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Extracellular Gelsolin Binds Lipoteichoic Acid and Modulates Cellular Response to Proinflammatory Bacterial Wall Components

This information is current as Robert Bucki, Fitzroy J. Byfield, Alina Kulakowska, of October 2, 2021. Margaret E. McCormick, Wieslaw Drozdowski, Zbigniew Namiot, Thomas Hartung and Paul A. Janmey J Immunol 2008; 181:4936-4944; ; doi: 10.4049/jimmunol.181.7.4936 http://www.jimmunol.org/content/181/7/4936 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Extracellular Gelsolin Binds Lipoteichoic Acid and Modulates Cellular Response to Proinﬂammatory Bacterial Wall Components1

Robert Bucki,2* Fitzroy J. Byﬁeld,* Alina Kulakowska,† Margaret E. McCormick,* Wieslaw Drozdowski,† Zbigniew Namiot,‡ Thomas Hartung,§ and Paul A. Janmey*

The various functions of gelsolin in extracellular compartments are not yet clearly defined but include actin scavenging and antiin- flammatory effects. Gelsolin was recently reported to bind endotoxin (LPS) from various Gram-negative bacteria with high affinity. In this study we investigate whether gelsolin also interacts with bacterial wall molecules of Gram-positive bacteria such as lipoteichoic acid (LTA) and whether gelsolin’s interaction with bacterial lipids from Gram-negative or Gram-positive bacteria affects their cellular inflammatory responses. A peptide based on the PPI binding site of gelsolin (160–169) binds purified LTA at the same molecular ratio that it binds phosphatidylinositol 4,5-bisphosphate. The OD of recombinant human plasma gelsolin was found to decrease Downloaded from following the addition of purified LTA, and the binding of gelsolin to LTA inhibits F-actin depolymerization by gelsolin. Simul- taneously, the ability of LTA to activate translocation of NF-␬B, E-selectin expression, and adhesion of neutrophils to LTA-treated human aortic endothelial cells was compromised by gelsolin. Gelsolin was able to partially inhibit LPS- or LTA-induced release of IL-8 from human neutrophils but was unable to prevent Gram-positive Bacillus subtilis or Gram-negative Pseudomonas aeruginosa growth and had no effect on the antibacterial activity of the cathelicidin-derived antibacterial peptide LL37. These data suggest that extracellular gelsolin is involved in the host immune recognition of LTA or LPS following release of these molecules http://www.jimmunol.org/ from the bacterial outer membrane during cell division or attack by drugs and immune components. The Journal of Immunology, 2008, 181: 4936–4944.

elsolin is an ϳ84-kDa actin-binding protein first identi- proposed is adult respiratory distress syndrome (ARDS)3 (8). Ac- fied in the cytoplasm. Intracellular gelsolin is involved tin release in excess of the actin scavenger molecules’ capacities G in the remodeling of actin filaments associated with affects the lung because of its size and rich blood flow through cell shape changes and movement (1, 2). Cells from gelsolin- narrow vessels, leading to the characteristic inflammatory

null mice exhibit a variety of motility and cytoskeletal defects. changes of ARDS. In established ARDS cases, plasma gelsolin by guest on October 2, 2021 Gelsolin-null fibroblasts have pronounced actin stress fibers, a levels were found to be on average 30% of normal values (4). phenotype consistent with an inability to sever and remodel Recently, repletion with exogenous plasma gelsolin was found actin filaments (3). The functions of extracellular gelsolin are to be beneficial in mice subjected to hyperoxia (9), a condition less well defined. Initially its involvement in scavenging F-actin that often results in ARDS development. Furthermore, the treat- polymers released into the circulation during cell death was the ment of mice with exogenous plasma gelsolin significantly only role considered (4, 5). This preventative nature of gelsolin blunted neutrophil recruitment to the lungs (9), and gelsolin was is an idea supported by observations that a reduction in plasma able to attenuate vascular permeability associated with burn in- gelsolin as well as the detection of circulating gelsolin-actin jury in rats (5). Reduction in plasma gelsolin levels was also complexes were reported in a variety of human and animal in- observed in patients with sepsis, myocardial infarction, hepati- jury states (4, 6, 7). tis, myonecrosis (10), and trauma (11, 12). However, an insuf- One of the most important clinical examples of secondary injury ficiency in the blood F-actin buffering system has not been ob- in which the pathological implication of circulating F-actin was served, and the involvement of plasma gelsolin in inflammatory mediator transport has been proposed (13). Plasma gelsolin binds LPS from various bacteria, and some LPS-induced cellular functions are neutralized by gelsolin and by *University of Pennsylvania, Institute for Medicine and Engineering, Philadelphia, PA 19104; †Department of Neurology and ‡Department of Physiology, Medical Uni- a peptide based on gelsolin residues 160–169 (14). The gelsolin P2 versity of Bialystok, Bialystok, Poland; §European Union Joint Research Centre, In- peptide (residues 150–169) binds LPS with greater affinity than it stitute for the Protection and Security of the Citizen, the Traceability, Risk and Vul- binds lysophosphatidic acid (LPA) (15), a potent extracellular ag- nerability Assessment Unit (TRiVA), Ispra, Italy; and Department of Biochemical Pharmacology, University of Konstanz, Konstanz, Germany onist that influences endothelial cell migration and proliferation Received for publication October 2, 2007. Accepted for publication July 24, 2008. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance 3 Abbreviations used in this paper: ARDS, adult respiratory distress syndrome; CSF, with 18 U.S.C. Section 1734 solely to indicate this fact. cerebrospinal fluid; DLS, dynamic light scattering; HAEC, human aorta endothelial 1 cells; LBP, LPS-binding protein; LPA, lysophosphatidic acid; LTA, lipoteichoic acid; This work was supported by the National Institutes of Health (Grant AR38910), the Malp-2, macrophage-activating lipopeptide-2; MIC, minimal inhibitory concentra- Cystic Fibrosis Foundation, and the Medical University of Bialystok Grant 3-44903L. tion; PAF, platelet-activating factor; PBP10, rhodamine B-QRLFQVKGRR; PIP2, 2 Address correspondence and reprint requests to Dr. Robert Bucki, University of phosphatidylinositol 4,5-bisphosphate; RhB, rhodamine B; rhGSN, recombinant hu- Pennsylvania, Institute for Medicine and Engineering, 1010 Vagelos Research Lab- man plasma gelsolin. oratories, 3340 Smith Walk, Philadelphia, PA 19104. E-mail address: buckirob@ mail.med.upenn.edu Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 4937

