Cellular Mechanism of Intraabdominal Abscess Formation by Bacteroides fragilis Frank C. Gibson III, Andrew B. Onderdonk, Dennis L. Kasper and Arthur O. Tzianabos This information is current as of September 27, 2021. J Immunol 1998; 160:5000-5006; ; http://www.jimmunol.org/content/160/10/5000

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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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cellular Mechanism of Intraabdominal Abscess Formation by Bacteroides fragilis1

Frank C. Gibson III,2* Andrew B. Onderdonk,† Dennis L. Kasper,* and Arthur O. Tzianabos*

We investigated the cellular mechanism by which Bacteroides fragilis promotes the development of intraabdominal abscesses in experimental models of sepsis. B. fragilis, as well as purified capsular polysaccharide complex (CPC) from this organism, adhered to primary murine mesothelial cells (MMCs) in vitro. The binding of CPC to murine peritoneal macrophage stimulated TNF-␣ production, which when transferred to monolayers of MMCs elicited significant ICAM-1 expression by these cells. This response resulted in enhanced polymorphonuclear leukocyte attachment to MMCs that could be inhibited by Abs specific for TNF-␣ or ICAM-1. Mice treated with TNF-␣- or ICAM-1-specific Abs failed to develop intraabdominal abscesses following challenge with Downloaded from purified CPC. These results illustrated the role of the CPC in promoting adhesion of B. fragilis to the peritoneal wall and coordinating the cellular events leading to the development of abscesses associated with experimental intraabdominal sepsis. The Journal of Immunology, 1998, 160: 5000–5006.

espite the plethora of bacteria that cause human disease, gilis or purified CPC, PSA, or PSB (15, 17–19). Early studies host responses to these organisms are comprised of three (20) showed that encapsulated B. fragilis bound to the perito- D pathologic mechanisms: tissue inflammation, granu- neal walls of rats better than unencapsulated Bacteroides spe- http://www.jimmunol.org/ loma, and abscess formation. Recent work has revealed a better cies, enabling B. fragilis to resist clearance from the peritoneal understanding of the mechanisms governing tissue inflammation cavity by the diaphragmatic lymph system (20). Several groups (1–3) and granuloma formation (4–6), while the processes under- have demonstrated that peritoneal mesothelium, a layer of cells lying abscess formation remain ill defined. Clinically, intraab- that constitutes a line of structural and immunologic defense in dominal abscesses are commonly formed following events that the abdominal cavity, potentiates the deposition of fibrin (21), lead to the perforation of the bowel and subsequent leakage of the and the production of an array of cytokines and cell adhesion colonic contents into the abdomen. Although Bacteroides fragilis molecules (21–28), plays an important role in abdominal sepsis. is among the least prevalent anaerobic species in the intestinal Despite the lack of information describing a role for peritoneal by guest on September 27, 2021 tract, it is responsible for the majority of all clinical cases of an- mesothelium during the formation of intraabdominal abscesses, aerobic sepsis and intraabdominal abscesses (7, 8). Studies inves- it is likely that inflammatory cells interact with this physical tigating the virulence properties associated with this organism barrier during the migration from host tissues to the peritoneal 3 have shown that the CPC of B. fragilis is responsible for abscess lumen. The processes governing accumulation of these cells in formation (9–14). The CPC is comprised of two distinctly charged the peritoneal cavity remain unclear (29); however, these mech- polysaccharides (PSA and PSB) coexpressed on the surface of this anisms most likely parallel those elucidated for migration of organism. Positive and negative charged groups on PSA and PSB immune cells from the vasculature to a focus of infection: a mediate the biologic properties of these polymers (15, 16). complex process regulated by cytokines, cell adhesion mole- Abscess formation is a complex host response that involves cules, and cell activation (23, 30, 31). the recruitment and accumulation of neutrophils, fibrin deposi- Several studies have shown that the host immune response is tion, and other incompletely defined processes. In experimental critical to abscess formation and that several cell types are im- models, abscesses develop following i.p. challenge with B. fra- portant in the development of intraabdominal abscesses (9, 19, 32, 33). Intraperitoneal challenge of animals with B. fragilis is Channing Laboratory, Departments of *Medicine and †Pathology, Brigham and followed by immune cell infiltration, with an initial influx of Women’s Hospital and Harvard Medical School, Boston, MA 02115 lymphocytes into the peritoneal cavity and the appearance of Received for publication September 30, 1997. Accepted for publication January neutrophils and macrophages approximately 4 days postchal- 13, 1998. lenge (9). Recent studies have shown that purified phagocytic The costs of publication of this article were defrayed in part by the payment of page cells from mice or humans cultured in vitro with CPC produce charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. TNF-␣, IL-1␣, IL-8, and IL-10 (33). It has been suggested that 1 This work was sponsored by National Institutes of Health Grants AI34073 and cytokines may be responsible for triggering the migration of AI39579. immune cells into the peritoneal cavity following contamination 2 Address correspondence and reprint requests to Dr. Frank C. Gibson III, Channing with B. fragilis (33); however, the source and role of these Laboratory, Departments of Medicine and Pathology, Brigham and Women’s Hos- cytokines remain undefined. pital and Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115. E- mail address: [email protected] The prevalence of isolation of encapsulated B. fragilis from clin- 3 Abbreviations used in this paper: CPC, capsular polysaccharide complex; GBSTIII, ical cases of abscess formation led us to hypothesize that associ- group B streptococcal type III capsular polysaccharide; MMC, murine mesothelial ated surface polysaccharides allow this organism to persist pref- cell; PGG, poly(1–6)-␤-glucotriosyl-(1–3)-␤-glucopyranose; PMNL, polymorphonu- clear leukocyte; pMo, murine peritoneal macrophage; PSA, polysaccharide A; PSB, erentially within the peritoneal cavity and initiate cellular events polysaccharide B. that lead to the formation of this pathobiologic host response. In

