Lancet. 1987;i:355-357 37. Napoleone E, di Santo A, Peri G, Mantovani A, de Gaetano G, Donati MB, Lorenzet R. The long pentraxin PTX3 up-regulates tissue factor in activated monocytes: another link between inflammation and clotting activation. J Leukoc Biol. 2004;76:203-209 38. Napoleone E, Di Santo A, Bastone A, Peri G, Mantovani A, de Gaetano G, Donati 15 MB, Lorenzet R. Long pentraxin PTX3 upregulates tissue factor expression in human endothelial cells: a novel link between vascular inflammation and clotting activation.

235 Arterioscler Thromb Vasc Biol. 2002;22:782-787 39. Jeannin P, Bottazzi B, Sironi M, Doni A, Rusnati M, Presta M, Maina V, Magistrelli G, Haeuw JF, Hoeffel G, Thieblemont N, Corvaia N, Garlanda C, Delneste Y, Mantovani A. Complexity and complementarity of outer membrane protein A recognition by cellular and humoral innate immunity receptors. Immunity. 2005;22:551-560

236 15

237

Tom Sprong, Martin den Heijer, Trees Jansen, Liesbeth Jacobs, Corrie de Kat-Angelino, Ina Klasen, Chris Neeleman, Anton K.M. Bartelink, Jos W.M. van der Meer, Marcel van Deuren Submitted

Mannose-binding lectin (MBL) and meningococcal disease, a case-parent study 16 Abstract

It is controversial whether mannose-binding lectin (MBL) deficiency constitutes a risk factor for meningococcal disease. We conducted an ICU based case-parent study in 120 survivors of meningococcal disease to investigate the role of MBL genotype, MBL plasma concentration and MBL/MASP2-activity in susceptibility to and clinical presentation of disease. Genotype for MBL was assessed using line probe assay. Transmission-disequilibrium test was performed for SNPs in the MBL2 gene, relative risks for effects of the exon 1 SNPs combined were determined using a log-linear model. No transmission disequilibrium was found for the SNPs in the MBL2 gene, the exon1 SNPs combined were also not associated with an increased risk for disease. Subgroup analysis showed increased risk for meningococcal meningitis associated with the exon 1 SNPs (RR 3.9, P = 0.03 and RR 3.77, P = 0.2, for heterozygousity and homozygousity, respectively). MBL plasma concentrations in meningitis patients were significantly lower than in patients with shock or bacteraemia. No patients were deficient for MASP2. Polymorphisms in the gene for MBL do not predispose for severe meningococcal disease. SNPs in exon 1 may be a risk factor for meningococcal meningitis. MASP2 deficiency is no frequent cause of meningococcal disease.

240 Introduction

Invasive meningococcal disease occurs when a pathogenic strain of Neisseria meningitidis penetrates the nasopharyngeal mucosa and invades the bloodstream. Restricted outgrowth of meningococci in the bloodstream and seeding into the subarachnoideal space leads to compartmentalized proliferation and signs of meningitis. Meningococcal septic shock, most often without signs of meningitis, develops when meningococci replicate rapidly in the bloodstream with systemic activation of the cytokine network, the coagulation and fibrinlolytic cascade and the kinin and complement system1. carriage rate of meningococci in dutch children is reported to be up to 26 % 2,3, although a recent study showed a lower prevalence of 1.5 % 4. Incidence of disease in the Netherlands between 1990 and 2002 was approximately 0.003 % - 0.004 % / year, dropping to approximately 0.002 % / year between 2002 and 2006 after the start of the vaccination campaign against group C meningococci.

Mannose-binding lectin (MBL) is a multimeric protein composed of 5 to 6 trimeric units complexed with MBL-associated serine protease-2 (MASP2)5. Binding of the MBL-MASP2 complex to Neisseria meningitidis activates complement via the lectin pathway 6-8. The magnitude of this process is determined by the quantity of bacteria, the MBL concentration and the functionality of MASP2. The MBL concentration is influenced by various single nucleotide polymorphisms (SNPs) localized in exon 1 and the promotor region of the MBL2 gene. The SNPs in exon 1, situated in codon 52, 54 and 57 designated D, B and C respectively (the wild-type allele for these positions is termed A) lead to impaired twisting of trimeric MBL-units and thereby to decreased MBL concentrations. The regulatory polymorphisms in the MBL2 promoter and 5’ untranslated region are at position -550, -221 and +4 and designated as L, X and Q (the wild type alleles are termed H, Y and P respectively). The 3 exon 1 SNPs and the 3 regulatory polymorphisms are in linkage disequilibrium, with 7 different common haplotypes reported in Caucasian populations: HYPA, LYPA, LXPA, LYQA, HYPD, LYPB and LYQC. As the MBL genotype is composed of two of these haplotypes, 28 different genotypes may occur 5,9.

Insufficient complement activation due to deficiency in complement components is a risk factor for invasive meningococcal disease. These complement deficiencies are very rare, and thus have low impact on the overall incidence of meningococcal disease 10,11. In contrast, MBL deficiency is rather common, with approximately 5 % of the population having undetectable MBL-plasma concentrations, and 35 - 40 % with low concentrations 12. 16

241 In 1999, Hibberd et al. reported, based on a case-control study, that polymorphisms in exon 1 of the MBL2-gene predispose to meningococcal disease 13. However, this study did not evaluate other MBL-genotypes nor MBL-plasma concentrations. Two other case-control studies were unable to confirm an association 14,15. This controversy is possibly due to differences in patient selection or case-control matching. To avoid matching bias we decided to perform a case-parent study that uses transmission disequilibrium testing 16, in order to asses whether susceptibility to meningococcal disease is related to MBL genotype. In addition, we were the first to investigate MBL functional concentration and MBL/MASP2 activity in 120 clinically well documented survivors of invasive meningococcal disease. This group included 12 patients with a positive family history of meningococcal disease.

Patients and Methods Ethics Patients and parents gave full informed consent prior to inclusion in the study. The study was approved by the local ethical committee.

Patients The Radboud University Nijmegen Medical Centre is a tertiary care hospital. Between 1990 and 2006, 238 patients with suspected meningococcal disease (based on clinical symptoms, purpueric rash and fever) were admitted. Twenty-two (9.2 %) of them died. From 179 of the 216 survivors the current address was known. These patients and their parents were invited to participate in the study; 59 patients did not respond, declined to participate or it was not possible to include the case and minimally one parent. Thus, 120 survivors (65 males) participated in the study, with 109 fathers and 118 mothers. Of 11 patients the father could not be included, and of 2 patients the mother. This yielded 107 complete trios. Of 115 children, 102 fathers and 113 mothers also MBL-plasma functional concentrations (MBL-P) and MBL/MASP2-activity (MBL-A) were measured. In addition to the patients, 6 affected sibs were also included.

Patients were median 3.4 years old at time of presentation to the ICU and 8.6 years years upon inclusion in the study. The fathers were aged 41.8 y (range 25.8 - 66.2) and the mothers were 39.6 y (range 24.2 - 69.0). Most individuals were Caucasian, 6 patients were of Asian origin, 1 patient was of South-American/African origin.

Disease type Patient data were collected by retrospective chart analysis when admission was before 2002 (n = 102) and by prospective analysis after this date (n = 18).

242 Table 1. Patient characteristics and clinical manifestation of meningococcal disease. Shock Shock + Bacteraemia Meningitis n = 46 Meningitis n = 24 n = 38 n = 12 M: 26 (57%) M: 6 (50%) M: 13 (54%) M: 20 (53%) Sex F: 20 (43%) F: 6 (50%) F: 11 (46%) F: 18 (47%) Age (yrs) 2.4 (0.5 - 14.7)* 2.5 (0.1 - 14.0) 3.4 (0.3 - 13.5) 4.8 (0.2 - 22.3) Proven 45 (98%) 12 (100%) 20 (83%) 32 (84%) Group B 30 (83%) 10 (83%) 12 (81%) 23 (88%) Group C 6 (17%) 2 (17%) 4 (25%) 3 (12%) PRISM-score 16 (6 - 35)*,# 13 (6 - 26)*,# 5 (0 - 25)* 4 (0 - 14) MAP (mmHg) 60 (35-90)*,# 60 (42 - 100) # 78 (53-141) 77 (57 - 112) Days in ICU 8 (1-30)*,# 7 (2 - 9)*,# 2 ( 1 - 9) 2 ( 1 - 4 ) Sequelae 34 (74%)*,# 5 (42%) 5 (21%) 8 (21%) Medians and ranges (between brackets) are presented or numbers and % (between brackets). PRISM = pediatric risk of mortality, MAP= mean arterial pressure, ICU = intensive care unit. Proven indicates N. meningitidis cultured from blood, CSF or skin-biopsy or Gram-negative diplococci found in the Gram-stain. Data on serogroup was available for 36 patients in the shock group, 12 patients in the shock and meningitis group, 16 patients in the bacteraemia group and 26 patients in the meningitis group. Differences were tested for significance using Mann-Whitney test or χ2-test when appropriate, and corrected for multiple testing using Bonferroni correction: P < 0.0083 was considered significant. * indicates significance for the comparison with the meningitis group, # indicates significance for comparison with the bacteraemia group.

Patients were divided in four groups based on clinical presentation: shock, shock + meningitis, bacteremia, or meningitis. Shock was defined as sustained hypotension despite volume expansion requiring prolonged (> 12 hours) vasopressive support. Meningitis was diagnosed when there were > 105 leukocytes/L in CSF or nuchal rigidity on clinical examination. Meningococcal bacteremia was diagnosed in patients having a typical presentation of meningococcal disease (sudden onset of fever and a purperic rash) in the absence of shock or meningitis. PRISM scores were calculated from data obtained during the first 24 hrs of ICU admission 17,18. Data on sequelae were obtained from the charts and during the interview at study entry. Table 1 shows the characteristics of the patients.

MBL-plasma functional concentration, MBL/MASP2 activity and MBL genotype. MBL-plasma functional concentration was assessed by enzyme-linked immunosorbent assay (ELISA) using immobilized mannan (Saccharomyces cerevisiae (M-7504, Sigma)) as ligand and murine monoclonal anti-MBL (HYB 131- 01, Statens Serum Institut) as detection antibody 19. An EDTA-pool from human volunteer blood donors was used as a standard, this standard was calibrated on the MBL Elisa kit (M1990, Sanquin, The Netherlands).

MBL/MASP2-activity was assayed as described previously 20. This assay measures the complete lectin pathway activity from binding of MBL to activation of C4. In brief, 0.1 mg/ml mannan in coatingbuffer (0.05 M carbonate buffer pH=9.5) was 16

243 incubated overnight at 4 ˚C. Plates were washed 3 times between each subsequent step. Blocking occurred with 1 % gelatine solution in TBS, where after EDTA plasma

samples diluted in binding buffer (20 mM Tris, 1 M NaCl, 10 mM CaCl2, 0.05% Tween, 0.1% gelatine; pH 7.4) were incubated for 1 hour at room temperature (RT). Pooled EDTA-plasma from 50 healthy donors was used as standard and set to 100 %, this plasmapool contains 775 AU/mL functional MBL. Next, plates were incubated with 1 μg/ml C4 (A-402, Quidell) diluted in GVB-Ca-Mg (3.2 mM Veronal,

1.8 mM Na-Veronal, 146 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% gelatine, pH=7.5 during 50 min at 37℃. Bound C4b was detected by incubating with rabbit anti-human C4 (A0065, Dako) diluted 20.000x in TBS/Tween/Ca for 45 min at RT and conjugation with alkaline phosphatase labelled goat-anti-rabbit immunoglobulin (D487, Dako), 1000x diluted in TBS/Tween/Ca for 1 hours at RT. Plates were developed using 1 mg/ml p-nitrophenylphosphate (Sigma) in diethanolamine buffer

(1M diethanolamine, 0.5 mM MgCl2, pH 9.8) for 15 minutes at 37℃, development was stopped using 2M NaOH, extinction was measured at 405 nm.

MBL-genotyping was done with a line probe assay (INNO-LiPA MBL2), commercially available from Innogenetics, Gent, Belgium. This assay determines the three SNPs in exon 1 (D,B and C) and the three common regulatory polymorphisms in the MBL2 promoter and 5’ untranslated region (L, X and ,Q) of the MBL2-gene.

Statistical analysis Combination of the genotype of the father and mother of complete trios yielded the expected genotype frequencies for the offspring. For the individual SNPs of the MBL2-gene standard transmission disequilibrium testing was performed. For calculation of the relative risk for contracting meningococcal disease due to the combined effects of exon 1 SNPs, we used an extension of the log-linear model as developed by Gjessing and Lie and publicly available HAPLIN software ((http://www. uib.no/smis/gjessing/genetics/software/haplin/) 21. Missing parents were accounted for by an expectation - maximisation (EM) algorithm. The effect of heterozygousity or homozygousity for YB, YC or YD was measured relative to the haplotypes YA and XA. Chi-square and Mann-Whitney test were used when appropriate.

Results MBL-plasma functional concentration and MBL/MASP2-activity in relation to MBL-genotype. MBL functional plasma concentration (MBL-P) and MBL/MASP2-activity MBL/ MASP2-A in the 330 study participants were strongly correlated (Spearman R = 0.92. P < 0.0001). There were no samples with detectable MBL-P but defective

244 Figure 1

1000

100

10 MBL/MASP2-A (%) MBL/MASP2-A

Figure 1. Correlation of MBL-plasma functional 0.1 concentration (MBL-P) and MBL/ 0.01 0.1 1 10 MASP2 activity (MBL-A) MBL-P (mg/L)

MBL/MASP2-A, which indicates that MASP2-deficiency in the absence of MBL- deficiency did not occur in our study population (Figure 1). Age at study entry did not affect MBL-P or MBL-A levels (Spearman correlation for MBL-P and age was R 0.07, P = 0.48 and for MBL-A and age R 0.05, P = 0.61).

In the total study population we identified 24 of the reported possible common 28 genotypes. The genotypes LYQC/LYPA, LYQC/LYPB, LYQC/LYQC/ and LYQC/ HYPD were not found. However, two rare SNP combinations were identified in a father and child with the genotypes HXPA/LYPD and HYPA/LYPD respectively.

Genotypes without exon 1 SNPs were the most frequent and had the highest MBL plasma concentrations (frequency 54 %, median 2.42 µg/L; range 8.6 - 0.31 µg/L). Decreased MBL concentrations occurred in homozygotes for the promoter region X polymorphism (frequency 5 %, median 0.86 µg/L; range 2.43 - 0.12 µg/L) and in heterozygotes for exon 1 SNPs (frequency 42 %, median 0.46 µg/L; range 0.04 - 2.68 µg/L). Interestingly, a D exon 1 SNP gave higher MBL plasma concentrations than a B or C exon 1 SNP. An X-polymorphism on one allele and an exon 1 SNP on the other allele gave very low MBL-plasma concentrations. Undetectable MBL levels were seen in individuals homozygous or compound heterozygous for exon 1 SNPs (frequency 4 %)(Figure 2).