(16). Plasma gelsolin was also found to interfere with platelet- (L4015), and Bacillus subtilis (L3265) were purchased from Sigma- activating factor (PAF)-induced cellular activation in vitro, sug- Aldrich. According to the manufacturer’s quality control, the preparation Ͻ gesting a protective mechanism for gelsolin in vivo (17). These of LTA contained 1 ng of LPS/1 mg of LTA, and therefore LPS would contribute Ͻ10 pg/ml culture medium in the highest LTA concentration findings suggest a new role for plasma gelsolin in inflammatory used. Purified LTA from S. aureus was prepared as described previously response and, in this context, actin may directly affect gelsolin (23, 31). To calculate molar concentrations of LPS and LTA, we have used binding to LPS, LPA, or PAF. the lowest range of their reported molecular mass in buffer without divalent LPS and lipoteichoic acid (LTA) represent the major virulence cations (stock solution was made in H2O). Recombinant human plasma gelsolin (rhGSN) was obtained from Biogen Idec. Solution of human al- factors of Gram-negative and Gram-positive bacteria, respectively. bumin was from Baxter Healthcare. Human aortic endothelial cells LTA concentrations can reach higher levels at infectious sites (HAECs) were obtained from BioWhittaker. ELISA kit for IL-8 determi- compared with LPS. Reported local tissue concentrations of LTA nation was from BioLegend. Heat-inactivated P. aeruginosa (PAO1) and can be as high as 26 ␮g/ml (18), which may be associated with the B. subtilis (American Type Culture Collection (ATCC) 6051) were ob- 8 7 ␮ tained by autoclaving their suspension (10 CFU/ml) in PBS for1hat fact that 10 Gram-positive bacteria contain as much as 1 gof ϳ 7 120°C. Monomeric G-actin was prepared from an acetone powder of LTA whereas 10 Gram-negative bacteria contain only 20 ng of rabbit skeletal muscle as described previously (32). LPS (19). The primary transmembrane proteins that are activated by proinflammatory bacterial moieties such as LPS and LTA be- Interaction of LTA with human plasma recombinant gelsolin long to the TLR family. Delivery of bacterial molecules from ex- and fluorescent peptides derived from gelsolin’s ternal fluids to the cell membrane and ultimately to TLR2 or TLR4 phosphatidylinositol 4,5-bisphosphate (PIP2) binding site (dominant receptors for LTA and LPS, respectively) is complex To determine potential effects of LTA and other TLR agonists on the struc- and involves a number of other factors such as sCD14, LPS-bind- ture of gelsolin, OD at 280 nm was measured in solutions containing varying protein (LBP), MD2, and moesin (20, 21). ing concentrations of LTA (from S. aureus) and Malp-2 added to 0.1 Downloaded from mg/ml of rhGSN in PBS. OD was also measured with PIP2 and phosphati- One important factor that determines the toxicity of bacterial dylcholine as positive and negative controls, respectively. A decrease in products to host cells is the geometry of their aggregation state. tyrosine and tryptophan fluorescence, due to decreased absorbance, has

