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Human Endothelial Cell Activation by Escherichia coli and Staphylococcus aureus Is Mediated by TNF and IL-1 β Secondarily to Activation of C5 and CD14 in Whole Blood This information is current as of September 30, 2021. Stig Nymo, Alice Gustavsen, Per H. Nilsson, Corinna Lau, Terje Espevik and Tom Eirik Mollnes J Immunol 2016; 196:2293-2299; Prepublished online 22 January 2016; doi: 10.4049/jimmunol.1502220 Downloaded from http://www.jimmunol.org/content/196/5/2293 Supplementary http://www.jimmunol.org/content/suppl/2016/01/22/jimmunol.150222 http://www.jimmunol.org/ Material 0.DCSupplemental References This article cites 42 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/196/5/2293.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 30, 2021 • No Triage! 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The Journal of Immunology Human Endothelial Cell Activation by Escherichia coli and Staphylococcus aureus Is Mediated by TNF and IL-1b Secondarily to Activation of C5 and CD14 in Whole Blood Stig Nymo,*,†,‡,x Alice Gustavsen,‡,x Per H. Nilsson,‡,x Corinna Lau, *,† Terje Espevik,{,‖ and Tom Eirik Mollnes*,†,‡,x,{ Endothelial cells (EC) play a central role in inflammation. E-selectin and ICAM-1 expression are essential for leukocyte recruitment and are good markers of EC activation. Most studies of EC activation are done in vitro using isolated mediators. The aim of the present study was to examine the relative importance of pattern recognition systems and downstream mediators in bacteria-induced EC activation in a physiological relevant human model, using EC incubated with whole blood. HUVEC were incubated with human whole blood. Escherichia coli– and Staphylococcus aureus–induced EC activation was measured by E-selectin and ICAM-1 Downloaded from expression using flow cytometry. The mAb 18D11 was used to neutralize CD14, and the lipid A analog eritoran was used to block TLR4/MD2. C5 cleavage was inhibited using eculizumab, and C5aR1 was blocked by an antagonist. Infliximab and canakinumab were used to neutralize TNF and IL-1b. The EC were minimally activated when bacteria were incubated in serum, whereas a substantial EC activation was seen when the bacteria were incubated in whole blood. E. coli–induced activation was largely CD14-dependent, whereas S. aureus mainly caused a C5aR1-mediated response. Combined CD14 and C5 inhibition reduced E-selectin and ICAM-1 expression by 96 and 98% for E. coli and by 70 and 75% for S. aureus. Finally, the EC activation http://www.jimmunol.org/ by both bacteria was completely abolished by combined inhibition of TNF and IL-1b. E. coli and S. aureus activated EC in a CD14- and C5-dependent manner with subsequent leukocyte secretion of TNF and IL-1b mediating the effect. The Journal of Immunology, 2016, 196: 2293–2299. irect bacterial tissue damage and bacteria-induced in- In recent years, the role of endothelial cells (EC) in inflammation flammation are important causes of both severe morbid- has been recognized both as central and undervalued (2–4). From D ity and mortality worldwide. Both Gram-negative and the basic point that EC activation is necessary for leukocyte re- Gram-positive bacteria can cause severe disease. Although his- cruitment to the recent discovery that inhibiting increased vascular torically Gram-negative bacteria have been the main cause of permeability in sepsis seems to alleviate most of the symptoms of by guest on September 30, 2021 severe infections, with Escherichia coli being the most common, the condition in murine models (5–7), it has been demonstrated there is an increasing prevalence of Gram-positive–induced in- that a better evaluation of EC activation has a central part in the fections, in particular with Staphylococcus aureus (1). quest for understanding and modulating inflammation. A large number of models have been used to examine EC ac- tivation, with HUVEC being a common surrogate. Using HUVEC, multiple potential activating factors have been found in in vitro *Research Laboratory, Nordland Hospital, 8092 Bodø, Norway; †Faculty of Health Sciences, K. G. Jebsen Thrombosis Research and Expertise Center, University of studies. However, as the field of sepsis has duly demonstrated, Tromsø, 9037 Tromsø, Norway; ‡Department of Immunology, Oslo University Hos- there is a long path from simple isolated cell models to the clinic, a pital, Rikshospitalet, University of Oslo, 0424 Oslo, Norway; xK. G. Jebsen Inflam- { path riddled with wrong turns and empty promises. mation Research