MBL genotype in all patients Table 2 shows the allele frequencies and the results of the transmission disequilibrium test for the 6 different MBL2 polymorphisms in complete trios (case, mother and father; n = 107). No significant transmission disequilibrium was observed for any of the individual alleles. Transmission disequilibrium at the H/L 16

245 promotor polymorphism approached significance, favouring transmission of the H allele. However, the MBL-plasma concentration is mainly determined by exon 1 polymorphisms either in homozygous or heterozygous form. Using the log-linear model (HAPLIN analysis) on all 120 patients, with missing parents accounted for by the EM algorithm, we tested whether the exon 1 polymorphisms were associated with an increased relative risk (RR) for meningococcal disease. We found a small increase in RR that was not significant (Table 3). Taken together these calculations Figure 2 10

1 MBL-P (mg/L) MBL-P

0.1

0.01

HYPA/HYPAHYPA/LYPAHYPA/LYQALYPA/LYPALYPA/LYQALYQA/LYQAHYPA/LXPALYPA/LXPALYQA/LXPALXPA/LXPAHYPA/HYPDLYPA/HYPDLYQA/HYPDHYPA/LYPBLYPA/LYPBLYQA/LYPBHYPA/LYQCLYQA/LYQCLXPA/HYPDLXPA/LYPBLXPA/LYQCLYPB/LYPBLYPB/HYPDHYPD/HYPD

Figure 2. MBL plasma functional concentration (MBL-P) in relation to genotype. Data from all participants are shown (n = 330). Bars indicate medians.

Table 2. Transmission-disequilibrium test results in the complete trios (n = 107) for the 3 common SNPs in exon 1 and the 3 regulatory polymorphisms in the promoter regions of the MBL2- gene. SNP Frequency Transmitted (n) Untransmitted (n) P D 9 % 22 13 0.13 B 11 % 20 26 0.38 C 2 % 5 3 0.48 L 60 % 43 63 0.05 X 22 % 35 43 0.37 Q 20 % 32 42 0.25 SNP = single nucleotide polymorphism

246 indicate that in our study population polymorphism in the MBL2 gene did not increase susceptibility to meningococcal disease.

MBL in relation to clinical presentation of meningococcal disease MBL-P was significantly lower in meningitis patients than in patients with another clinical manifestation (Figure 3). Accordingly, for meningitis we found more often (53%) one or two exon 1 SNPs than patients in the other groups, although this was not significant (39%; P = 0.13 χ2 test).

In meningitis patients, the observed frequency of exon 1 SNPs was higher than expected from the genotype of their parents (53 % observed vs. 43 % expected); this was not the case for patients with septic shock (36% observed vs. 34% expected) or bacteraemia (29% observed vs. 38% expected). Using the log-linear model, we found that for this subgroup, the presence of 1 or 2 exon 1 SNPs was associated with an increased relative risk for disease (Table 3). This was significant in the case of heterozygousity for an exon 1 SNP. For homozygous exon 1 SNPs this was not significant, possibly due to the low number of patients. In the patients that had presented with septic shock or meningococcal bacteraemia, the presence

Table 3. Relative risk for meningococcal disease associated with an exon 1 SNP heterozygous P homozygous P all patients (n = 120) 1.66 (.91-3.0) 0.10 1.78 (.58-5.3) 0.31 shock n = (46) 1.13 (.43-3.0) 0.82 1.8 (.33-9.9) 0.51 bacteraemia n = (24) 1.07 (.29-4.3) 0.92 0.72 (.55-9.4) 0.80 meningitis n = (38) 3.9 (1.2-13) 0.03 3.77 (.49-29) 0.2 Relative risk for disease of haplotypes (+ 95% CI) associated with the D, B and C SNPs (YD, YB and YC, respectively) in exon 1 of the MBL2 gene, for all patients combined and for the patient groups shock, bacteraemia and meningitis. A reciprocal reference was used, in this case the combination of the Figure 3 haplotypes YA and XA. The patients that had presented with meningococcal septic shock in combination with meningitis could not be included in this analysis because of a too small number.

100

Figure 3. MBL plasma functional concentration (MBL-P) 1 in relation to disease type. MBL-P (mg/L) S indicates the patients that presented with shock, S+M the patients that presented with shock in combination with meningitis, B 0.1 indicates patients with bacteraemia and M indicates patients with meningitis. P = .13, .19 and .04 for the comparison of S, S+M and B with meningitis, respectively and P = .04 for 0.01 the comparison of S, S+M and B combined S S+M B M with meningitis. 16

247 of an exon 1 polymorphism was not associated with an increased risk for disease. This indicates that MBL-deficiency may predispose for meningococcal meningitis, but not for other clinical manifestations of meningococcal disease.

Frequency of exon 1 polymorphism in patients with group B meningococci was similar to that in patients with group C disease. Patients with group B disease had a similar non-significant relative risk for meningococcal disease as the complete group of patients (RR for heterozygousity 2.08 (95% CI 0.96-4.54) and RR for homozygousity 3.12, 95% CI 0.75-12.8). Additionally, the MBL genotype did not affect disease susceptibility in patients between 0.5 and 3.0 years (n = 48, RR for heterozygousity 1.18 95% CI 0.45-3.07 and RR for homozygousity 1.28, 95% CI 0.25-6.33).

MBL genotype in families with meningococcal disease Twelve families (10%), had a history of an additional first, second or third degree relative with meningococcal disease. In 8 families, at least one first degree relative had been affected; in 6 families this was a brother or sister, in 1 family this was the mother, and in one family both a sister and mother. Of these, 3 cases were co-primary or secondary. One family had a history of meningococcal disease in a second degree relative, 3 families had a history in a third degree relative. Frequency of exon 1 SNPs in patients with a positive family history was similar (heterozygousy 33 %, homozygousy 0%) to that seen in patients without a positive family history. MBL functional plasma concentration in patients with a positive family history was median 2.3 µg/L (range 0.15 - 4.50 µg/L). From 8 affected first degree relatives (6 sibs, 2 mothers) material was also available for genotyping, here frequencies of exon 1 SNPs were 37.5 % heterozygous and 0 % homozygous.

Discussion

In the present study we found that polymorphisms in the gene for MBL do not predispose for severe meningococcal disease necessitating ICU-treatment. However, SNPs in exon 1 may be a risk factor for meningococcal meningitis. MBL- deficiency is unlikely to be an important cause of familial meningococcal disease. MASP2 deficiency is not a frequent cause of meningococcal disease.

In recent years, it has been questioned whether MBL-deficiency should be seen as a factor of importance in susceptibility to infectious disease or that it should be considered as a redundant molecule with little impact on susceptibility 22,23. Evidence for the latter notion stems from contradictory results in case-control studies 13- 15,24,25, a large prospective trial in adults that failed to show an association of MBL

248 deficiency with infectious disease 12 and the high worldwide prevalence of multiple MBL2 deficiency or low-producing alleles 26. However, this does not exclude an effect of MBL-deficiency on specific infectious diseases or in particular patient or age groups 27-31. Our study does not support a role for MBL-deficiency in susceptibility for disease in a group of children with meningococcal infection admitted to a tertiary care ICU. Of the individual SNPs, only the regulatory polymorphism H showed a (non-significant) trend for preferential transmission to the patients. However, the H variant is not linked to MBL-deficiency, in contrast it is associated with higher plasma concentrations of MBL (median 1.97, range 0.04-5.99 for HH genotype, median 1.53 range 0.04-8.6 for H/L genotype and median 0.95 range 0.04-5.05 for LL genotype). In addition, we found no evidence that exon 1 polymorphisms, that lead to with deficiency of MBL from the plasma, are associated with increased susceptibility to meningococcal infection. Our findings correspond to the findings in two case-control studies concerning meningococcal disease 14,15 and is further supported by the observation that the frequency of exon 1 SNPs in our patient group (36% heterozygous and 5% homozygous) was equal to that in an unselected population of 9425 adult Danish patients (37% heterozygous and 5% homozygous) 12. Our study contrasts with the study of Hibberd et al., who studied the role of exon 1 polymorphisms in meningococcal disease in a group of patients similar in age and race. Of interest, frequency of the exon 1 polymorphisms in our patient group was significantly different from the control group in the study of Hibberd (22.1 % heterozygotes, P = 0.002 and 1.5 % homozygotes, P = 0.04; χ2-test) 13. This indicates that, had we used the same control group, we would have confirmed the results in the Hibberd study. However, the control group in the Hibberd study may have been subject to selection bias as only patients without infectious disease were chosen 13. The design of our study precludes this kind of selection, giving stronger support to our conclusions.

We classified our patients in four clinically relevant groups, based on the presence or absence of shock or meningitis, and confirmed that this classification reliably distinguishes patients with severe disease from those with mild disease 32. In a subgroup analysis, we found that exon 1 SNPs were associated with a significantly increased relative risk for meningitis. In these patients, also a lower median plasma concentration of MBL in comparison with the other groups was found, which supports this observation and indicates that the presence of these exon 1 SNPs directly affected MBL plasma concentration. Reportedly, terminal complement pathway deficiencies predispose to less severe manifestations of meningococcal disease 11. It is tempting to speculate that MBL-deficiency, similar to these terminal complement deficiencies, predisposes to less severe disease. Of note, this finding was done in a subgroup (n = 38) and should be confirmed in a larger cohort of 16

249 patients with only meningococcal meningitis.

MBL might be especially important in young children where it functions as an ante- antibody 29,33. Therefore, we analysed whether exon 1 SNPs were associated with susceptibility for disease in patients between 0.5 and 3 years old, but we found no increase in risk.

The group of patients included in this study is clinically highly relevant, as ICU patients have more severe disease and higher mortality rates than patients not necessitating ICU treatment, and comprises the complete spectrum of meningococcal disease 1. However, it is skewed on two sides. Patients with mild disease not necessitating ICU treatment and - for obvious reasons - patients with a fatal course were not included. Because exon 1 polymorphisms did not predispose to disease in the most severely ill patients (the shock patients) it is unlikely that inclusion of fatalities and their parents would have changed the results. As we found that exon 1 SNPs increased susceptibility to meningococcal meningitis, inclusion of non-ICU patients with meningitis might have strengthened our results for this group.

We confirmed earlier observations that MBL-plasma concentration is mostly determined by exon 1 SNPs, and that there is a moderate effect of the ‘X’ SNP in the promoter region 34. MBL/MASP2-activity (the ability of MBL to bind mannan and activate C4 via MASP2) was completely dependent on MBL-plasma concentration. The observation that there were no discrepancies between plasma concentration and activity suggests that there were no MASP-2 deficient patients in our population 35. This indicates that MASP-2 deficiency is not a frequent cause of meningococcal disease.

Previous reports have implicated MBL as a predisposing factor for familial meningococcal disease 36,37. We found a high number of patients (10 %) with a positive family history, from which 66 % had a first degree relative with meningococcal infection. Frequency of exon 1 SNPs in these patients with familial meningococcal disease was equal to that in the other patients, no patients were homozygous. This indicates that MBL-deficiency is not a major cause of familial meningococcal disease in patients admitted to an ICU. However, the high number of patients with a positive family history suggests that there are other, not yet defined, genetic factors that predispose to meningococcal infection, in addition to environmental and exposure factors.

In conclusion, mutations in the gene for MBL do not predispose for severe

250 meningococcal disease necessitating ICU-treatment. Exon 1 polymorphism in our study predisposed to development of meningococcal meningitis, it is conceivable that MBL-deficiency is a susceptibility factor for mild meningococcal disease in children.

251 References 1. Van Deuren M, Brandtzaeg P, Van der Meer JWM. Update on meningococcal disease with emphasis on pathogenesis and clinical management. Clin Microbiol Rev. 2000;13:144- 166 2. Reintjes R, Conyn-van Spaendonck MA. Carriage of meningococci in contacts of patients with meningococcal disease. Age and other risk factors need to be taken into account. BMJ. 1999;318:665-666 3. Conyn-van Spaendonck MA, Reintjes R, Spanjaard L, van Kregten E, Kraaijeveld AG, Jacobs PH. Meningococcal carriage in relation to an outbreak of invasive disease due to Neisseria meningitidis serogroup C in the Netherlands. J Infect. 1999;39:42-48 4. Bogaert D, Hermans PW, Boelens H, Sluijter M, Luijendijk A, Rumke HC, Koppen S, van Belkum A, de Groot R, Verbrugh HA. Epidemiology of nasopharyngeal carriage of Neisseria meningitidis in healthy Dutch children. Clin Infect Dis. 2005;40:899-902 5. Turner MW. Mannose-binding lectin: the pluripotent molecule of the innate immune system. Immunol. Today. 1996;17:532-540 6. Jack DL, Dodds AW, Anwar N, Ison CA, Law A, Frosch M, Turner MW, Klein NJ. Activation of complement by mannose-binding lectin on isogenic mutants of Neisseria meningitidis serogroup B. J. Immunol. 1998;160:1346-1353 7. Jack DL, Jarvis GA, Booth CL, Turner MW, Klein NJ. Mannose-binding lectin accelerates complement activation and increases serum killing of Neisseria meningitidis serogroup C. J Infect Dis. 2001;184:836-845. 8. Drogari-Apiranthitou M, Kuijper EJ, Dekker N, Dankert J. Complement activation and formation of the membrane attack complex on serogroup B Neisseria meningitidis in the presence or absence of serum bactericidal activity. Infect Immun. 2002;70:3752-3758 9. Turner MW, Hamvas RM. Mannose-binding lectin: structure, function, genetics and disease associations. Rev Immunogenet. 2000;2:305-322 10. Sjoholm AG, Jonsson G, Braconier JH, Sturfelt G, Truedsson L. Complement deficiency and disease: an update. Mol Immunol. 2006;43:78-85 11. Figueroa JE, Densen P. Infectious diseases associated with complement deficiencies. Clin.Microbiol. Rev. 1991;4:359-395 12. Dahl M, Tybjaerg-Hansen A, Schnohr P, Nordestgaard BG. A population-based study of morbidity and mortality in mannose-binding lectin deficiency. J Exp Med. 2004;199:1391- 1399 13. Hibberd ML, Sumiya M, Summerfield JA, Booy R, Levin M, and, the, Meningococcal, Research, Group. Association of variants of the gene for mannose-binding lectin with susceptibility to meningococcal disease. Lancet. 1999;353:1049-1053 14. Garred P, Michaelsen TE, Bjune G, Thiel S, Svejgaard A. A low serum concentration of mannan-binding protein is not associated with serogroup B or C meningococcal disease. Scand. J. Immunol. 1993;37:468-470 15. Tully J, Viner RM, Coen PG, Stuart JM, Zambon M, Peckham C, Booth C, Klein N, Kaczmarski E, Booy R. Risk and protective factors for meningococcal disease in adolescents: matched cohort study. Bmj. 2006;332:445-450 16. Spielman RS, McGinnis RE, Ewens WJ. Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am J Hum Genet. 1993;52:506-516 17. Pollack MM, Ruttimann UE, Getson PR. Pediatric risk of mortality (PRISM) score. Crit Care Med. 1988;16:1110-1116. 18. van Brakel MJ, van Vught AJ, Gemke RJ. Pediatric risk of mortality (PRISM) score in meningococcal disease. Eur J Pediatr. 2000;159:232-236. 19. Roestenberg M, McCall M, Mollnes TE, van Deuren M, Sprong T, Klasen I, Hermsen CC, Sauerwein RW, van der Ven A. Complement activation in experimental human malaria infection. Trans R Soc Trop Med Hyg. 2007;101:643-649 20. Petersen SV, Thiel S, Jensen L, Steffensen R, Jensenius JC. An assay for the mannan- binding lectin pathway of complement activation. J Immunol Methods. 2001;257:107-116