Low and high toxicity of LPS was associated with packing into been previously documented as an assay for PIP2 binding to gelsolin (33). lamellar and hexagonal phases, respectively (22). LTA and LPS The fluorescence of rhodamine B-QRLFQVKGRR (PBP10) or rhodamine B (RhB)-QRL (␭ 565 nm, ␭ 590 nm) was measured 15 min after obtained from different bacteria species induce the release of in- ex em addition of various concentrations of PIP or LTA from different bacterial ␣ ␤ 2 http://www.jimmunol.org/ flammatory cytokines such as TNF- , IL-1 , IL-6, and IL-8 (19, strains to 2 ␮M peptide solutions (PBP10 or RhB-QRL) in buffer A (10 23). All TLR signaling pathways elicit MyD88- or TRIF (TIR mM Tris, 10 mM MES (pH 7.0)). The expectation was that peptides bound domain-containing adaptor-inducing IFN-␤)-dependent activation to lipids would alter rhodamine B fluorescence similarly to previous ob- of the transcription factor NF-␬B. Those signaling cascades in- servation with LPS (14) and PIP2 (34). volve recruitment of different proteins such as IL-1R-associated Immunoblotting analysis kinase (IRAK), TNFR-associated factor 6 (TRAF6), TGF-␤-acti- Samples of cerebrospinal fluid, saliva, bile, and blood after collection were vated kinase-1 (TAK1), IKK complex, and MAPK (24). In RAW immediately centrifuged (2000 ϫ g, 20 min), subjected to total protein 264.7 macrophages, LTA was also found to activate the PI3K/ analysis, and frozen. The study was approved by the Medical University of AKT pathway and p38 MAPK-kinase, which in turn initiates Bialystok Ethics Committee for Research on Humans and Animals, and by guest on October 2, 2021 NF-␬B activation (25). TLR2 activation, contrary to earlier obser- written consent was obtained from all subjects. After being thawed, gel sample buffer was added to the samples, which were then boiled and sub- vation (25), was also proposed to function as a serum-independent jected to electrophoresis on 10% polyacrylamide gels in the presence of factor, indicating significant differences between LTA- and LPS- SDS. After electrophoresis, proteins were transferred to polyvinylidene mediated host cell activation (26, 27). LTA is also a potent patho- difluoride membrane (Amersham Biosciences), which were blocked by in- genicity factor that causes cardiac dysfunction in Gram-positive cubation in 5% (w/v) nonfat dry milk dissolved in TBS-T (150 mM NaCl, ϭ sepsis (28), may cause neuronal death (29), and determines clinical 50 mM Tris, 0.05% Tween 20 (pH 7.4)). Following transfer, proteins were probed using a monoclonal anti-human gelsolin Ab (Sigma-Aldrich, outcome in patients with pneumococcal meningitis (18). G4896) used at a 1/10,000 dilution in TBS-T. HRP-conjugated secondary In this study we report that in addition to the capability to bind Abs were used at a 1/20,000 dilution in TBS-T. Immunoblots were devel- endotoxin (14), extracellular gelsolin binds LTA from different oped with the FujiFilm LAS-300 system using an ECL Plus HRP-targeted Gram-positive bacteria strains. The result of this binding is the chemiluminescent substrate (Amersham Biosciences). The detection limit for gelsolin in our experimental condition for Western blot analysis was 5 inhibition of gelsolin’s F-actin depolymerizing activity and com- ng, and the highest volume of samples applied was 20 ␮l. Data revealed the promised ability of LTA to activate endothelial cells, as measured presence of gelsolin in all plasma and cerebrospinal fluid (CSF) samples. by E-selectin expression, activation of the transcription factor NF- Gelsolin was not detected in saliva and bile samples within the detection ␬B, and neutrophil adhesion. Gelsolin was also found to inhibit the limit for this study (data not shown). release of IL-8 from human neutrophils subjected to LTA, LPS, Severing activity of gelsolin and heat-inactivated bacteria treatment. To prepare F-actin, 150 mM KCl and 2 mM MgCl2 were added to G-actin solutions and allowed to incubate for1hatroom temperature. The severing Materials and Methods activity of rhGSN (50 nM), blood serum (2.5 ␮l), and cerebrospinal fluid Materials (25-50␮l) was measured in 0.4 ␮M pyrene-labeled F-actin samples in the