Center, University of Oslo, 0424 Oslo, Norway; Center of Molecular Inflammation Research, Norwegian University of Science and Technology, To narrow this gap, we have developed a novel model to look at ‖ 7491 Trondheim, Norway; and Department of Cancer Research and Molecular human EC activation in a more complex system using an already Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway established whole blood model, that is, coincubating lepirudin anticoagulated whole blood with HUVEC monolayers (8). ORCIDs: 0000-0003-2276-766X (A.G.); 0000-0002-7192-5794 (P.H.N.); 0000- 0003-1750-8065 (C.L.). Recognition of bacteria by the innate immune system is me- Received for publication October 15, 2015. Accepted for publication December 22, diated by pattern recognition receptors. There are several different 2015. classes of these, where TLRs play a central role, particularly in This work was supported by the Research Council of Norway, the Norwegian Coun- detection of LPS from Gram-negative bacteria through TLR4, but cil on Cardiovascular Disease, the Northern Norway Regional Health Authority, the also motifs on Gram-positive bacteria, particularly lipoproteins, Southern and Eastern Norway Regional Health Authority, the Odd Fellow Founda- tion, and by European Community Seventh Framework Programme Grant 602699 recognized by TLR2 (9). CD14 is a promiscuous coreceptor to (DIREKT). several of the TLRs, and it plays a central role in recognition both Address correspondence and reprint requests to Prof. Tom Eirik Mollnes, Depart- by TLR4 and TLR2 (10). ment of Immunology, Oslo University Hospital, Rikshospitalet, 0424 Oslo, Norway. The complement system is an important recognition system E-mail address: [email protected] comprising both fluid-phase and cell-bound components. There are The online version of this article contains supplemental material. three pathways of pathogen recognition (classical, lectin, and al- Abbreviation used in this article: EC, endothelial cell. ternative pathways) that all lead to activation of the C3 convertases, This article is distributed under The American Association of Immunologists, Inc., which proteolyze C3 into C3a, an anaphylatoxin with a range of Reuse Terms and Conditions for Author Choice articles. effector functions, and C3b, which acts as an opsonin on bacteria Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 and other exogenous or endogenous structures. With further ac- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502220 2294 BACTERIA-INDUCED WHOLE BLOOD ACTIVATION OF EC tivation of C3, the C5 convertase is constructed, which activates C5, E.coli (105 bacteria/ml unless specified otherwise) or heat-inactivated 7 leading to the release of C5a, the most potent anaphylatoxin in this S. aureus (10 bacteria/ml, unless specified otherwise). Samples were then system, and assembly of the terminal C5b-9 complex. This com- incubated at 37˚C with 5% CO2 for 4 h with gentle shaking. Thereafter, whole blood, serum, or medium was removed and EDTA (20 mM) was plex can either be formed in the fluid phase as sC5b-9 or assembled added before centrifugation for 15 min at 3000 3 g at 4˚C. Plasma or on a membrane as the membrane attack complex, which may lyse supernatant was stored at 270˚C until analysis. Gram-negative bacteria. Complement system activation is strictly EC activation markers regulated by endogenous inhibitors at different steps of the cascade (11–13). After removal of medium, serum, or whole blood, HUVEC monolayers We have earlier shown that combined inhibition of CD14 and were gently washed twice with ice-cold PBS, fixed with 0.5% parafor- maldehyde, and incubated at 4˚C for 2.5 min, as described previously (21). one of the key complement components, at the level of either C3 or After gentle washing with PBS, anti–ICAM-1-FITC and anti–E-selectin- C5, can more or less completely attenuate cytokine release and PE, or their isotype controls, and anti–MCAM-PerCP were added and leukocyte activation in whole blood after bacteria-induced acti- plates were incubated for 30 min at 4˚C. Cells were washed twice with vation both with Gram-negative and Gram-positive bacteria (14– PBS, briefly trypsinated, and transferred to 5-ml polypropylene tubes 16). Furthermore, in a murine sepsis model, we have recently (Sarstedt, Nuernbrecht, Germany), washed with PBS with 0.1% BSA, and run on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). shown that combined inhibition reduces both inflammation and HUVEC were gated as MCAM-positive cells. Median values were used for mortality (17).