252 21. Gjessing HK, Lie RT. Case-parent triads: estimating single- and double-dose effects of fetal and maternal disease gene haplotypes. Ann Hum Genet. 2006;70:382-396 22. Casanova JL, Abel L. Human Mannose-binding Lectin in Immunity: Friend, Foe, or Both? J Exp Med. 2004;199:1295-1299 23. Klein NJ. Mannose-binding lectin: do we need it? Mol Immunol. 2005;42:919-924 24. Roy S, Knox K, Segal S, Griffiths D, Moore CE, Welsh KI, Smarason A, Day NP, McPheat WL, Crook DW, Hill AV. MBL genotype and risk of invasive pneumococcal disease: a case-control study. Lancet. 2002;359:1569-1573 25. Kronborg G, Weis N, Madsen HO, Pedersen SS, Wejse C, Nielsen H, Skinhoj P, Garred P. Variant mannose-binding lectin alleles are not associated with susceptibility to or outcome of invasive pneumococcal infection in randomly included patients. J Infect Dis. 2002;185:1517-1520 26. Verdu P, Barreiro LB, Patin E, Gessain A, Cassar O, Kidd JR, Kidd KK, Behar DM, Froment A, Heyer E, Sica L, Casanova JL, Abel L, Quintana-Murci L. Evolutionary insights into the high worldwide prevalence of MBL2 deficiency alleles. Hum Mol Genet. 2006;15:2650-2658 27. Turner MW. Mannose-binding lectin (MBL) in health and disease. Immunobiology. 1998;199:327-339 28. Eisen DP, Minchinton RM. Impact of mannose-binding lectin on susceptibility to infectious diseases. Clin Infect Dis. 2003;37:1496-1505 29. Koch A, Melbye M, Sorensen P, Homoe P, Madsen HO, Molbak K, Hansen CH, Andersen LH, Hahn GW, Garred P. Acute respiratory tract infections and mannose-binding lectin insufficiency during early childhood. Jama. 2001;285:1316-1321 30. Neth O, Hann I, Turner MW, Klein NJ. Deficiency of mannose-binding lectin and burden of infection in children with malignancy: a prospective study. Lancet. 2001;358:614-618 31. Vekemans M, Robinson J, Georgala A, Heymans C, Muanza F, Paesmans M, Klastersky J, Barette M, Meuleman M, Huet F, Calandra T, Costantini S, Ferrant A, Mathissen F, Axselsen M, Marchetti O, Aoun M. Low mannose-binding lectin concentration is associated with severe infection in patients with haematological cancer who are undergoing chemotherapy. Clin Infect Dis. 2007;44 32. Bovre K, Froholm LO, Gaustad P, Holten E, Hoiby EA. Some agent characteristics and their coexistence related to occurrence and severity of systemic meningococcal disease in Norway, Winter 1981-1982. NIPH Ann. 1983;6:75-84 33. Turner MW. Deficiency of mannan binding protein--a new complement deficiency syndrome. Clin. Exp. Immunol. 1991;86 Suppl 1:53-56 34. Madsen HO, Garred P, Thiel S, Kurtzhals JA, Lamm LU, Ryder LP, Svejgaard A. Interplay between promoter and structural gene variants control basal serum level of mannan- binding protein. J. Immunol. 1995;155:3013-3020 35. Stengaard-Pedersen K, Thiel S, Gadjeva M, Moller-Kristensen M, Sorensen R, Jensen LT, Sjoholm AG, Fugger L, Jensenius JC. Inherited deficiency of mannan-binding lectin- associated serine protease 2. N Engl J Med. 2003;349:554-560 36. Bax WA, Cluysenaer OJ, Bartelink AK, Aerts PC, Ezekowitz RA, van Dijk H. Association of familial deficiency of mannose-binding lectin and meningococcal disease [letter]. Lancet. 1999;354:1094-1095 37. Bathum L, Hansen H, Teisner B, Koch C, Garred P, Rasmussen K, Wang P. Association between combined properdin and mannose-binding lectin deficiency and infection with Neisseria meningitidis. Mol Immunol. 2006;43:473-479

253

Tom Sprong, Johanna van der Ven-Jongekrijg, Chris Neeleman, Jos W. M. van der Meer and Marcel van Deuren Submitted

Innate cytokine production capacity and clinical manifestation of meningococcal disease 17 Abstract

We analyzed the role of the innate production capacity for tumor necrosis factor (TNF), interleukin 1β (IL-1β), IL-12 and IL-10 in the clinical presentation and severity of meningococcal disease. Whole blood cultures from survivors of severe meningococcal disease obtained median 5.4 years after hospitalization were stimulated with meningococcal lipopolysaccharide and heat-killed Neisseria meningitidis bacteria. 113 survivors of meningococcal disease were included. We classified these patients according to clinical manifestation in four groups: shock (n = 43), both shock and meningitis (n = 11), bacteraemia (neither shock nor meningitis, n = 24) and distinct meningitis (n = 35). The classification into four groups stratifies these patients according to disease severity. No differences in whole blood cytokine production were found between the patients in these four groups. However, within the group of patients that had presented with shock, interleukin 1β and the interleukin 1β / interleukin 10 ratio were negatively correlated with disease severity (R = -0.35, P = 0.03 and R = -0.33, P = 0.04, respectively, PRISM score). Clinical manifestation of meningococcal disease cannot be explained by the innate production capacity of whole blood cultures for the cytokines TNF, IL-1β, IL-10 and IL-12. In patients that presented with shock, a low production capacity for interleukin 1β and a low interleukin IL-1β / interleukin 10 production ratio was associated with more severe disease.

256 Introduction

Invasive meningococcal disease occurs when, after penetration of the nasopharyngeal mucosa, a pathogenic strain of Neisseria meningitidis invades the bloodstream. As published over 20 years ago 1, invasive disease can present with distinct clinical manifestations defined by the presence or absence of shock or meningitis. This classification is pathophysiologically based on the rapidity of, and the compartment in which, meningococcal proliferation occurs. During meningococcal septic shock, meningococci multiplicate seemingly unimpeded in the bloodstream. As a consequence, a systemic inflammatory response (SIRS) is provoked by massive and systemic activation of a range of inflammatory systems. Among these the cytokine network is of particular interest. When outgrowth in the bloodstream is restricted, meningococci seeded into the subarachnoideal space can proliferate within that compartment. In this case, meningococcal meningitis with a localized inflammatory response develops 2.

Various components of the innate immune response are assumed to play a role in the control of outgrowth of meningococci in the bloodstream. One of these is the capacity to mount a cytokine profile that brings an adequate cellular defence mechanism into action. Cytokines influence meningococcal disease in a complex fashion during the early stage of infection, throughout clinically overt disease as well as during repair and recovery. Early studies found evidence that high levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF) and interleukin- 1β (IL-1β) elicited shock and disseminated intravascular coagulation (DIC) 3-5, but these cytokines were difficult to measure because of their short half-life. Later studies showed that other cytokines like IL-6, IL-12 and particularly IL-10, an anti- inflammatory cytokine responsible for the inactivation of monocytes 6, were more accurately measured and reflected the severity of disease better 7-9. Nowadays, the concept is that the balance between anti- and pro-inflammatory cytokines is a reliable marker for outcome 10,11.

Due to various polymorphisms in the genes for cytokines, their receptors, the intracellular pathways leading to cytokine production and post-transcriptional events, there are significant inter-individual differences in the amounts of cytokines produced after a certain stimulus. For meningococcal disease, various polymorphisms in the genes for cytokines or their receptors have been associated with altered risk for disease or disease severity and outcome 12. We hypothesised that upon contact with meningococci, the innate capacity to produce high amounts of pro-inflammatory cytokines or low amounts of anti-inflammatory cytokines, protects against the rapid outgrowth of bacteria in the bloodstream and determines

257 17 the clinical manifestation and severity of disease that develops.

To test this hypothesis we measured the in-vitro production of TNF, IL-1β, IL-12 and IL-10 in whole blood cultures stimulated with purified meningococcal LPS and with heat-killed (HK) meningococci in 113 survivors of meningococcal disease.

Patients and Methods Ethics Studies were done after approval of the local ethical committee and informed consent was obtained from and for all patients and parents.

Patients Between 1990 and 2005, 236 patients aged between 0.1 and 22.7 years with meningococcal disease were admitted to the ICU of the Radboud University Nijmegen Medical Centre, either transferred from other hospitals or as a primary admission. Diagnosis of meningococcal disease was made on typical presentation. Infection with N. meningitidis was considered proven when there was a positive culture of blood, skin biopsy or CSF or when Gram-negative diplococci were seen in Gram-stained biopsies or CSF.

Of the 236 patients, 22 (9.3 %) died. From 37 survivors the address could not be retrieved. The remaining 177 patients (or their parents) were invited to participate in the study; 62 of them did not respond or declined to participate, in two patients the whole blood stimulation was unsuccessful. Thus, 113 patients (63 males, 50 females) took part in the study. The median age of the patients at admission to the ICU was 3.4 years (range 0.1 – 22.7 years). The delay between admission and participation was median 5.4 years (range 0.4 – 14.3 years). The median age at inclusion in the study was 8.9 years (range 1.3 - 36.4 years).

Patient data were collected by retrospective chart analysis when admission was before 2002 (n = 102) and by prospective analysis after this date (n = 11). Patients were divided in four groups based on clinical manifestation (shock, shock plus meningitis, bacteremia, or meningitis). Shock was defined as systolic arterial pressure below the fifth percentile for age despite adequate volume expansion requiring prolonged (>12 hours) vasopressive (i.e. (nor)epinephrine or dopamine) support 13. Meningitis was diagnosed when there were > 105 leukocytes/L in CSF or when there were clinical signs of meningitis. Meningococcal bacteremia was diagnosed in patients having a typical presentation of meningococcal disease (sudden onset of fever and a purperic rash) in the absence of shock or meningitis.

258 PRISM scores were calculated from data during the first 24 hrs of ICU admission 14,15. Data on sequelae were also obtained.

Whole blood cultures Sterile, endotoxin-free heparinized blood from all 113 patients was diluted 1:1 in RPMI 1640 and incubated in polypropylene cryotubes (Nalgene NUNC, Roskilde, Denmark) with RPMI (blank), with 1x107/mL heat-killed (HK) serogroup B N. meningitidis H44/76 and with 10 ng/mL purified LPS (matching the amount of LPS present on HK bacteria) isolated by phenol extraction from this strain 16. Meningococci and meningococcal LPS were a kind gift of Dr. P. van der Ley, at the Netherlands Vaccine Institute. Samples were incubated for 20 hrs at 37 °C, centrifuged at 3500 RPM whereafter the plasma was collected and stored for analysis at –80 °C. In the control (unstimulated) samples median cytokine concentrations were equal to the lower detection limit of the multiplex cytokine assay.

Cytokine analysis TNF, IL-1β, IL-10 and IL-12 were measured using commercially available Bio-Plex protein array system (Bio-Rad Laboratories, Inc.) 17. Lower limits of detection were 8 pg/mL for TNF, IL-1β, IL-12 and IL-10.

Statistical analysis Tests were performed using Graph Pad Prism 4 software. χ2 and Mann-Whitney test for non-parametrical data were used to compare the different patient groups. Spearman correlation was used to calculate correlation coefficients and R-square values. Bonferroni correction for multiple testing was used for the analyses in Table 1; P < 0.083 was considered significant.

Results Patient characteristics The clinical characteristics of the patients according to their clinical manifestation are shown in table 1. Patients with meningococcal septic shock (S) were most severely ill, as evidenced by the lowest blood pressure, the highest lactate concentrations, the most severe DIC with lowest platelet numbers and lowest fibrinogen concentrations, the highest PRISM-score, the longest stay in the ICU and the highest number of sequelae. Of note, patients with shock also had the lowest CRP concentrations at admission and the shortest time from onset of symptoms to admission. The latter two parameters indicate a very rapid course and fulminant disease. Patients with shock plus meningitis (S+M) had a lower disease severity and less fulminant disease than the patients with shock alone. Disease

259 17 Table 1. Patient data according to presentation shock Shock and bacteraemia meningitis meningitis N 43 11 24 35 m / f (n) 26/17 5/6 13/11 18/17 Age admission (y) 2.4 (0.5-15)* 2.5 (0.1-14) 3.4 (0.3-8)* 5.1 (0.4-22) Age inclusion (y) 8.4 (1.4-29) * 9.2 (4.5-22) 7.4 (2.1-14) * 12.4 (2.5-36) T1 (h) 12 (3.5-20)* 14 (6-24)* 14 (2.8-48)* 23 (10-72) T2 (h) 16 (6-38)* 18 (11-27)* 18 (3-51)* 25 (12-44) Proven N. men (%) 95 % 100 % 83 % 86 % Group B (%) 82 % 81 % 81 % 88 % PRISM 16 (6-34)#,* 12 (6-26)#,* 6 (0-25)* 3 (0-14) MAP (mmHg) 60 (35-89)#,* 59 (42-100)* 75 (53-141) 78 (57-112) ) CRP (mg/l) 61 (9-154)µµ 78 (34-150) 87 (2-174) 147 (37-237) Lactate (mmol/l) 4.1 (1.5-10.8)#,* 2.3 (1.2-6.9) 2.0 (1.0-5.3) 2.8 (0.9-4.8) Fibrinogen (g/l) 1.6 (0.1-3.8)#,* 2.1 (0.8-4.6)* 2.9 (0.8-6.3) 4.1 (2.2-6.8) Platelets (109/l) 70 (10-276)&,#,*, 159 (39-284)& 159 (24-291) 188 (69-432) Days in ICU 7 (1-30)#,* 6 (2-9)#,* 2 (1-9) 1 (1-4) Sequelae (%) 77 % (40 %)#,* 36 % (0 %) 33 % (0 %) 20 % (0 %) Patients were dived into four groups, based on clinical presentation. Presented are medians and ranges, unless otherwise indicated). Group B indicates percentage of serogroup group B N. meningitidis, rest was group C, no other serogroups isolated. Sequelae indicate all sequelae reported by patients or parents; severe sequelae, defined as severe scars, amputations, paralysis or prolonged hypocortisism is shown between brackets. m = males, f = females, T1 = time from first symptom to admission to the initial hospital, T2 = time from first symptom to admission at the ICU of the Radboud University Nijmegen Medical Centre, N. men = Neisseria meningitidis, PRISM = pediatric risk of mortality, MAP = mean arterial pressure, CRP = C-reactive protein. Differences were tested for significance using Mann-Whitney test or χ2-test when appropriate, and corrected for multiple testing using Bonferroni correction: P < 0.0083 was considered significant. * indicates significance for the comparison with the meningitis group, # indicates significance for comparison with the bacteraemia group, & indicates significance for the comparison with the shock and meningitis group

severity in this group was higher than in the patients with bacteraemia (B), but the time from first symptom to presentation was similar. Patients with meningococcal meningitis (M) had the lowest disease severity and the longest duration between onset of symptoms to admission, reflecting less fulminant disease (Table 1, Figure 1). These data indicate that the simple classification of patients in these four groups is clinically relevant and reflects severity and fulminancy of the disease. No differences were found in any of the clinical characteristics between the male and female patients (not shown).