presence of LTA (S. aureus), LPS (E. coli), LPA, or PIP2. The fluorescence QRLFQVKGRR (gelsolin residues 160–169) and QRL peptides from gel- ␭ ␭ solin were prepared by solid phase peptide synthesis and fluorescently intensity of pyrene F-actin was monitored for 5 min ( ex 365 nm, em 386 labeled at their N termini by reaction with succinimidyl esters of rhoda- nm) using an SL-5B spectrofluorometer (PerkinElmer). Calculation of sev- mine B as previously described (30). The cathelicidin-derived antibacterial ering activity based on the rate of fluorescence decrease was performed as peptide LL37 peptide was purchased from Bachem. Ultrapure LPS from previously described (35). Prophyromonas gingivalis (tlrl-pglps), which is a ligand for TLR2, was Cell culture and NF-␬B activation from InvivoGen. Macrophage-activating lipopeptide-2 (Malp-2) corre- sponding to the isomer originally isolated from Mycoplasma fermentans, HAECs were grown in an incubator at 37°C and 5% CO2 in endothelial cell which signals via TLR2 and TLR6, was from Alexis Biochemicals. Human basal medium-2 with supplements (Cambrex Bio Science). NF-␬B trans- LBP peptide (86–99 aa) that binds lipid A and neutralizes LPS was from location was measured after a 2-h incubation with 10 ng/ml TNF-␣ (pos- Hycult Biotechnology. LPS from Escherichia coli (serotype O26:B6), itive control), 10 ␮g/ml Malp-2, 0.1 ␮g/ml LPS from P. gingivalis,10 Pseudomonas aeruginosa 10 (L9143), and Klebsiella pneumoniae (L4268) ␮g/ml purified LTA, or with these TLR agonists that had been preincu- and LTA from Staphylococcus aureus (L2551), Streptococcus faecalis bated with 1–10 ␮M of rhGSN. The intracellular location of NF-␬B was 4938 GELSOLIN INTERACTION WITH LPS AND LTA

observed using a mAb to the NF-␬B/subunit p65 (Molecular Probes), and cell nuclei were detected by counterstaining with 4Ј,6-diamidino-2-phe- nylindole dihydrochloride (Sigma-Aldrich). Individual cells were counted to assess NF-␬B localization as nuclear if the two stains colocalized or as cytoplasmic if they did not (14). E-selectin expression on HAEC surface HAECs were placed in four chamber slides at 1 ϫ 105 cells/ml and allowed to adhere to the surface for 24 h. Cells were then incubated in serum-free media for 6 h and placed in either no serum, 10 ␮g/ml LTA, 10 ␮g/ml LTA ϩ 4 ␮M gelsolin, or 10 ng/ml TNF-␣ for 24 h. Cells were then fixed in 4% paraformaldehyde for 20 min at room temperature, incubated in ammonium chloride for 20 min, and treated with a mouse anti-human E-selectin Ab (BD Pharmingen, 1/100 dilution in 0.5% BSA), followed by a rabbit anti-mouse secondary Ab (1/100 dilution in 0.5% BSA). Cells were then rinsed three times in PBS and viewed with a ϫ63 lens. Adhesion of neutrophils to LTA-activated HAECs To determine whether gelsolin is able to affect the adhesivity of neutrophils to LTA-stimulated HAECs, we measured the adhesion of calcein-AM- labeled neutrophils to confluent cultures of HAECs treated with LTA with or without gelsolin using a previously employed method (36). Endothelial cells were placed in 24-well plates and treated at 37°C for 8 h with 1–10 Downloaded from ␮g/ml of LTA or LTA ϩ 2 ␮M of gelsolin. Neutrophils were isolated from human blood using the endotoxin-free Lympholyte-poly kit (Cedarlane Laboratories) and resuspended in RPMI 1640 media (Invitrogen), labeled for 30-min with 2 ␮M calcein-AM, resuspended with RPMI 1640 media, and incubated with HAECs. After 30 min, unbound neutrophils were washed off and the number of bound cells was determined fluorometrically

using a Fluoroskan Ascent FL multiple plate reader (Labsystems). Percent- http://www.jimmunol.org/ age adhesion was expressed as: [(fluorescence after washing the plates Ϫ background fluorescence)/(fluorescence before washing the plates Ϫ background fluroescence)] ϫ 100. Bound neutrophils were viewed with a Leica microscope using a ϫ40 objective. Images were acquired using a Cool- SNAP HQ camera. Microscopy Neutrophils treated with E. coli LPS (100 ng/ml) or S. aureus LTA (5 ␮g/ml) with or without 2 ␮M gelsolin aftera2hincubation were viewed ϫ using a Leica microscope with a 40 objective. by guest on October 2, 2021

Determination of IL-8 concentration in the cell supernatant FIGURE 1. Interaction of PBP10 peptide and plasma gelsolin with Neutrophils (5 ϫ 106 cells/ml) suspended in RPMI 1640 buffer containing LTA. A, Rhodamine B fluorescence changes of PBP10 (10 ␮M) dissolved 2% human albumin were activated with highly purified LTA from S. au- in 10 mM Tris, 0.1 mM EGTA (pH 7.4) buffer in the presence of PIP2 and reus (0.1–10 ␮g/ml), LPS from E. coli (10 ng/ml), conventionally purified LTA from different bacteria strains. B, OD of rhGSN in solutions contain- LTA from S. aureus (5 ␮g/ml), or dilutions of autoclaved bacterial sus- ing varying amounts of purified LTA (S. aureus), LPS from P. gigivalis ␮ ␮ pension (1 l/ml) with or without rhGSN (0.5–4 M) or LBP peptide (10 (TLR2 ligand), Malp-2 (TLR2 and TLR6 ligand), PIP , and phosphatidyl- ␮ 2 M). Cell-free neutrophil supernatants were collected by centrifugation at choline (PC). Error bars represent SDs from three to four measurements. 5000 ϫ g for 5 min and stored at Ϫ80°C until cytokine determination. IL-8 was measured using a sandwich ELISA, according to the manufacturer’s instructions. The detection limit was 30 pg/ml. then added. After incubation for 18 h at 37°C, the bacterial concentration Antimicrobial activity was measured as the OD at 595 nm, and the MIC was read as the lowest concentration resulting in inhibition of detectable bacterial growth. To test the hypothesis that binding of gelsolin to the bacterial wall components LPS or LTA will prevent LL37 membrane insertion, we evaluated Evaluation of gelsolin effect on LPS and LTA aggregation state LL37 antibacterial activity in the presence of rhGSN. The bactericidal ac- by dynamic light scattering (DLS) tivities of the LL37 peptide against Gram-negative kanamycin-resistant P. aeruginosa (PAO1) and Gram-positive B. subtilis (ATCC 6051) was mea- LPS and LTA molecules are amphipathic and form aggregates of varying sured as previously described (37). Bacteria were grown to mid-log phase sizes. These aggregates can be evaluated using DLS spectroscopy (39). In at 37°C (controlled by the evaluation of OD at 600 nm) and resuspended the absence of surface-active agents and divalent cations, LPS and LTA in PBS. The bacteria suspensions were then diluted in 100 ␮l of solutions self-assemble into micellar structures. The LPS or LTA aggregate size containing antibacterial agents by themselves or with 2 ␮M rhGSN. After (hydrodynamic diameter) was determined using a DynaPro 99 DLS instru- a 1-h incubation at 37°C, the suspensions were placed on ice and diluted ment. The method measures the diffusion constant of the aggregates from 10- to 1000-fold. Then, 10 ␮l aliquots of each dilution were spotted on P. the autocorrelation function of scattered light intensity. The diameter is ϭ ␲␩ aeruginosa isolation agar or Luria-Bertani agar plates for overnight culture calculated from the relation D kT/6 Rh, where D is the translational at 37°C. The number of colonies at each dilution was counted the following diffusion constant, ␩ is the solvent viscosity, k is Boltzman’s constant, and morning. The CFU per milliliter of the individual samples were determined Rh is the hydrodynamic radius. To determine whether gelsolin affects the using the dilution factor. aggregation state of LPS and LTA, solutions of bacterial wall products were evaluated before and after 20 min of incubation with either gelsolin Evaluation of minimal inhibitory concentration (MIC) or BSA (62 ␮M of each). The MIC of the LL37 peptide was determined by a microbroth dilution Statistical analysis method (38) with Mueller-Hinton broth (MH) or MH supplemented with 2 Ϯ mM MgCl2 with or without the addition of rhGSN. A series of 2-fold Data are reported as means SD from three to six experiments. Differ- dilutions of LL37 in 0.25ϫ MH broth were prepared from a stock solution ences between means were evaluated using the unpaired Student’s t test, and placed in 96-well plates to which dilutions of B. subtilis bacteria were with p Ͻ 0.05 being taken as the level of significance. The Journal of Immunology 4939 Downloaded from http://www.jimmunol.org/

FIGURE 3. Translocation of NF-␬B from cytoplasm to nucleoplasm of HAEC (in endothelial cell basal medium-2 with supplements). A, Images show translocation induced with TNF-␣ or Malp-2 that was not prevented by recombinant human plasma gelsolin (arrows indicate nuclei without and with bright spots in control- and Malp-2-treated samples, respectively). Scale bar ϭ 20 ␮M. B, Quantiﬁcation of gelsolin-mediated inhibition when

translocation of NF-␬B was induced with LPS from P. gigivalis and pu- by guest on October 2, 2021 riﬁed LTA from S. aureus. Data are shown from one representative experiment performed in triplicate.

creased, so did the level of peptide fluorescence, suggesting insertion of the peptide-bound rhodamine B into a more hydro- phobic environment (30). At the molar ratios tested, only the first stage of decreased fluorescence was seen with LTA or

PIP2. LTA from different Gram-positive bacteria, including purified LTA from S. aureus, had similar effects on PBP10 fluorescence. There was no significant fluorescence change after adding LTA to a control peptide with the sequence RhB-QRL (data not shown). Binding of LTA to intact gelsolin was evident from a change in UV absorbance shown in Fig. 1B. Purified LTA decreases the absorbance of gelsolin, with a maximal decrease of ϳ20%, similar to that seen with LPS from P. gingi- FIGURE 2. Effect of LTA (S. aureus), LPA, PIP , and LPS (E. coli)on 2 valis and PIP . Malp-2 and zwitterionic phosphatidylcholine the pyrene F-actin (0.4 ␮M) filament-severing activity of rhGSN (0.05 2 had no effect on gelsolin absorbance. This result indicates that ␮M) (A), human blood serum (B), and CSF (C) (samples from patients diagnosed with facial nerve palsy or ischialgia due to discopathy). Error in addition to gelsolin’s ability to bind LPS (14, 40) from Gram- bars represent SDs from four measurements. negative bacteria, gelsolin is also able to interact with LTA, a Gram-positive bacterial wall component.