Cytokine production in whole blood cultures In unstimulated whole blood cultures no or only limited cytokine production occurred. After stimulation with purified meningococcal LPS, substantial amounts of TNF, IL-1β, IL-12 and IL-10 were produced. As expected 18, HK N. meningitidis was a more potent inducer of cytokines (Table 2). The production of TNF, IL-1β, IL-12 and IL-10 after stimulation with meningococcal LPS was highly correlated to cytokine production by HK N. meningitidis (Spearman R respectively 0.84, 0.76, 0.81 and 0.74; all P < 0.0001). There was no relation between age at inclusion

260 40 40 SHOCK SHOCK AND 35 35 MENINGITIS

30 30

25 25

20 20 PRISM 15 15

10 10

5 5

0 0 0 12 24 36 48 0 12 24 36 48 40 40 BACTERAEMIA MENINGITIS 35 35

30 30

25 25

20 20 PRISM 15 15 Figure 1 10 10 Pediatric risk of mortality 5 5 (PRISM) score plotted against time between onset 0 0 of symptoms and admission 0 12 24 36 48 0 12 24 36 48 for the different patient time from disease onset to admission time from disease onset to admission groups.

Table 2. Cytokine production by whole blood cultures stimulated for 20 hrs with purified meningococcal LPS or HK Neisseria meningitidis TNF (pg/mL) IL-1β (pg/mL) IL-12 (pg/mL) IL-10 (pg/mL) Meningococcal LPS 3660 8075 27 1063 (237-15473) (940-30147) (8-257) (118-3225) HK N. meningitidis 7259 19624 44 1566 (990-23665) (3478-90653) (12-323) (297-10326) Presented are medians and ranges, differences between LPS and N. meningitidis were all significant (P < .001, by Mann-Whitney test). and the amounts of TNF, IL-1β and IL-12 produced; there was a weak negative correlation between IL-10 and age at inclusion (Spearman R = -0.21, P = .03). Induction of IL-1β production after stimulation with LPS in female patients was 73 % lower than male patients (P = 0.02), no significant differences were seen between males and females for the other cytokines or for HK N. meningitidis.

The production of TNF, IL-1β and IL-12 after stimulation with LPS and HK meningococci was strongly correlated. In contrast, IL-10 production correlated only weakly with the production of these cytokines (Table 3), suggesting a different regulation mechanism for the production of IL-10 as compared to that for TNF, IL- 1β or IL-12. No significant differences in whole blood cytokine production, stimulated either with meningococcal LPS (Figure 2) or with HK N. meningitidis (data not shown), were found between patients stratified according to their clinical manifestation. There was also no difference for the TNF/IL-10 ratio and the IL-1β/IL-10 ratio.

Similarly, no significant significant correlation existed between the production of

261 17 Table 3. Spearman correlation coefficient for production of TNF, IL-1β, IL-12 and IL-10 by whole blood cultures stimulated for 20 hrs with meningococcal LPS TNF (pg/mL) IL-1β (pg/mL) IL-12 (pg/mL) IL-1β R = 0.59 (P < .0001) R2 = 0.35 IL-12 0.60 (P < .0001) 0.48 (P < .0001) R2 = 0.36 R2 = 0.23 IL-10 0.27 (P = .006) 0.26 (P = .008) 0.25 (P = .009) R2 = 0.07 R2 = 0.07 R2 = 0.06

the measured cytokines or the ratio of TNF-to-IL-10 or IL-1β-to-IL-10, and disease severity as expressed by the PRISM-score for all patients combined. However, for the group of patients with shock, the severity of disease correlated negatively and significantly with the IL-1β production (R = -0.35, P = 0.03, R = -0.21, P = 0.17, for stimulation with LPS and HK N. meningitidis, respectively). In this group no correlation was found between PRISM and IL-10 (R = -0.02 P = 0.89 and R = 0.23, P = 0.14 for LPS and HK N. meningitidis, respectively). However, there was a significant negative correlation between disease severity and the ratio of IL-1β/ IL-10 production (R = -0.33, P = 0.04 and R = -0.36, P = 0.02 for LPS and HK N. meningitidis respectively). The TNF- and IL-12 production, and the TNF/IL10 ratio were not correlated with disease severity.

100000 100000

10000 10000 (pg/mL) β IL-1 TNF (pg/mL) 1000 1000

100 100 S S+M B M S S+M B M

1000 10000

100 1000

100

10 IL-10 (pg/mL) IL-12 (pg/mL)

1 10 S S+M B M S S+M B M

Figure 2 Cytokine production after stimulation with meningococcal LPS by whole blood cultures of survivors of meningococcal disease according to patient groups shock (S), shock and meningitis (S+M), bacteraemia (B) and meningitis. Bars indicate medians. Differences between groups were tested using Mann-Whitney test, no significant differences for any of the cytokines were found.

262 25000 25000 25000 R = - 0.35 R = 0.38 R = - 0.33

20000 P = 0.03 20000 P = 0.02 20000 P = 0.06

15000 15000 15000 (pg/mL)

β 10000 10000 10000 IL1

5000 5000 5000

0 0 0 0 10 20 30 40 0 50 100 150 200 250 300 0 2 4 6 8 10 12

60 60 60 R = - 0.33 R = 0.46 R = - 0.43 50 P = 0.04 50 P = 0.003 50 P = 0.01

/IL-10 40 40 40 β

30 30 30

Ratio IL1 20 20 20

10 10 10

0 0 0 0 10 20 30 40 0 100 200 300 0 4 8 12

PRISM platelets lactate Figure 3 Correlation of IL-1β and the IL-1β/IL-10 ratio with PRISM, platelet count (x109/L) and lactate (mmol/L) concentrations, after stimulation of whole blood cultures with meningococcal LPS. R indicates Spearman correlation coefficient. For visual purposes, one outlier was removed from the graphs showing the IL-1β/IL- 10 ratio, this outlier was not excluded from the correlation analysis. PRISM = Pediatric risk of mortality.

The IL-1β production and the IL-1β-to-IL-10 ratio in shock patients showed also a significant correlation with other parameters of disease severity, like platelet count and serum lactate concentration (Figure 3). No significant correlation was found with admission values of blood pressure, fibrinogen or the number of days spent in ICU.

Discussion

In the present study we made two major observations. First, in contrast to our hypothesis, we found that the clinical manifestation of meningococcal disease cannot be explained by the innate production capacity of the cytokines TNF, IL- 1β, IL-10 and IL-12 in whole blood cultures. Second, in patients that presented with shock, a low production capacity for IL-1β and a low ratio of IL-1β-to-IL-10 production was associated with more severe disease.

In 1983, Bøvre and Høiby introduced a simple system that classified patients based on the absence or presence of shock and meningitis 1. We confirmed that this classification reliably distinguishes patients with a rapid evolution of disease due to the rapid outgrowth of meningococci in the bloodstream, from those who had a less fast evolution thanks to a better control 19-21. This makes this classification ideally

263 17 fitted for the study of factors that determine early outgrowth of meningococci.

Our study population is clinically highly relevant and reflects the complete spectrum of invasive meningococcal disease. However, it is skewed on two sides. On one side, patients with meningitis or bacteremia and a mild course are not included because these patients are not referred to an ICU. On the other side, we miss - for obvious reasons - the most severely ill shock patients, i.e. those who died. Nonetheless, we excellently characterized the patients with regard to clinical manifestation and severity of disease and therefore believe that the conclusions drawn are solid. The age of the patients, at the time the stimulation tests were performed, was median 5.4 years older than the age at disease onset. This gap however, probably did not affect the results because age had only a minor effect on cytokine production.

In order to mimic the in vivo situation as closely as possible we used for the in vitro whole blood stimulation experiments, HK N. meningitidis and purified meningococcal LPS concentrations comparable to those encountered in vivo during disease 19-21. In addition, we chose to incubate for 20 hrs to allow measurement of TNF, IL-1β, IL-12 and IL-10 in the same culture. We confirmed that HK N. meningitidis is more potent stimulus than purified meningococcal LPS 18. In addition, we found that cytokine production after stimulation with HK N. meningitidis and LPS are strongly correlated, suggesting that LPS is the dominant cytokine-inducing moiety of meningococci 18,22. Furthermore, we found a positive correlation between the amount of TNF, IL-1β and IL-12 produced, suggesting that production of these cytokines shares at least some regulatory pathways. In contrast, IL-10 did not follow this pattern, indicating that IL- 10 production is regulated by different mechanisms 23.

Our results differ from those of Westendorp et al. who studied TNF and IL-10 production in relatives of 61 patients with meningococcal disease and found low TNF and high IL-10 production correlating with severe disease 24. Westendorp however, used LPS derived from E.coli in concentrations (1000 ng/mL) far exceeding those encountered in genuine disease. In contrast, we studied patients and used HK meningococci and meningococcal LPS in clinically relevant concentrations 22. We believe that these modifications and the larger number of patients (n = 113) evaluated, give stronger support to our conclusions.

Our in vitro whole blood culture system has two limitations. First, it does not contain cells not present in blood (such as endothelial cells, and Kupfer cells in the liver) that in vivo may have been responsible for cytokine production 25,26. Second, information about the early kinetics of cytokine production is missed as only the amount (and ratio) of cytokines accumulated in the culture after 20 hrs is measured. However,

264 we speculate that other non-cytokine components of the innate immune system, either alone or in synergy with bacterial factors, may be more relevant for the early outgrowth of meningococci and hence the type of disease that develops. Of interest, whole blood killing of meningococci is critically dependent on a functional complement system 27,28.

Once meningococcal septic shock has been established, multiple factors including the delay to effective treatment 29-31, bacterial properties 32 and host polymorphisms 12 influence disease severity. We show in the present study, that a low production capacity for IL-1β and a low ratio of IL-1β-to-IL-10 production is associated with more severe disease. This finding is supported by the studies of Read et al. 32,33 who found that variations in the promotor gene for IL-1β, causing less production, are associated with increased mortality. Moreover, a genetically high production capacity for IL-1 receptor antagonist (IL-1Ra), the major inhibitor of IL-1β action, has also been shown to predispose to more severe disease 32,34,35. Taken together, these findings are consistent with a crucial and protective role for IL-1β in the progression of meningococcal septic shock.

When produced in excessive amounts, IL-1β is believed to be deleterious 5,36. This seems to contrast with our finding of a harmful effect of less IL-β production and a lower ratio of IL-1β-to-IL-10 production as assessed in-vitro. This paradox has puzzled various authors before 24,32,37,38. One of the explanations is that the ineffective early immune response results in higher bacterial outgrowth and hence more severe disease. However, our finding that outgrowth of meningococci (as defined by type of presentation) is not influenced by the capacity to mount a certain pro- or anti-inflammatory cytokine response, refutes this opinion. Rather, our findings underscore the importance of a balanced pro- and anti-inflammatory reaction not only during the very early stage after bacterial invasion, but also during the subsequent progression of disease 10,11,39. When at any moment during the course of the disease this balance is lost, the inflammatory response runs out of bounds and high plasma concentrations of cytokines accompany severe disease irrespective of the innate production capacity for these cytokines.

In sum, our study indicates that the clinical manifestation of meningococcal disease is not determined by the innate production capacity for the cytokines TNF, IL-1β, IL-10 and IL-12 as measured in whole blood cultures. However, disease severity in patients with meningococcal septic shock is positively influenced by the capacity to mount an adequate IL-1β response. This latter finding may possibly explain the failure of adjunctive therapies in sepsis aimed at inhibiting IL-1β 40,41.

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266 meningococcal disease. Prog Clin Biol Res. 1995;392:219-233 20. Hackett SJ, Guiver M, Marsh J, Sills JA, Thomson APJ, Kaczmarski EB, Hart CA. Meningococcal bacterial DNA load at presentation correlates with disease severity. Arch Dis Child. 2002;86:44-46 21. Ovstebo R, Brandtzaeg P, Brusletto B, Haug KB, Lande K, Hoiby EA, Kierulf P. Use of robotized DNA isolation and real-time PCR to quantify and identify close correlation between levels of Neisseria meningitidis DNA and lipopolysaccharides in plasma and cerebrospinal fluid from patients with systemic meningococcal disease. J Clin Microbiol. 2004;42:2980-2987 22. Sprong T, Moller ASWM, Bjerre A, Wedege E, Kierulf P, van Der Meer JW, Brandtzaeg P, van Deuren M, Mollnes TE. Complement activation and complement-dependent inflammation by Neisseria meningitidis occurs independent of lipopolysaccharide. Infect Immun. 2004;72:3344-3349 23. Netea MG, Sutmuller R, Hermann C, Van der Graaf CA, Van der Meer JW, van Krieken JH, Hartung T, Adema G, Kullberg BJ. Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells. J Immunol. 2004;172:3712-3718 24. Westendorp RG, Langermans JA, Huizinga TW, Elouali AH, Verweij CL, Boomsma DI, Vandenbroucke JP. Genetic influence on cytokine production and fatal meningococcal disease. Lancet. 1997;349:170-173 25. Giroir BP, Johnson JH, Brown T, Allen GL, Beutler B. The tissue distribution of tumor necrosis factor biosynthesis during endotoxemia. J Clin Invest. 1992;90:693-698 26. Huston JM, Ochani M, Rosas-Ballina M, Liao H, Ochani K, Pavlov VA, Gallowitsch- Puerta M, Ashok M, Czura CJ, Foxwell B, Tracey KJ, Ulloa L. Splenectomy inactivates the cholinergic antiinflammatory pathway during lethal endotoxemia and polymicrobial sepsis. J Exp Med. 2006;203:1623-1628 27. Figueroa JE, Densen P. Infectious diseases associated with complement deficiencies. Clin.Microbiol. Rev. 1991;4:359-395 28. Sprong T, Brandtzaeg P, Fung M, Pharo AM, Hoiby EA, Michaelsen TE, Aase A, van der Meer JW, van Deuren M, Mollnes TE. Inhibition of C5a-induced inflammation with preserved C5b-9-mediated bactericidal activity in a human whole blood model of meningococcal sepsis. Blood. 2003;102:3702-3710 29. Welch SB, Nadel S. Treating meningococcal infections in children. Hosp Med. 2002;63:268-273 30. Nadel S, Britto J, Booy R, Maconochie I, Habibi P, Levin M. Avoidable deficiencies in the delivery of health care to children with meningococcal disease. J Accid Emerg Med. 1998;15:298-303 31. Ninis N, Phillips C, Bailey L, Pollock JI, Nadel S, Britto J, Maconochie I, Winrow A, Coen PG, Booy R, Levin M. The role of healthcare delivery in the outcome of meningococcal disease in children: case-control study of fatal and non-fatal cases. Bmj. 2005;330:1475 32. Read RC, Cannings C, Naylor SC, Timms JM, Maheswaran R, Borrow R, Kaczmarski EB, Duff GW. Variation within genes encoding interleukin-1 and the interleukin-1 receptor antagonist influence the severity of meningococcal disease. Ann Intern Med. 2003;138:534-541 33. Read RC, Camp NJ, di Giovine FS, Borrow R, Kaczmarski EB, Chaudhary AG, Fox AJ, Duff GW. An interleukin-1 genotype is associated with fatal outcome of meningococcal disease. J Infect Dis. 2000;182:1557-1560. 34. Endler G, Marculescu R, Starkl P, Binder A, Geishofer G, Muller M, Zohrer B, Resch B, Zenz W, Mannhalter C. Polymorphisms in the interleukin-1 gene cluster in children and young adults with systemic meningococcemia. Clin Chem. 2006;52:511-514 35. Balding J, Healy CM, Livingstone WJ, White B, Mynett-Johnson L, Cafferkey M, Smith OP. Genomic polymorphic profiles in an Irish population with meningococcaemia: is it possible to predict severity and outcome of disease? Genes Immun. 2003;4:533-540 36. Okusawa S, Gelfand JA, Ikejima T, Connolly RJ, Dinarello CA. Interleukin 1 induces a shock-like state in rabbits. Synergism with tumor necrosis factor and the effect of cyclooxygenase inhibition. J. Clin.Invest. 1988;81:1162-1172

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268 269 17

Summary and General Discussion 18 The dreadful consequences of meningococccal septic shock are due to the breakdown of homeostasis by a massive inflammatory response with the cytokine network and the complement system as key players. Understanding what happens and how this happens is a prerequisite to find treatments that are able to modulate this response in a way that preserves the beneficial bactericidal effects of the inflammatory response while attenuating the detrimental systemic effects.