LTA inhibits the actin filament severing activity of recombinant Results gelsolin, human serum, and CSF LTA interacts with gelsolin’s PPI-binding sequence (residues When added to 0.05 ␮M rhGSN, 5 and 10 ␮M LTA inhibit 40% 160–169) and 90% of gelsolin’s severing activity, respectively (Fig. 2A). On The effect of LTA on the fluorescence of the gelsolin-derived a molar basis, LTA has potency similar to LPA, another known PBP10 peptide is shown in Fig. 1A. As previously reported, upon gelsolin inhibitor. When added to whole human serum (Fig. 2B)or interaction of PBP10 with LPS (14), there is an initial decrease in CSF (Fig. 2C), LTA and PIP2 were also able to inhibit actin- fluorescence at low LPS/peptide ratios; as the amount of LPS in- severing activity, although larger amounts of lipids were needed 4940 GELSOLIN INTERACTION WITH LPS AND LTA

FIGURE 4. Gelsolin prevents LTA-induced activation of HAEC evaluated by E-selectin expression (A) and human neutrophil adhesion (B). C, Quantification of neutrophil adherence calculated from the fluorescence of calcein-AM. A and B, Data from one representative experiment are shown. C, Error bars represent SD from Significantly different from LTA ,ء .four measurements (10 ␮g/ml)-activated samples. Downloaded from http://www.jimmunol.org/

compared with inhibition of pure gelsolin in aqueous solution. The adhesion to the cell surface. The adhesion of neutrophils to LTA ability of LTA to inhibit the actin-depolymerizing activity in se- (1–10 ␮g/ml)-treated HAECs for8hisshown in Fig. 4, B and C. rum is specific to gelsolin, because the actin-sequestering activity Recombinant plasma gelsolin effectively prevents LTA-induced by guest on October 2, 2021 of Gc-globulin (vitamin D-binding protein), the other component activation of HAECs that translates to a decrease in neutrophil of the plasma actin scavenger system (35, 41), was not affected by adhesion. Quantification of fluorescence from calcein-AM-labeled LTA (data not shown). neutrophils documents a significant decrease in neutrophil adhesion to HAEC activated with 10 ␮g/ml LTA in the presence of 2 ␬ Inhibition of NF- B translocation to the nuclei of HAECs in the ␮M rhGSN. presence of rhGSN Translocation of NF-␬B to the nucleoplasm represents another consequence of bacterial wall product interaction with cell mem- Exposure of neutrophils to LTA or LPS results in changes in brane TLRs. As shown in Fig. 3A, TNF-␣ and Malp-2 induce cell morphology NF-␬B translocation from cyto- to nucleoplasm of HAECs, but the Neutrophil activation is associated with marked changes in cellular effect was not inhibited in the presence of rhGSN. On the other morphology. Typically, shortly after activation by LPS or LTA, hand, NF-␬B translocation induced with LPS from P. gingivalis or neutrophils adopt an elongated shape with a rough surface and purified LTA (Fig. 3B) was almost completely prevented by 2 and form protrusions and aggregates (Fig. 5). After2hofincubation 10 ␮M rhGSN, respectively. with LPS or LTA in the presence of rhGSN, the morphological predictors of neutrophil activation were less pronounced and were Expression of E-selectin on HAECs limited to a lower population of cells (Fig. 5). This result suggests LPS and LTA have been reported to induce E-selectin expression that gelsolin may modulate neutrophil activation by sequestering on HUVECs, with a maximum increase between 4 and 8 h (42). bacterial wall products and buffering their interaction with TLRs. Similarly, we detected an increase in E-selectin expression on HAECs after activation with LTA. As shown in Fig. 4A,2␮M gelsolin partially prevents LTA-mediated E-selectin expression on Gelsolin partially prevents IL-8 release from LPS, LTA, or HAECs. Fluorescence quantification revealed that the average in- lysed bacteria-activated neutrophils tensity of E-selectin markers was 180 Ϯ 75, 250 Ϯ 95, 355 Ϯ 180, Unstimulated human neutrophils produce very low, but detectable, and 260 Ϯ 120 for control, TNF-␣, LTA, and LTA ϩ gelsolin amounts of cytokines, and after activation they produce and release samples, respectively. several cytokines, including IL-8, TNF-␣, and G-CSF at levels 10–50 times higher compared with the resting state. Although IL-8 Adhesion of neutrophils to LTA-treated HAECs is produced by a variety of cell types, neutrophils are the major Similar to HUVECs (42) and human lung microvascular endothe- source of this proinflammatory cytokine (19). In the presence of lial cells (36), treatment of HAECs with LTA increases neutrophil LTA, time-dependent induction of IL-8 release was observed, with The Journal of Immunology 4941