In textbooks on infectious diseases and host defence, it is generally accepted that the inflammatory events during meningococcal and other Gram-negative septic shock are primarily elicited by the lipopolysaccharide (LPS) component of the bacterial outer membrane. In chapters 2 - 4, we used a genetically modified meningococcus (H44/76lpxA or H44/76[pLAK33]) that is deficient for LPS to study the relative importance of LPS versus non LPS components of meningococci in the induction of cytokines and the activation of complement.

In chapter 2 we demonstrated that non-LPS components of meningococci are responsible for a substantial part of tumor necrosis factor (TNF)-α and interleukin (IL)-1β production and virtually all interferon (IFN)-γ production in human peripheral blood mononuclear cells. In addition, experiments with mice showed that LPS- deficient meningococci were able to induce cytokine production and death, albeit that approximately 100-fold more LPS-deficient meningococci were needed to cause mortality in mice than 'wild-type' LPS containing meningococci (Neisseria meningitidis H44/76). We conclude that N. meningitidis can induce cytokines and disease independent of LPS.

CD14 and TLR4 are reported to be the principle cellular receptors for LPS, therefore, in chapter 3, we investigated the role of the CD14-TLR4 receptor complex for cytokine induction by meningococcal LPS and non-LPS components of meningococci. WT14, a monoclonal antibody against CD14, abolished TNFα and IL-1β induction by Escherichia coli LPS in human mononuclear cells, while cytokine induction by meningococcal LPS or LPS-containing N. meningitidis H44/76 and LPS-deficient H44/76lpxA meningococci was only partly inhibited by anti-CD14. Experiments with macrophages isolated from TLR4-deficient mice, revealed that TLR4 is crucial for cytokine production by meningococcal LPS, but LPS-containing or LPS-deficient meningococci were able to induce a significant cytokine response in TLR4-deficient macrophages, similar to that of TLR4-containing murine macrophages. Thus, N. meningitidis induced cytokines in human PBMCs or murine peritoneal macrophages by pathways independent from the LPS receptors CD14 and TLR4.

272 In chapter 4, we investigated the involvement of LPS and non-LPS components of meningococci in complement activation and complement-dependent inflammatory effects in a human whole blood model of meningococcal sepsis. The LPS containing H44/76 and LPS deficient H44/76lpxA N. meningitidis strains both activated complement effectively and to a similar extent. In contrast, purified meningococcal LPS, used at a concentration matched to the amount present in whole bacteria, did not induce any complement activation. Both complement receptor-3 up-regulation and oxidative burst in neutrophils and monocytes were induced by N. meningitidis independent of LPS. Thus, in the whole blood model of meningococcal sepsis, complement activation and the immediate complement-dependent inflammatory effects of CR3 up-regulation and oxidative burst induced by meningococci occurred independent of LPS.

The conclusions from chapters 2 - 4 imply that therapeutic strategies designed to block only the effects of LPS or the LPS-receptors CD14 and TLR4 will have a partial effect on the induction of cytokines, and will not stop complement activation at all. Therefore, it is doubtful that such therapies will modify the course of disease. Our findings may thus explain the failure of earlier anti-endotoxin therapies to influence morbidity or mortality in a positive way.

In chapter 5 we investigated whether lipoproteins - compounds which had recently gained much attention as a promising treatment option for Gram-negative sepsis because they are able to inhibit cytokine production induced by E. coli LPS - had an effect on cytokine production by meningococcal LPS or whole Gram-negative bacteria. Therefore, we assessed the neutralizing effect of low-density lipoproteins (LDL), high-density lipoproteins (HDL), and very low-density lipoproteins (VLDL) on LPS- or whole bacteria-induced cytokine production in human mononuclear cells. A strong inhibition of E. coli LPS-induced cytokines by LDL and HDL was seen, whereas VLDL had a less pronounced effect. In contrast, N. meningitidis LPS, in similar concentrations, was neutralized much less effectively than E. coli LPS. Effective neutralization of meningococcal LPS required a prolonged interaction time, a lower concentration of LPS, and higher concentrations of lipoproteins. Minimal neutralizing effects of the lipoproteins were observed on whole E. coli or N. meningitidis bacteria under all conditions tested. These findings raise questions about the rationale for adjuvant treatment of meningococcal and other types of Gram-negative sepsis with lipoproteins.

An unexpected, but interesting approach to inhibit TLR4 dependent cytokine production comes from the LPS-molecule itself. In chapter 6, we investigated novel LPS species derived from N. meningitidis H44/76 by insertional inactivation of the

273 18 lpxL1 and lpxL2 gene that have a lipid A portion consisting of five (pentaacylated lpxL1) or four (tetraacylated lpxL2) fatty acids. We found that these novel LPS species functioned as LPS antagonists in humans by inhibiting cytokine production dependent on toll-like receptor 4 (TLR4). In contrast to the situation in humans, lpxL1 LPS was an agonist for cytokine production in peritoneal macrophages of mice, and exacerbated arthritis in a murine collagen induced arthritis model. Although - in the light of the studies presented earlier in this thesis - it is not expected that inhibiting LPS or TLR4 alone will have a considerable beneficial effect on the course of meningococcal septic shock, the LPS antagonists might be useful in other inflammatory diseases that are TLR4 dependent such as rheumatoid arthritis, asthma or and inflammatory bowel disease.

From chapters 2 - 6 we have learned that it may be unwise to focus only on LPS as a disease inducing element of N. meningitidis. The ultimate strategy to stop the systemic inflammation induced by the meningococcus would neutralise all elements of the bacterium that provoke inflammation. Future research should focus on identifying the non-LPS meningococcal components and their receptors that are responsible for the production of cytokines and the activation of complement. It may not be possible to find one magic bullit to stop the inflammatory response at the level of the bacterium or immune receptors. Maybe we need something more like a cluster bomb - multiple molecules that have an effect on multiple components of meningococci or multiple cellular and plasmatic receptors.

In chapter 7 we studied whether cytokine production induced by meningococci is influenced by mannose-binding lectin (MBL), a key molecule in the lectin pathway of complement activation. We found that MBL significantly augmented IL-1β and IL-10 production after stimulation with LPS-deficient meningococci, in a dose-dependent manner. In contrast, TNFα, IL-6 and IFNγ productions were unaffected. No effect of MBL was observed on cytokine induction by meningococcal LPS. We concluded that MBL interacted with non-LPS components of N. meningitidis and in this way selectively modulated the cytokine response. Our findings link the complement system to cytokine production. Whether the augmented cytokine production by MBL contributes to the defensive function of MBL to meningococcal infections is subject to future research.

Chapters 8 - 12 of this thesis focus on complement activation by N. meningitidis in vitro as well as in vivo. The importance of the complement system in meningococcal disease is emphasized by several observations. Complement deficiencies are defined risk factors for meningococcal infections, indicating that complement is crucial in the initial defence against this bacterium. On the other hand, during

274 fulminant meningococcal sepsis, disease severity, tissue damage, and outcome are closely related to the degree of complement activation. Thus, with respect to the pathogenesis of meningococcal disease, the complement system has been aptly named a double-edged sword.

In chapter 8 we studied the effect of complement inhibition on inflammation and bacterial killing in a novel human whole blood model of meningococcal sepsis. We found that the mannose-binding lectin and alternative complement pathway were crucial for complement activation by N. meningitidis and oxidative burst induced in granulocytes and monocytes. Inhibition of C5a, using mAb 137-26 binding the C5a moiety of C5 before cleavage, prohibited CR3 up-regulation, phagocytosis, and oxidative burst but had no effect on C5b-9 (TCC) formation, lysis, and bacterial killing. A monoclonal antibody that blocks cleavage of and in this way prevents C5a and TCC formation, showed the same effect on CR3, phagocytosis, and oxidative burst as the anti-C5a mAb but additionally inhibited TCC formation, lysis, and bacterial killing, consistent with a C5b-9-dependent killing mechanism. In conclusion, the anti-C5a mAb 137-26 inhibited the potentially harmful effects of N meningitidis-induced C5a formation while preserving complement-mediated bacterial killing. We suggest that this is an attractive approach for the treatment of meningococcal sepsis.

Chapter 9 highlights the importance of the alternative pathway of complement in the defence against meningoccoci. We describe 2 cases of meningococcal septic shock, 1 of them fatal, in 2 children of a Turkish family. In the surviving patient, alternative pathway activation was absent and factor D plasma concentrations were undetectable. Mutation analysis of the factor D gene revealed a T638 > G (Val213 > Gly) and a T640 > C (Cys214 > Arg) mutation in the genomic DNA from the patient, both in homozygous form. In vitro incubation of factor-D deficient plasma of the boy with serogroup B N. meningitidis showed normal MBL-mediated complement activation but no formation of the alternative pathway C3-convertase C3bBbP, and severely decreased C3bc formation and terminal complement activation. The defect was restored after supplementation with factor D. Thus, Factor D deficiency leads to reduced alternative-pathway dependent complement activation by meningococci, and in this way to an increased susceptibility to invasive disease.

In Chapter 10 we investigated in patients with meningococcal septic shock and meningococcal disease without shock to what extent the alternative and lectin pathways of complement contribute to the systemic activation of complement and the ensuing tissue damage. In the patients with meningcoccal septic shock, high systemic concentrations of complement activation products C3bc (indicating

275 18 common pathway activation) and TCC (terminal pathway activation) were seen. Systemic activation of complement excellently correlated with disease severity and parameters of DIC. We also found high concentrations of C3bBbP, indicating alternative pathway activation. Two of the shock patients were MBL-deficient and had much lower circulating values of C3bc and TCC than MBL-sufficient patients that presented with shock. Interestingly, these MBL-deficient patients had relatively low disease severity and mild disseminated intravascular coagulation (DIC). This indicates that MBL is critical for the systemic activation of complement during meningococcal septic shock.

In chapter 11 the restriction fragment length polymorphism genotyping method we used for determination of MBL genotypes is presented. We developed a PCR-RFLP method for the X/Y promoter polymorphism of the MBL gene that works with the previously described PCR-RFLP method for the three exon 1 variants to detect the mutations in the MBL gene that cause MBL deficiency.

Chapter 12 provides a concise review of the diverse actions of the complement system in sepsis, emphasising on the defence against infection and the damage done by uncontrolled systemic activation of the complement system.

The data from chapters 8 - 12 show that MBL the and alternative pathway are crucial for the activation of complement during meningococcal septic shock. Inhibition of C5a may be an attractive approach to diminish the devastating effects of septic shock especially because it preserves complement-mediated bacterial killing. It is interesting that the activation of complement was closely related to the development of disseminated intravascular coagulation (DIC) and thrombcytopenia. DIC is caused by activation of the coagulatory system and causes much of the severe sequelae associated with meningococcal disease such as amputations. This may be mediated by C5a-induced upregulation of tissue factor, but is incompletely understood. The pathogenesis of DIC and ways to influence the morbidity associated with it deserves further exploration.

In chapters 13 - 15 we studied various new mediators in patients with meningococcal septic shock.

Vascular endothelial growth factor (VEGF) is a central factor in angiogenesis and is an important mediator of vascular permeability. In chapter 13, patients with meningococcal infection were investigated in the early phase of invasive meningococcal disease. VEGF plasma concentrations were increased and were highest in the presence of shock compared with patients presenting without shock.

276 VEGF concentration at admission correlated with the severity of disease and the amount of fluids administered in the first 24 h. In all patients, a decrease in VEGF was associated with a decrease in fluid intake during t = 24 to 48 h. The results suggest that microvascular permeability in sepsis is closely linked to the plasma concentration of VEGF. The role of VEGF in sepsis-associated increased microvascular permeability needs further exploration and may represent a new therapeutic target.

In chapter 14 we studied macrophage migration inhibitory factor (MIF), a mediator of innate immunity and important in the pathogenesis of septic shock in-vivo in patients with meningococcal disease, in human experimental endotoxaemia and in whole blood cultures. Using a newly developed sensitive and specific enzyme- linked immunosorbent assay (ELISA), we found that MIF was increased in patients with meningococcal disease and highest in the presence of shock. MIF could not be detected in a human endotoxaemia model although high concentrations of TNFα were found. In addition, MIF was not produced by whole blood cells incubated with LPS or meningococci. Therefore, it can be questioned whether MIF in human sepsis is induced by LPS or TNFα. Our observations suggest that MIF plays a role in the development of shock in patients with sepsis, mediated in concert with other pathological events.

Chapter 15 studied the long pentraxin PTX3, an important element of the innate immune system and a potential candidate as a diagnostic tool in inflammatory conditions. We found high plasma concentrations of PTX3 at admission in patients with meningococcal disease. Concentrations were significantly higher in patients with shock compared to patients without shock. In contrast, CRP at admission was lower in the shock group as compared to the no shock group. High PTX3 or low CRP concentration at admission discriminated well between presence and absence of shock, but PTX3 did not correlate with disease severity (PRISM) and days spent in ICU. In conclusion, PTX3 was an early indicator of shock in patients with severe meningococcal disease that followed a pattern of induction distinct from CRP.

In the part of the thesis presented in chapters 13 - 15 we identified 2 mediators, VEGF and MIF - that had not been previously studied in meningococcal disease - as possible mediators of tissue damage, both mediating different disease mechanisms. Because both VEGF and MIF are induced very early in the course of disease, they are primary mediators, which hold a promise for therapeutic interventions. However, it should be noted that the pathogenesis of sepsis and septic shock is complex and involves multiple mediators at multiple time points. Thus, therapies aimed at modulating the course of sepsis would most likely require a multi-target approach

277 18 and should attack mediators upstream in the inflammatory reaction.

Although it is a possible prognostic factor, The pathophysiological role of PTX3 in meningococcal disease is unknown. PTX3 binds to meningococci and the Gram- negative outer membrane protein OmpA. Future research will be aimed to explore whether PTX3 is important in the early defence against meningcococci.

One of the most intriguing aspects of meningococcal disease is that although carriage rates for meningcococci in some age groups exceed 20 %, only a minority of people ever get disease. Even more peculiar is perhaps that clinical manifestation of disease is so diverse.

In Chapter 16 we studied the role of MBL genotype, MBL plasma concentration and MBL/MASP2-activity in susceptibility to and clinical presentation of disease in a case-parent study including 120 patients with meningococcal disease and parents. Genotype for MBL was assessed using line probe assay. No transmission disequilibrium was found for the single nucleotide polymorphisms (SNPs) in the MBL2 gene using transmission disequilibrium testing. The exon1 SNPs combined were also not associated with an increased risk for disease. However, subgroup analysis showed increased risk for meningococcal meningitis associated with the exon 1 SNPs. In addition, MBL plasma concentrations in meningitis patients were significantly lower than in patients with shock or bacteraemia. We concluded that polymorphisms in the gene for MBL do not predispose for invasive severe meningococcal disease, but SNPs in exon 1 may be a risk factor for meningococcal meningitis.

Our findings contrast with previous studies on MBL and susceptibility to meningococcal disease. We believe that these findings of an association between MBL-deficiency and susceptibility to meningococcal disease can be explained by the use of flawed control groups with unusual low numbers of MBL-deficient individuals. The case-parent design of our study precludes such a matching bias, but has the problem of lower power. Thus, we cannot exclude that MBL-deficiency has a small effect on susceptibility to meningococcal disease which is only apparent in patients with meningococcal meningitis.

In Chapter 17 we analyzed the role of the innate production capacity for TNF, IL-1β, IL-12 and IL-10 in the clinical presentation and severity of meningococcal disease. Whole blood cultures from 113 survivors of meningococcal disease obtained median 5.4 years after hospitalization were stimulated with meningococcal lipopolysaccharide and heat-killed Neisseria meningitidis bacteria. Patients were

278 classified according to clinical manifestation in four groups: shock, both shock and meningitis, bacteraemia (neither shock nor meningitis) and distinct meningitis. No differences in whole blood cytokine production were found between the patients in these four groups. However, within the group of patients that had presented with shock, interleukin 1β (IL-1β) and the IL-1β / IL-10 ratio were negatively correlated with disease severity. Thus, patients with meningococcal septic shock had a more severe course of disease when the production capacity for IL-1β was lower. This latter finding may possibly explain the failure of adjunctive therapies in sepsis aimed at inhibiting IL-1β.

We could not explain the differences in clinical manifestation of meningococcal disease by the innate cytokine response in whole blood. Other non-cytokine components of the innate immune system, such as the complement system, either alone or in synergy with bacterial factors, may be more relevant for the early outgrowth of meningococci and hence the type of disease that develops.

Future research should focus on identifying the hosts genetic and functional factors that determine susceptibility to and clinical course of meningococcal disease. In this way we may be able to predict the clinical course of a patient in an early stage of the infection and select the patients at high risk for a deleterious course. In addition, if we could identify patients at high risk for developing meningococcal disease, e.g. by national screening programs, and would be able to vaccinate only people at risk, large scale vaccination campaigns may become unnecessary.

A final remark. Meningococcal disease is a serious problem in the Netherlands and other industrialised countries, and every effort should be made to save all the patients we can. However, the core of the problem lies in the third world, especially Africa - the ‘meningitis belt’ - and Asia. In these countries, meningococcal disease is a disease of the underprivileged. The questions in these countries are different: are there enough vaccines, are there enough antibiotics, is there a hospital, is there any help at all? Although the inequity between the western and third world lies at the base of this problem and is not easily solved, we should also make every effort we can to help the people in those countries with resources and research.

279 18

Nederlandse Samenvatting en Discussie 18 Meningokokken septische shock is een ernstige infectieziekte die vaak, in korte tijd, fataal afloopt. De heftige ontstekingsreactie tijdens deze ziekte wordt aangezet door complement-activatie en cytokine-productie en heeft primair tot doel bacteriegroei te remmen. De ontstekingsreactie leidt echter ook tot ernstige verstoring van de homeostase met hierbij gedissemineerde intravasale stolling (DIS)en shock en daardoor uitval van meerdere organen. Hoe deze reactie precies verloopt, is slechts ten dele bekend. Betere behandeling van de ziekte door beteugeling van de ontstekingsreactie zodanig dat de gunstige effecten van deze reactie intact blijven, vereist meer kennis over wat er precies gebeurt en hoe dit wordt veroorzaakt.

Leerboeken over infectieziekten vertellen ons dat de ontstekingsreactie bij meningokokken septische shock wordt veroorzaakt door lipopolysaccharide (LPS), een component van de bacteriële buitenmembraan. Door gebruik te maken van een genetisch gemodificeerde meningokok (H44/76lpxA of H44/76[pLAK33]) die geen LPS in zijn buitenmembraan heeft, onderzochten we (in hoofdstuk 2-4) het relatieve belang van het ‘LPS deel’ en van de ‘niet-LPS delen’ van de meningokok bij de activatie van complement en inductie van cytokine productie.

In hoofdstuk 2 laten we zien dat een belangrijk deel van de tumor-necrosis factor (TNF)-α en interleukine (IL)-1β productie en vrijwel alle interferon (IFN)-γ productie van menselijke mononucleaire bloedcellen na contact met meningokokken op rekening komt van de niet-LPS delen van de meningokok. De LPS-deficiente meningokokken konden ook cytokine productie en mortaliteit veroorzaken in een muis-model van meningokokken sepsis. Echter, de dosis die hiervoor nodig was, lag honderd maal hoger dan wanneer ‘wild-type’ LPS bevattende meningokokken werden gebruikt. Hieruit concluderen we dat N. meningitidis cytokine-productie en ziekte kan induceren, onafhankelijk van LPS.

Volgens de literatuur zijn CD14 en TLR4 belangrijkste cellulaire receptoren voor het LPS; dit was de reden om in hoofdstuk 3 te onderzoeken wat de rol is van CD14 en TLR4 bij de cytokine inductie door niet-LPS componenten van meningokokken. Een monoklonaal antilichaam gericht tegen CD14 bleek de TNFα en IL-1β productie door menselijke mononucleaire bloedcellen na stimulatie met E. coli LPS volledig te blokkeren. Cytokine inductie door LPS van meningokokken, LPS-bevattende H44/76 meningokokken en LPS-deficiente H44/76lpxA meningokokken werd echter slechts gedeeltelijk geremd. Experimenten met macrofagen geïsoleerd van TLR4- deficiënte muizen wezen uit dat TLR4 cruciaal is voor de inductie van cytokinen door meningokokken LPS. Echter, complete LPS bevattende meningokokken en LPS-deficiënte meningokokken gaven in TLR4-deficiënte muizen macrofagen beiden een krachtige cytokine respons, vergelijkbaar met de respons in macrofagen

282 van TLR4 bevattende muizen. Dit betekent dat N. meningitidis cytokine productie kan induceren in humane mononucleaire bloedcellen of muizen macrofagen onafhankelijk van de LPS receptoren CD14 en TLR4.

In hoofdstuk 4 onderzochten we het belang van LPS en van de niet-LPS bestanddelen van de meningokok bij de activatie van complement en de complement-afhankelijke ontstekingsreactie in menselijk bloed. We vonden dat de LPS bevattende H44/76 meningokokken en de LPS-deficiente H44/76lpxA meningokokken beiden krachtig complement activeerden. Gezuiverd LPS van meningokokken, gebruikt in hoeveelheden vergelijkbaar met de hoeveelheid aanwezig op complete meningokokken, was een relatief zwakke activator van complement. Opregulatie van de complement receptor-3 op leukocyten en productie van vrije zuurstofradicalen door leukocyten gebeurde onafhankelijk van LPS. Complement activatie en de complement afhankelijke leukocyten activatie uitgelokt door de meningokok geschiedt in menselijk bloed dus onafhankelijk van LPS.

De resultaten van de onderzoeken in hoofdstuk 2-4, impliceren dat behandeling- strategieën gericht op het blokkeren van LPS of de LPS-receptoren CD14 en TLR4, slechts een gedeeltelijk effect zullen hebben op de productie van cytokinen, en geen effect op de activatie van complement. Om deze reden valt het te betwijfelen dat zulke therapieën het ziekte beloop kunnen modificeren. Het is aannemelijk dat dit één van de oorzaken is waardoor anti-endotoxine therapieën er niet in geslaagd zijn de afloop van meningokokken septische shock te verbeteren.

In hoofdstuk 5 onderzochten we of lipoproteinen, stoffen die door hun remmende effect op E.coli LPS geïnduceerde cytokine productie aandacht hebben gekregen als eventuele behandelingsmogelijkheid van Gram-negatieve sepsis, ook een remmend effect hebben op cytokine inductie door meningokokken LPS of complete Gram-negatieve bacteriën. Hiertoe bestudeerden we het effect van low-density (LDL), high density (HDL) en very low density (VLDL) lipoproteinen op cytokine productie geïnduceerd door LPS of complete bacteriën in humane mononucleare cellen. Het bleek dat de cytokine productie na stimulatie met E. coli LPS krachtig werd geremd door LDL en HDL, en iets minder door VLDL. Cytokine productie na stimulatie met meningokokken LPS werd echter veel minder effectief geremd. Voor effectieve neutralisatie van meningokokken LPS was een langere interactie tijd, een lagere concentratie van LPS of hogere concentratie van lipoproteinen nodig. De cytokine productie na stimulatie met complete Gram-negatieve bacteriën, E.coli zowel als N. meningitidis, werd slechts minimaal geneutraliseerd door de lipoproteinen. Onze resultaten zetten vraagtekens bij de rationale om lipoproteinen te gebruiken als adjuvante behandeling voor Gram-negatieve sepsis.

283 18 Een onverwachte, maar interessante benadering om TLR4-afhankelijke cytokine productie te remmen komt vanuit het LPS molecuul zelf. In hoofdstuk 6 hebben we meningokokken LPS varianten onderzocht met een lipid A deel dat bestaat uit vijf (pentageacyleerd lpxL1 LPS) of vier (tetrageacyleerd lpxL2 LPS) vetzuren. Deze nieuwe LPS varianten zijn afkomstig van genetisch gemanipuleerde meningokokken waarbij lpxL1 of het lpxL2 gen is geïnactiveerd. Deze LPS mutanten bleken LPS- geïnduceerde TLR4-afhankelijke cytokine productie door menselijke bloedcellen uitstekend te remmen, ze fungeerden als LPS-antagonisten. In tegenstelling tot de situatie in mensen was lpxL1 LPS een agonist voor cytokine productie in peritoneaal macrofagen van muizen en het zorgde voor een exacerbatie van arthritis in een arthritis model in muizen. Hoewel het niet verwacht kan worden dat het remmen van LPS of TLR4 alleen een gunstig effect heeft op het beloop van meningokokken septische shock, is het wellicht mogelijk dat de onderzochte LPS-varianten bruikbaar zijn bij de behandeling van andere TLR4 afhankelijke ontstekingsziekten zoals rheumatoide arthritis, asthma of inflammatoire darmziekten.

Uit de hoofdstukken 2-6 kunnen we afleiden dat het voor beteugeling van de ontstekingsreactie bij meningokokkenziekte, onverstandig lijkt de pijlen alleen op LPS te richten. Willen we de onwenselijke effecten van de ontstekingsreactie remmen dan zullen we de rol hierin van de niet-LPS onderdelen van de meningokok beter moeten kennen: wat is de exacte bijdrage, hoe verloopt de herkenning en de inductie van ontstekingsstoffen? Het is misschien niet mogelijk om een enkele zilveren kogel te vinden die de inflammatoire reactie op het niveau van de bacterie kan stoppen; misschien hebben we iets nodig als een clusterbom, dat wil zeggen meerdere moleculen die een effect hebben op meerdere bestanddelen van meningokokken en hun cellulaire of plasmatische receptoren, om het beloop van meningokokkenziekte gunstig kunnen beïnvloeden.

In hoofdstuk 7 bestudeerden we het effect van mannose-bindend lectine (MBL), het sleutelmolecuul voor complement-activatie via de lectine-route, op de cytokine productie door menselijke mononucleaire bloedcellen na contact met meningokokken. We ontdekten dat MBL de IL-1β- en IL-10-productie uitgelokt door LPS-deficiente meningokokken versterkt, maar dat er geen effect was op de TNFα-, IL-6- en IFNγ-productie. Ook was er geen effect van MBL op de, veel sterkere, cytokine productie na contact met meningokoken LPS. We concludeerden dat MBL waarschijnlijk een interactie aangaat met de niet-LPS bestanddelen van de meningokok en hierbij de productie van specifieke cytokines beïnvloedt. Of de door ons ontdekte verbinding tussen het complement-systeem en het cytokine-netwerk ook bijdraagt aan de bescherming tegen meningokokkenziekte is onderwerp voor toekomstig onderzoek.

284 De hoofdstukken 8–12 van dit proefschrift wordt, zowel in-vitro als in-vivo, complement-activatie door meningokokken bestudeerd. Complement-activatie is een evolutionair oud beschermingsmechanisme tegen extracellulaire infecties. Mensen met complement-deficiënties hebben veel grotere kans op meningokokkenziekte. Aan de andere kant is, bij mensen met een normaal complementsysteem, de ernst van meningokokkenziekte nauw gerelateerd aan de mate van complement- activatie. Daarom wordt het complement systeem bij meningokokkenziekte ook wel een ‘dubbelzijdig zwaard’ genoemd.

In hoofdstuk 8 bestudeerden we het effect van remming van complement-activatie op ontsteking en de bacteriële ‘killing’ in een in-vitro model met menselijk bloed. We vonden dat de lectine- en de alternatieve-route essentieel zijn voor complement- activatie door meningokokken en de complement afhankelijke productie van vrije zuurstofradicalen in neutrofiele granulocyten en monocyten. Blokkade van complementfactor C5a, door het monoklonaal antilichaam (mAb) 137-26, dat het C5a-deel van C5 bindt en blokkeert nog voordat C5 gesplitst wordt in C5a en C5b, remt de opregulatie van Complement Receptor-3 (CR3), de fagocytose en de productie van vrije zuurstofradicalen volledig. Daarentegen is er geen effect op de vorming van C5b-9 (= Terminaal Complement Complex (TCC)), noch op lysis en killing van meningokokken. Een ander mAb dat de splitsing van C5 remt, had hetzelfde effect op de opregulatie van CR3, de fagocytose en de productie van vrije zuurstofradicalen, maar remde ook TCC-formatie en lysis en killing van bacteriën. Dit betekent dat het anti-C5a mAb 137-26 de schadelijke effecten van N. meningitidis geïnduceerde vorming van C5a kan remmen terwijl het de complement afhankelijke bacteriële killing intact houdt. Dit belooft nieuwe mogelijkheden voor de behandeling van meningokokken sepsis.