FIGURE 5. Neutrophil morphology following a 2-h exposure to LPS (100 ng/ml) or LTA (10 ␮g/ml) with or without 2 ␮M rhGSN. Results are shown from one experiment performed in triplicate. a maximum reached at 24 h (data not shown). IL-8 secretion induced by addition of purified LTA was also concentration dependent. The amount of IL-8 released after exposure to 10 ng/ml of Downloaded from LPS was comparable to that observed after neutrophil activation with 5–10 ␮g/ml of LTA or 1 ␮l/ml of heat-inactivated bacteria FIGURE 7. B. subtilis (Fig. 6). Neutrophils coincubated with LPS, purified and nonpu- Bactericidal activity of LL37 against (ATC 6051) and P. aeruginosa (PAO1) alone or in the presence of 2 ␮M rhGSN. rified LTA, or lysed bacteria in the presence of rhGSN released Error bars represent SDs from three measurements (A). LL37 MIC value significantly lower amounts of IL-8. In the case of activation with (␮g/ml) for B. subtilis (ATC 6051) evaluated in Mueller-Hinton broth purified LTA, we observed a partial but progressive decrease of (MH) did not change in the presence of rhGSN (B). http://www.jimmunol.org/ released IL-8 when an increased concentration of rhGSN was added. In agreement with previous observations, gelsolin may function as an inhibitor of LTA/LPS-induced IL-8 synthesis (19). The inhibition of IL-8 was also observed after LPS addi- 30 min after LPS or LTA treatment did not prevent IL-8 release tion in the presence of LBP peptide (aa 86–99) that binds lipid to the same extent as when gelsolin was added together with the A and neutralizes LPS. Gelsolin addition to neutrophil samples stimuli (data not shown). This result supports the hypothesis by guest on October 2, 2021

FIGURE 6. A, Purified LTA from S. aureus-induced IL-8 release from human neutrophils in a concentration- dependent manner. Gelsolin prevents IL-8 release from neutrophils treated with purified LTA (B), LPS (E. coli) or nonpurified-LTA (S. aureus)(C), and neutrophils treated with heat-inactivated Gram-negative P. aeruginosa PAO1 or Gram-positive B. subtilis ATCC 6051 (1 ␮l of each) (D). Error bars represent SDs from three Significantly ,ء .measurements performed in duplicate different from control neutrophil samples or those treated with LPS, LTA, or heat-inactivated bacteria. 4942 GELSOLIN INTERACTION WITH LPS AND LTA

LL37 to kill B. subtilis or P. aeruginosa (Fig. 7A). The MIC value for the LL37 peptide with or without gelsolin (Fig. 7B) was un- changed. These data suggest that gelsolin is unable to interact with LTA molecules within the intact bacterial wall in the same way as takes place upon LTA release from dividing or dying bacteria.

Gelsolin affects aggregation stage of LPS and LTA molecules Amphiphilic molecules such as LPS, lipid A, and LTA form aggregates in aqueous environments above a critical micellar concentration. The actual structure of these aggregates is not a constant, but depends on concentration, solvent conditions, and the effects of other solutes that can co-assemble in the micelles (20, 22). Accordingly, the average size of LPS and LTA molecules was observed to decrease as their solutions were diluted (Fig. 8A). Pre- vious analyses of aqueous LPS suspensions by negative staining and platinum shadowing revealed the presence of small globular aggregates (diameter 20–80 nm) and short filaments (43, 44). Upon the addition of serum proteins, mainly albumin, LPS aggregates become larger (Ͼ200 nm) and form structures several mi- Downloaded from crometers in length (44). In our study we observed a decrease in LPS and LTA aggregate size (Fig. 8) in the presence of purified gelsolin. This effect is consistent with a gelsolin-specific interaction with LPS and LTA. http://www.jimmunol.org/ Discussion Many of the biological activities of Gram-negative bacterial endotoxin are shared by LTA, a complex glycolipid from the outer wall of Gram-positive bacteria (45). Whereas most LPS- or LTA- mediated cellular effects are similar, LPS and LTA share few steps in their activation mechanisms and signal transduction pathways (27, 42, 46). The finding that gelsolin binds to LPS (14), LPS from P. gingivalis that acts as a specific agonist of TLR2, and purified by guest on October 2, 2021