Hoofdstuk 9 laat zien hoe belangrijk de alternatieve route is voor de afweer tegen meningokokken. We beschrijven twee kinderen met meningokokken septische shock uit één familie. Een van de twee kinderen overleed. In het patiëntje dat overleefde bleek er geen alternatieve route activatie. Dit kwam doordat er geen factor D (FD) was. Mutatie analyse van het FD-gen liet een T636>G (Val 213 > Gly) en een T640>C (Cys 214 > Arg) mutatie zien in het DNA van het patiëntje, beide in homozygote vorm. In-vitro incubatie van het Deficiënte plasma met meningokokken toonde normale lectine-route activatie, maar geen vorming van het alternatieve route C3-convertase C3bBbP, en daardoor ook veel minder vorming van C3bc- en TCC. Dit defect herstelde na toevoeging van extra FD. Aldus leidt FD-deficientie tot een sterk verminderde alternatieve route activatie na contact met meningokokken, en zorgt dit voor een verhoogde vatbaarheid voor deze ziekte.

285 18 In hoofdstuk 10 onderzochten we bij patiënten met meningokokkenziekte in welke mate de alternatieve- en de lectine-route bijdragen aan de activatie van complement en de hierop volgende weefselschade. In patiënten met meningokokken septische shock bleek de plasma concentratie van de complement activatie producten C3bc (wijzend op algemene route activatie) en TCC (wijzend op terminale route activatie) sterk verhoogd. Deze systemische activatie van het complement systeem correleerde met de ziekte-ernst en parameters voor gedissemineerde intravasale stolling (DIS). Activatie van de alternatieve route werd aangetoond door het vinden van verhoogde C3bBbP concentraties. Twee van de patiënten met shock bleken MBL-deficiënt. De twee hadden lagere concentraties van C3bc en TCC dan de overige MBL-sufficiënte shock patiënten. Opvallend genoeg hadden deze twee MBL-deficiënte patiënten ook minder DIS, en was bij hen de ziekte ernst ook minder. Deze observaties laten zien dat ook in-vivo MBL van cruciaal belang is voor de systemische complement activatie tijdens meningokokken septische shock.

In hoofdstuk 11 presenteren we een ‘restriction fragment length polymorphism (RFLP)’ methode voor het genotyperen van de verschillende MBL genotypes. We hebben een PCR-RFLP methode ontwikkeld voor het X/Y promoter polymorphisme in het MBL2 gen om zo de mutaties in het MBL2 gen te kunnen detecteren die MBL-deficiëntie veroorzaken.

Hoofdstuk 12 geeft een literatuur overzicht van de acties van het complement systeem tijdens sepsis. Hierin belichten we met name de rol van het complement systeem als ‘dubbelzijdig zwaard’.

De resultaten gepresenteerd in hoofdstukken 8 - 12 laten zien dat de alternatieve en de lectine-route beide van cruciaal belang zijn voor het activeren van het complementsysteem bij meningokokken septische shock. Het blokkeren van C5a zou een aantrekkelijke benadering kunnen zijn om de schadelijke effecten van complement activatie te verminderen, vooral omdat anti-C5a mAb de complement gemediëerde bacteriële ‘killing’ intact houdt. Interessant is verder dat er een nauwe relatie bestaat tussen complement-activatie en de DIS en thrombocytopenie. DIS, dat wordt veroorzaakt door activatie van - onder andere - tissue factor, is bij meningokokken septische shock de oorzaak van veel ernstige restverschijnselen, zoals necrose van extremiteiten. Bij het ontstaan van DIS speelt mogelijk C5a- gemedieerde opregulatie van tissue factor een rol, maar het proces is tot nu toe onvoldoende begrepen. De rol van complement-activatie bij het ontstaan van DIS, en de mogelijkheden dit te beïnvloeden, verdienen daarom ook meer onderzoek.

In de hoofdstukken 13 tot en met 15 hebben we verschillende, tot dusver nog niet

286 onderzochte, ontstekingsmediatoren bij meningokokkenziekte bestudeert.

‘Vascular Endothelial Growth Factor’ (VEGF) is een belangrijke factor bij angiogenese en vasculaire permeabiliteit. In hoofdstuk 13 onderzochten we patiënten met invasieve meningokokkenziekte in een vroege fase van de opname. De plasma concentratie van VEGF was bij hen verhoogd en de hoogste waardes werden gevonden in de patiënten met shock. De VEGF-concentratie bij opname correleerde met de ernst van de ziekte en de hoeveelheid vocht toegediend in de eerste 24 uur. Bij alle patiënten werd de normalisering van de VEGF-concentratie na 24 – 48 uur, gevolgd door een afname in de vocht behoefte. Deze resultaten suggereren dat microvasculaire permeabiliteit tijdens sepsis mogelijk in verband staat met de VEGF-concentratie. Wellicht vormt VEGF een nieuw mikpunt voor toekomstige therapieën voor meningokokken septische shock.

In hoofdstuk 14 bestudeerden we ‘Macrophage Migration Inhibitory Factor’ (MIF), in-vivo bij patiënten met meningokokkenziekte en vrijwilligers die in een experimentele setting LPS kregen toegediend, en in-vitro in een vol bloed model. We gebruikten een nieuw ontwikkelde, zeer gevoelige en specifieke ‘enzyme linked immunosorbent assay’ (ELISA), en vonden dat de plasmaconcentratie van MIF verhoogd was in patiënten met meningokokkenziekte, waarbij de hoogste waarden werden gevonden in patiënten met septische shock. MIF kon daarentegen niet aangetoond worden tijdens experimentele humane endotoxinaemie, hoewel bij deze vrijwilligers wel zeer hoge TNFα concentraties werden gevonden. Ook kon er geen MIF productie worden gevonden na stimulatie met LPS of meningokokken in het in-vitro model waarbij ook TNFα werd geproduceerd. Onze bevindingen tonen aan dat de productie van MIF tijdens sepsis niet afkomstig is van bloedcellen en dat deze – in tegenstelling tot wat werd gedacht – niet bepaald wordt door de TNFα plasma concentratie.

In hoofdstuk 15 keken we naar het pentraxine 3 (PTX3). PTX3 lijkt qua vorm en functie op C-reactief proteïne (CRP) en heeft een functie bij de klaring van celresten en de aangeboren afweer. PTX3 wordt geproduceerd door endotheel cellen, terwijl CRP wordt gemaakt door levercellen na stimulatie met IL-6. Een verhoogde PTX3 concentratie kan gebruikt worden bij de diagnostiek van ontstekingsziekten. Bij patiënten met meningokokkenziekte vonden we hoge PTX3 concentraties op het moment van opname. De PTX3 concentraties waren het hoogst in de patiënten met shock, dit in tegenstelling tot de CRP-concentratie die het hoogste was bij patiënten met meningitis. Aldus is PTX3 een vroege indicator voor shock bij patiënten met meningokokkenziekte.

287 18 We beschreven in hoofdstuk 13-15 het patroon van VEGF, MIF en PTX3. VEGF en MIF zijn twee mediatoren die bij meningokokkenziekte nog niet eerder zijn bestudeerd. Omdat beiden vroeg in het ziekteproces worden geproduceerd en geassocieerd zijn met ernstige weefselschade, spelen ze mogelijk een rol in het ontstaan van deze weefselschade en vormen ze hierdoor een mogelijk doel van therapie. Echter, we weten uit de literatuur dat veel andere mediatoren bij meningokokken septische shock een vergelijkbaar patroon volgen. Daarom zullen therapieën die bedoeld zijn om het dramatische beloop van meningokokken septische shock te keren, waarschijnlijk gericht moeten zijn op meerdere van deze mediatoren. De pathofysiologische rol van PTX3 in het beloop van meningokokkenziekte dient verder te worden uitgezocht. Uit de literatuur is bekend dat PTX3 aan het buitenmembraan proteïne OmpA, van meningokokken bindt. Aldus is PTX3, naast een indicator van ziekte, mogelijk ook van belang voor de afweer tegen meningokokken.

Een van de meest intrigerende aspecten van meningokokkenziekte is dat, hoewel er veel mensen zijn die meningokokken in hun neus huisvesten (in sommige leeftijdsgroepen is dit zelfs 20 %), slechts een klein deel van deze mensen ziek wordt. Evenzo merkwaardig is dat dezelfde bacterie bij verschillende individuen verschillende ziektebeelden kan veroorzaken.

In hoofdstuk 16 bestudeerden we de rol van MBL op de vatbaarheid voor de ziekte en het type ziektebeeld dat zich ontwikkelt. Hiertoe deden we een ‘case-parent’ studie in 120 patiënten met meningokokkenziekte en hun ouders. MBL genotype werd bepaald met behulp van een ‘line probe assay’. We vonden geen transmissie disequilibrium voor de verschillende SNPs in het MBL2 gen, hetgeen betekent dat MBL2 SNPs geen verhoogd risico geven op ernstige meningokokkenziekte. De combinatie van de verschillende SNPs in exon 1 was ook niet geassocieerd met een verhoogd risico op meningokokkenziekte. In een subgroep analyse vonden we echter wel een verhoogd risico voor meningokokken meningitis geassocieerd met de exon 1 SNPs. Ook was de plasmaconcentratie van MBL in de patiënten met meningitis significant lager dan bij patiënten met bacteriemie of shock. Aldus concluderen we dat polymorphismen in het MBL2 gen niet bijdragen aan een verhoogde kans op het krijgen van ernstige invasie meningokokkenziekte, hoewel de SNPs in exon 1 wel een risico factor voor meningitis zouden kunnen zijn.

Deze bevindingen verschillen van eerdere studies die wel lieten zien dat exon 1 polymorphismen in het MBL2 gen geassocieerd waren met een grotere kans op het krijgen van meningokokkenziekte. Deze studies hadden als nadeel dat er controle groepen werden gebruikt met ongebruikelijke weinig MBL-deficiënte personen. Het

288 ‘case-parent’ ontwerp van onze studie voorkomt deze ‘matching bias’, maar heeft wel als nadeel dat de statistische ‘power’ minder is. Om deze reden kunnen we niet geheel uitsluiten dat MBL-deficiëntie een klein effect heeft op vatbaarheid voor meningokokkenziekte met name bij patiënten met meningitis.

In hoofdstuk 17 analyseerden we of de aangeboren productiecapaciteit voor de cytokinen TNFα, IL-1β, IL-12 en IL-10 de klinische manifestatie van meningokokkenziekte bepaald. Bloed van 113 overlevenden van meningokokkenziekte, afgenomen mediaan 5.4 jaar na presentatie op de intensive care, werd gestimuleerd met N. meningitidis LPS en hitte gedode N. meningitidis bacteriën. De patiënten werden geclassificeerd naar aanleiding van ziekte manifestatie in vier groepen: shock, shock met meningitis, meningitis zonder shock en bacteriemie (geen shock en geen meningitis). We vonden geen enkel verschil in cytokine productie in bloed tussen deze 4 verschillende groepen. In de groep van patiënten die zich hadden gepresenteerd met shock vonden we echter wel een negatieve correlatie van de IL-1β en de IL-1β / IL-10 ratio met ziekte ernst. Dit betekent dat wanneer de aangeboren productiecapaciteit voor IL-1β lager is, patiënten met meningokokken septische shock een ernstiger beloop van de ziekte hebben. Deze laatste bevinding is wellicht een verklaring voor het falen van therapieën die gericht zijn op het remmen van de IL-1β productie tijdens sepsis.

De verschillende klinische manifestaties van meningokokkenziekte konden niet verklaard worden door verschillen in aangeboren productie capaciteit voor de verschillende cytokinen die we bestudeerd hebben. Andere, niet-cytokine onderdelen van het aangeboren immuunsysteem van de gastheer, zoals het complement systeem, alleen dan wel in synergie met bacteriële factoren, zijn misschien meer relevant voor het type ziekte dat zich ontwikkelt.

Toekomstig onderzoek moet zich richten op de genetische en functionele gastheerfactoren die vatbaarheid voor en klinische manifestatie van meningokokkenziekte bepalen. Wellicht kunnen we dan het klinische beloop beter voorzien, en de patiënten selecteren die een grote kans hebben op een ernstig beloop. Als we patiënten met een hoog risico op het krijgen van meningokokkenziekte kunnen identificeren, bijvoorbeeld via nationale screeningsprogramma’s, en we zouden alleen die mensen hoeven te vaccineren, dan zouden grootschalige landelijke vaccinatie campagnes overbodig kunnen worden. Een laatste opmerking. Meningokokkenziekte is een ernstig probleem in Nederland en andere geïndustrialiseerde landen; we moeten alles doen om zo veel mogelijk patiënten te redden. De kern van het probleem ligt echter ergens anders, namelijk in de derde wereld, en dan vooral Afrika - de ‘meningitis gordel’ - en Azië. In deze

289 18 landen is meningokokkenziekte een ziekte van minderbedeelden. De vragen in deze landen zijn heel anders: zijn er genoeg vaccins, zijn er genoeg antibiotica, is er een ziekenhuis, is er sowieso enige vorm van hulp. Hoewel de ongelijkheid tussen het westen en de derde wereld de bron van dit probleem vormt, moeten we ons uiterste best doen om ook de mensen in die landen te helpen met alle middelen die we hebben.

290 291 18 Dankwoord - word of thanks

Geen proefschrift zonder dankwoord. Dé kans om mijn waardering voor iedereen die belangrijk is geweest in de totstandkoming van dit proefschrift vast te leggen voor de eeuwigheid, hoewel het een schier onmogelijk taak is om iedereen zoveel lof toe te schrijven als ze verdienen ..!

Dr. Ir. M. van Deuren, beste Marcel, je bent erg belangrijk geweest in zowel de totstandkoming van dit proefschrift, als in mijn opleiding tot internist en de persoon die ik nu ben. Daarnaast ben je ook nog een vriend. Ik kan je hiervoor niet genoeg bedanken.

Prof. Dr. J.W.M. van der Meer, beste Jos, in de eerste plaats bedankt voor het herkennen van mijn mogelijkheden en de kansen die je me gegeven hebt. Je bent een icoon als wetenschapper en internist. Ik ben er trots op dat je zowel mijn promotor als ook mijn opleider was. Bedankt.

Prof. Dr. T.E. Mollnes, dear Tom, thank you for giving me the opportunity to work in your lab and perform research together. I have learned so much from you, besides this you are a fantastic person and by far the quickest responding co- author involved in this thesis. Prof. Dr. P. Brandtzaeg, beste Petter, thanks for your kindness in Norway and your input in our research. You are one of the giants on whose shoulders I hope to stand.

Prof. Dr. N. Klein, dear Nigel and Prof. Dr. M. Turner, dear Malcolm, the stay at the lab at the Immunobiology unit was a defining time for me, as well for my future professional life as for my personal life. I am proud that I am able to say that I have worked in your lab.

Dr. C. Neeleman, beste Chris, bedankt voor al je hulp op de kinder IC en je betrokkenheid bij de patiënten. Dr. P. Pickkers, beste Peter, ik liep mijn eerste co-schap bij je en de manier waarop je onderzoek en kliniek combineert is een voorbeeld voor me. Bedankt. Prof. dr M.G.N. Netea, beste Mihai, je bent een fantastische wetenschapper en ook nog één van de aardigste mensen die ik ken. Dát is prettig om mee samen te werken. Bedankt. Dr P. vd Ley, beste Peter en Dr. L Steeghs, beste Liana, Als niet-artsen hebben jullie een andere kijk op het doen van goed onderzoek. Hier heb ik veel van geleerd. Bedankt.

Beste Liesbeth, Trees en Johanna, bedankt voor jullie hulp bij mijn eerste stapjes in het lab en voor alle andere dingen waar jullie me bij geholpen hebben, inclusief

292 alle gezelligheid. Sorry dat ik niet vaker op de koffie ben gekomen. Ook Ineke, Anneke, Heidi, Helga, Berry, Pierre en Magda: bedankt! Dear Anne (Pharo), Gunni and Anna (Bjerre), thank you for helping around in Oslo (especially with the mountain bike), the lab in Norway and your incredible hospitality. Dear Marina, Clare, Liz, Lindsey, Helli, Abby and all the other people in the lab in London, thank you so much for an excellent time! Beste Collega’s in het lab en op de fellow-kamer (Anna, Alieke, Jeroen, Evelien, Chantal, Matthew, Gerben, Corine, Chantal, Jolande en Ralph), bedankt voor jullie gezelligheid en vriendschap.

Bedankt voor jullie bijdrage en fantastische samenwerking: Nike Stikkelbroeck, Loek van Alphen, Wil Tax, Anton Stalenhoef, Shahla Abdollahi-Roodsaz, Leo Joosten, Dirk Roos, Corrie Weemaes, Christel Geesing, Dorine Swinkels, Rian Roelofs, Jacques de Kok, Lucas van Eijk, Hans van der Hoeven, Paul Smits, Anneke Geurts-Moespot, Fred Sweep, Martin den Heijer, Corrie de Kat-Angelino, Ina Klasen en Ton Bartelink. Thanks for your input and the excellent cooperation: Anne-Sophie Møller, Elisabeth Wedege, Peter Kierulf, Dominic Jack, Michael Fung, Ernst-Arne Høiby, Terje Michalesen, Audun Aase, Marlies Knaup, Didier Leroy, Thierry Calandra, Giuseppe Peri, Alberto Mantovani and Stefano Signorini.

Martijn, veel dank voor de fantastische vormgeving van kaft en proefschrift.

Beste drs. Wilco (wanneer nou eens dr. ?!) en broer Niels (Ome Niels nu !), ik ben blij jullie mijn paranimfen willen zijn. Marieke, je bent een leuke en lieve zus. Pap en Mam, bedankt voor … alles eigenlijk. Esther, lieve schat, zonder jou was dit proefschrift vast nog veel dikker geweest en jaren eerder af! Bedankt dat je het zover niet hebt laten komen! Hou van je. Kas en Siem, ik ben zo blij dat jullie er zijn!

Als laatste wil ik alle kinderen en ouders bedanken die mee hebben gedaan aan het onderzoek. Zonder jullie was dit alles niet mogelijk geweest.

293 294 Curriculum Vitae

Tom Sprong werd op 20 september 1975 geboren in s’ Hertogenbosch. Het VWO examen werd behaald in 1993 aan het Jeroen Bosch College, waarna hij geneeskunde ging studeren aan de Katholieke Universiteit in Nijmegen.

Tijdens zijn studie was hij actief als bestuurslid van de Medische Faculteits Vereniging Nijmegen en hoofdredacteur van het faculteitsblad ‘Impuls’. Daarnaast was hij werkzaam als assistent auteur/redacteur op de afdeling medische uitgeverijen van Wolters-Noordhoff. Het doctoraal examen geneeskunde werd in 1998 behaald en het artsexamen in augustus 2000. Vanaf 2000 werkte hij eerst als junior onderzoeker en later als arts niet in opleiding tot specialist (AGNIO) op de afdeling Algemeen Interne Geneeskunde van het Universitair Medisch Centrum St Radboud. In mei 2001 begon hij met het promotietraject van dit proefschrift en de opleiding tot internist (opleider prof dr. J.W.M. van der Meer en dr J. de Graaf) als Assistent Geneeskundige in Opleiding tot Klinisch Onderzoeker (AGIKO). Tijdens de opleiding tot internist was hij actief als voorzitter van de assistenten vereniging Interne Geneeskunde UMC St Radboud.

Voor aanvang van alsook tijdens de opleiding werden een aantal stages gevolgd. Te weten in 2000 aan de Immunobiology Unit van ‘The Institute of Child Health, University College Londen’, Verenigd Koninkrijk, bij prof. dr. Malcolm Turner en prof. dr. Nigel Klein en in 2002 aan the ‘Institute of Immunology’, Ullevål University Hospital te Oslo, Noorwegen bij prof. dr. Tom Eirik Mollnes en prof. dr. Petter Brandtzaeg. In 2003 werd een stage gedaan aan het Rijksinstituut voor Volksgezondheid en Milieu (nu Nederlands Vaccin Instituut) te Utrecht bij dr. Peter van der Ley.

De perifere stage interne geneeskunde werd gevolgd in het Canisius Wilhelmina Ziekenhuis te Nijmegen (opleider dr. A.S.M. Dofferhoff) van juni 2006 tot en met december 2007. Hier werd ook een aanvang gemaakt met het aandachtsgebied infectieziekten, welke vanaf januari 2008 tot en met januari 2009 zal worden voltooid in het Universitair Medisch Centrum St Radboud (opleider prof. dr. B.J.Kullberg). Sinds febuari 2009 is hij geregistreerd als internist, de registratie tot internist- infectioloog zal kort daarop volgen.

Tom Sprong is getrouwd met Esther Bodde en heeft twee hele leuke kinderen, Kas en Siem.

295 List of Publications

1. M.P. Hendriks, G.O.R.J. Janssens, T. Sprong, C.P. Bleeker-Rovers, C.M.L. van Herpen, M.A.W. Merx Agressieve fibromatose in het hoofd-halsgebied: een benigne tumor met mutilerende effecten. Nederlands tijdschrift voor Oncologie. 2008; 5(7):311-16

2. Sprong T, Peri G, Neeleman C, Mantovani A, Signorini S, van der Meer JW, van Deuren M. PTX3 and C-reactive protein in severe meningococcal disease. Shock. 2008 Jun 27. [Epub ahead of print]

3. Sprong T, van Deuren M. Mannose-binding lectin: ancient molecule, interesting future. Clin Infect Dis. 2008 Aug 15;47(4):517-8.

4. Popa C, Abdollahi-Roodsaz S, Joosten LA, Takahashi N, Sprong T, Matera G, Liberto MC, Foca A, van Deuren M, Kullberg BJ, van den Berg WB, van der Meer JW, Netea MG. Bartonella quintana lipopolysaccharide is a natural antagonist of Toll-like receptor 4. Infect Immun. 2007 Oct;75(10):4831-7. Epub 2007 Jul 2.

5. Roestenberg M, McCall M, Mollnes TE, van Deuren M, Sprong T, Klasen I, Hermsen CC, Sauerwein RW, van der Ven A. Complement activation in experimental human malaria infection. Trans R Soc Trop Med Hyg. 2007 Jul;101(7):643-9. Epub 2007 May 3.

6. Sprong T, Pickkers P, Geurts-Moespot A, van der Ven-Jongekrijg J, Neeleman C, Knaup M, Leroy D, Calandra T, van der Meer JW, Sweep F, van Deuren M. Macrophage migration inhibitory factor (MIF) in meningococcal septic shock and experimental human endotoxemia. Shock. 2007 May;27(5):482-487.

296 7. Sprong T, Mollnes TE, van Deuren M The double-edged sword of complement activation during sepsis. Netherlands Journal of Critical Care. 2007 february; 11(1):19-23

8. van Deuren M, Sprong T. Diagnostic image (283). A man with a swollen tongue Ned Tijdschr Geneeskd. 2006 Oct 28;150(43):2400-1; author reply 2401

9. van Eijk LT, Nooteboom A, Hendriks T, Sprong T, Netea MG, Smits P, van der Hoeven JG, Pickkers P. Plasma obtained during human endotoxemia increases endothelial albumin permeability in vitro. Shock. 2006 Apr;25(4):358-62.

10. Sprong T, Roos D, Weemaes C, Neeleman C, Geesing CL, Mollnes TE, van Deuren M. Deficient alternative complement pathway activation due to factor D deficiency by two novel mutations in the Complement Factor D gene in a family with meningococcal infections. Blood. 2006 Jun 15;107(12):4865-70. Epub 2006 Mar 9.

11. Pickkers P, Sprong T, Eijk LV, Hoeven HV, Smits P, Deuren MV. Vascular endothelial growth factor is increased during the first 48 hours of human septic shock and correlates with vascular permeability. Shock. 2005 Dec;24(6):508-512.

12. Roelofs MF, Joosten LA, Abdollahi-Roodsaz S, van Lieshout AW, Sprong T, van den Hoogen FH, van den Berg WB, Radstake TR. The expression of toll-like receptors 3 and 7 in rheumatoid arthritis synovium is increased and costimulation of toll-like receptors 3, 4, and 7/8 results in synergistic cytokine production by dendritic cells. Arthritis Rheum. 2005 Aug;52(8):2313-22.

297 13. Sprong T, Jack DL, Klein NJ, Turner MW, van der Ley P, Steeghs L, Jacobs L, van der Meer JW, van Deuren M. Mannose binding lectin enhances IL-1beta and IL-10 induction by non- lipopolysaccharide (LPS) components of Neisseria meningitidis. Cytokine. 2004 Oct 21;28(2):59-66.

14. Netea MG, Kullberg BJ, de Jong DJ, Franke B, Sprong T, Naber TH, Drenth JP, Van der Meer JW. NOD2 mediates anti-inflammatory signals induced by TLR2 ligands: implications for Crohn’s disease. Eur J Immunol. 2004 Jul;34(7):2052-9.

15. Sprong T, Moller AS, Bjerre A, Wedege E, Kierulf P, van der Meer JW, Brandtzaeg P, van Deuren M, Mollnes TE. Complement activation and complement-dependent inflammation by Neisseria meningitidis are independent of lipopolysaccharide. Infect Immun. 2004 Jun;72(6):3344-9.

16. Sprong T, Netea MG, van der Ley P, Verver-Jansen TJ, Jacobs LE, Stalenhoef A, van der Meer JW, van Deuren M. Human lipoproteins have divergent neutralizing effects on E. coli LPS, N. meningitidis LPS, and complete Gram-negative bacteria. J Lipid Res. 2004 Apr;45(4):742-9. Epub 2004 Feb 1.

17. Roelofs RW, Sprong T, de Kok JB, Swinkels DW. PCR-restriction fragment length polymorphism method to detect the X/Y polymorphism in the promoter site of the mannose-binding lectin gene. Clin Chem. 2003 Sep;49(9):1557-8. No abstract available.

18. Sprong T, Brandtzaeg P, Fung M, Pharo AM, Hoiby EA, Michaelsen TE, Aase A, van der Meer JW, van Deuren M, Mollnes TE. Inhibition of C5a-induced inflammation with preserved C5b-9-mediated bactericidal activity in a human whole blood model of meningococcal sepsis. Blood. 2003 Nov 15;102(10):3702-10. Epub 2003 Jul 24.

298 19. Sprong T, van der Ley P, Steeghs L, Taw WJ, Verver-Janssen TJ, Netea MG, van der Meer JW, van Deuren M. Neisseria meningitidis can induce pro-inflammatory cytokine production via pathways independent from CD14 and toll-like receptor 4. Eur Cytokine Netw. 2002 Oct-Dec;13(4):411-7.

20. Netea MG, Kullberg BJ, Joosten LA, Sprong T, Verschueren I, Boerman OC, Amiot F, van den Berg WB, Van der Meer JW. Lethal Escherichia coli and Salmonella typhimurium endotoxemia is mediated through different pathways. Eur J Immunol. 2001 Sep;31(9):2529-38.

21. Sprong T, Stikkelbroeck N, van der Ley P, Steeghs L, van Alphen L, Klein N, Netea MG, van der Meer JW, van Deuren M. Contributions of Neisseria meningitidis LPS and non-LPS to proinflammatory cytokine response. J Leukoc Biol. 2001 Aug;70(2):283-8.

299 Awards

2008 Winner quiz ‘acute interne geneeskunde’ - Junior Nederlandse Internisten Vereniging (JNIV) Conferentie ‘acute interne geneeskunde’

2004 International Sepsis Forum award for best research on a sepsis subject - European Conference on Clinical Microbiology and Infectious Diseases (ECCMID)

2000 Posterprize Surgical Infection Society - Europe - Joint Meeting of the European Shock Society (ESS) and Surgical Infection Society –Europe (SIS-E)

300 Presentations on national and international conferences

2008 European Conference on Clinical Microbiology and Infectious Diseases (ECCMID) PTX3 and C-reactive protein in severe meningococcal disease

2007 European Society of Pediatric infectious diseases (ESPID) 2007 Innate cytokine production capacity and clinical manifestation of meningococcal disease and Mannose binding lectin and meningococcal disease : a case parent study (poster)

2006 Annual Meeting of the European Society of Intensive Care Medicine (ESICM) Thematic session : sepsis related cellular dysfunctions – complement related pathways

2005 Internistendagen Nederlandse Internisten Vereniging Complement activation during meningococcal sepsis

2004 3rd International Conference on Innate Immunity N. meningitidis lipid A variant lipopolysaccharides (LPS) function as LPS antagonists by inhibiting toll-like receptor 4 (TLR4) dependent cytokine production (poster)

European Conference on Clinical Microbiology and Infectious Diseases (ECCMID) Complement activation during meningococcal sepsis

2003 Najaarsvergadering Vereniging voor Infectieziekten (VIZ) Inhibition of C5a-mediated inflammation with preserved C5b-9 mediated bactericidal activity in a human whole blood model of meningococcal sepsis

2003 Wintermeeting Nederlandse Vereniging voor Immunologie (NVI) Cytokine induction by Streptococcus pneumonia : polysaccharide capsule impedes TLR‑dependent cytokine production

301 2003 Landsteiner Masterclass Mannose-binding lectin The role of the lectin pathway in complement activation by Neisseria meningitidis

2002 Joint Meeting of the Belgian and Dutch Societies for Immunology Complement activation and complement-dependent inflammation occur independently of lipopolysaccharide (LPS)

2001 41st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) Enhancement of Cytokine Induction after Association of Neisseria meningitidis lipopolysaccharide (LPS) with the Outer Membrane (poster)

Voorjaarsvergadering Vereniging voor Infectieziekten (VIZ) Cytokine induction by Neisseria meningitides

Sixth conference of the International Endotoxin Society (IES) Inhibition of LPS induced cytokine production by polymyxin B is also dependent on the saccharide tail of LPS (poster) Cytokine induction by various LPS-types isolated from different meningococcal mutants (poster) Mannose binding lectin enhances the production of IL-1β but not that of TNFα after stimulation with non-LPS components of Neisseria meningitidis (poster)

2000 Joint Meeting of the European Shock Society (ESS) and Surgical Infection Society –Europe (SIS-E) The induction of tumour necrosis factor-α and Interleukin-1β by lipopolysaccharide (LPS) and non-LPS components of Neisseria meningitidis (poster) Determination of KDO, as simple method used for the quantitative comparison of cytokine induction by meningococcal LPS and that of other bacteria (poster)

302 303