LTA with approximately the same strength as LPA and PIP2 suggests that gelsolin may be involved in both LTA and LPS presentation to TLR4 and TLR2, acting potentially as a scavenger or delivery promoter of immunogenic bacterial wall components. This proposed function is similar to gelsolin’s involvement in the presentation of LPA to cellular receptors (13). The specificity of gelsolin’s effects on acidic bioactive lipids is enforced by lack of gelsolin’s effect on TNF-␣ and Malp-2 activation of NF-␬B translocation in endothelial cells. As LTA and LPS are potent mediators of the innate immune response, efficacy of the host recognition may determine survival during bacterial infection. It is also possible that the binding of gelsolin to LTA or LPS determines their interaction with other proteins such as LBP, CD14, or MD2 and factors such as lipoprotein involved in host cell detection and elim- FIGURE 8. The size distribution (filled symbols) and light scattering ination of bacterial products. Gelsolin may have a buffering effect intensity (open symbols) (A) with progressive dilutions of LPS (squares) on the availability of LTA/LPS for these targets. Therefore, LPS and LTA (triangles) in PBS (EC, E. coli; PA, P. aeruginosa, KP, K. pneu- (29) and LTA binding to gelsolin may determine the immune re- moniae; SA, S. aureus; SF, S. faecalis). DLS evaluations are shown of 1 sponse at different steps of the signaling pathway. LTA, LPS, LPA, mM LPS (B) and 0.5 mM LTA (C) aggregation states in PBS solution and PAF bind to at least one common site within the gelsolin without rhGSN (open column) and 5 min after rhGSN addition (62 ␮M, gray column). molecule (14, 47, 48), indicating the possibility for competition among these lipids. However, the possible role of gelsolin in LPS and LTA recognition may also differ from the role of LBP or CD14 that gelsolin acts as an LTA/LPS buffer preventing their ago- protein. In healthy subjects, blood gelsolin is present at much nistic effect on TLRs, although gelsolin may also interfere with higher concentrations than the other high-affinity ligands, and, un- other mediators involved in the regulation of neutrophil syn- like LBP and CD14, gelsolin is not an acute-phase protein (49, 50). thesis or release of IL-8. Gelsolin levels are decreased in several pathological inflammatory states, and lowered gelsolin levels have potential for identi- Gelsolin does not interfere with bacterial killing by LL37 fying patients at risk for adult respiratory distress syndrome or rhGSN by itself had no effect on bacterial growth or the ability of multiple organ failure in some settings. Repletion of plasma gel- the synthetic human cathelicidin-derived antibacterial peptide solin was shown to be beneficial in murine hyperoxic lung injury The Journal of Immunology 4943

(51) and endotoxemia (52), and gelsolin was able to attenuate vas- plasma gelsolin infusion attenuates burn-induced pulmonary microvascular dys- cular permeability associated with burn injury in rats (5). The hy- function. J. Appl. Physiol. 96: 25–31. 6. Smith, D. B., P. A. Janmey, and S. E. Lind. 1988. Circulating actin-gelsolin pothesis that plasma gelsolin may have utility in clinical settings complexes following oleic acid-induced lung injury. Am. J. Pathol. 130: such as ARDS, burn, and sepsis is supported by the results of this 261–267. 7. Smith, D. B., P. A. Janmey, J. A. Sherwood, R. J. Howard, and S. E. Lind. 1988. study. In this context, prevention of a decrease in blood gelsolin Decreased plasma gelsolin levels in patients with Plasmodium falciparum ma- during sepsis (10) may protect the host from bacterial wall prod- laria: a consequence of hemolysis? Blood 72: 214–218. uct-mediated increase of cytokines and systemic complications 8. Ware, L. B., and M. A. Matthay. 2000. The acute respiratory distress syndrome. N. Engl. J. Med. 342: 1334–1349. that can lead to septic shock and multiorgan failure. The first an- 9. Christofidou-Solomidou, M., A. Scherpereel, C. C. Solomides, J. D. Christie, imal study in which gelsolin was used to prevent septic shock (52) T. P. Stossel, S. Goelz, and M. J. DiNubile. 2002. Recombinant plasma gelsolin indicated an increase in animal survival when an injection of LPS diminishes the acute inflammatory response to hyperoxia in mice. J. Investig. Med. 50: 54–60. was followed by rhGSN administration. The mechanism of gelso- 10. Suhler, E., W. Lin, H. L. Yin, and W. M. Lee. 1997. Decreased plasma gelsolin lin’s role in this effect is poorly understood, and it is still an open concentrations in acute liver failure, myocardial infarction, septic shock, and question as to which of gelsolin’s activities—F-actin severing, in- myonecrosis. Crit. Care Med. 25: 594–598. 11. Dahl, B., F. V. Schiodt, P. Ott, R. Gvozdenovic, H. L. Yin, and W. M. Lee. 1999. activation of LPS/LTA, or buffering of other lipid mediators— Plasma gelsolin is reduced in trauma patients. Shock 12: 102–104. translates to an increase in survival during endotoxemia or in other 12. Mounzer, K. C., M. Moncure, Y. R. Smith, and M. J. Dinubile. 1999. Relation- ship of admission plasma gelsolin levels to clinical outcomes in patients after critical care stages. major trauma. Am. J. Respir. Crit. Care Med. 160: 1673–1681. The presence of gelsolin in CSF (53) combined with the ob- 13. Goetzl, E. J., H. Lee, T. Azuma, T. P. Stossel, C. W. Turck, and J. S. Karliner. served decrease in chronic immune-inflammatory diseases such as 2000. Gelsolin binding and cellular presentation of lysophosphatidic acid. J. Biol. Chem. 275: 14573–14578. multiple sclerosis (54) and the previously reported ability of LTA

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