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 5001 this work, we present data that demonstrate the preferential bind- lected by centrifugation and washed extensively to remove enzyme. Cells ing of B. fragilis, as well as purified surface polysaccharides from were grown in DMEM with 12% FCS supplemented with 2-hydrocortisone this organism, to MMCs in vitro. These polysaccharides stimulate and epidermal growth factor until confluent (4–6 days), then subcultured ␣ into 24-well or 96-well collagen-coated plates, grown for 24 to 48 h, and TNF- production by peritoneal macrophages that in turn elicited used upon reaching confluency (approximately 2.5 ϫ 105 and 3.3 ϫ 104 the production of ICAM-1 by MMCs. ICAM-1 expression on cells/well, respectively, and Ͼ98% pure by morphology and immunoflu- MMCs served as a functional ligand that supports increased orescent assay). Bacteroides sp. were added at various multiplicities of PMNL binding to these cells. mAb-blocking experiments in mice infection, polysaccharide Ags were added at various concentrations, or ␣ supernatant fluids from Ag-stimulated macrophage were added to these demonstrated that both TNF- and ICAM-1 expression are nec- cells. The number of MMCs per monolayer was determined for each assay. essary for the development of intraabdominal abscesses in vivo. Murine peritoneal macrophage. pMo were elicited in C57BL/6 mice by These studies define cellular events critical for intraabdominal ab- peritoneal injection of thioglycolate broth. After 3 days, cells were har- scess formation by B. fragilis. vested by lavage, washed, added to 24-well tissue culture plates at 1 ϫ 106 cells/ml, and allowed to adhere to plastic for2hat37°C. The monolayers were washed to remove nonadherent cells and cultured (Ͼ90% macro- Materials and Methods phage) with 1 ml culture medium, or medium containing 10 ␮g/ml CPC, Cultivation and preparation of bacterial strains CPC treated with 20 ␮g/ml polymyxin B, PGG-glucan (Alpha-Beta Tech- B. fragilis NCTC 9343 was obtained from National Culture Type Collec- nologies, Worcester, MA), or GBSTIII. After 24 h, supernatants were har- vested, centrifuged at 1000 ϫ g for 20 min to remove cells, and stored tion, Rockville, MD. Bacteroides thetaiotaomicron strain 491909 and Bac- Ϫ teroides distasonis strain 8503 are clinical strains obtained from the Chan- frozen at 80°C. ning Laboratory’s (Brigham and Women’s Hospital, Boston, MA) PMNLs. To isolate PMNLs, peripheral blood was taken from healthy hu- anaerobe stock culture collection, and were identified to the species level man donors and layered over a bed of Mono-Poly resolving medium (ICN Downloaded from by long chain fatty acid analysis and conventional biochemical reactions. Biomedicals, Palo Alto, CA). Following separation by centrifugation, the PMNL fraction was collected (Ͼ95% pure), washed with ice-cold DMEM Each strain was passaged once on Brucella agar supplemented with 5% 7 Ϫ to remove separation medium, resuspended in DMEM to 2 to 3 ϫ 10 defibrinated sheep’s blood and stored frozen at 80°C in peptone-yeast Ͻ extract broth. When needed, frozen aliquots were thawed, grown overnight cells/ml, and maintained on ice ( 30 min). A 1-ml sample of these cells at 37°C in an anaerobic chamber. Bacterial growth was collected and re- was warmed to 37°C and added to monolayers of MMCs in 24-well plates. suspended in DMEM without serum to the desired multiplicity of infection Bacteroides- and CPC-binding assays and blocking experiments before bacterial binding experiments. http://www.jimmunol.org/ Monolayers of MMCs were cocultured with B. fragilis, B. thetaiotaomi- Isolation of CPC; purification of PSA and group B cron,orB. distasonis for1hat37°C with 5% CO2. Monolayers were Streptococcus type III capsular polysaccharide washed extensively to remove unbound bacteria, and an equivalent volume of sterile water was added to the monolayers. Following lysis, vigorous The CPC used in these studies was isolated from B. fragilis grown in aspiration-expulsion cycles were performed with a pipet to evenly distrib- proteose-peptone yeast extract broth supplemented with hemin and mena- ute bacteria. The lysate was serially diluted in 1% peptone, plated on Bru- dione in a 20-L pH-controlled (pH 7.2) batch culture overnight at 37°C, as cella agar, and grown for 48 h for viable count (CFU/ml) determination. described previously (34). Additional experiments involved the addition of B. fragilis CPC, PSA, PSA was generated from pure CPC by isoelectric focusing with a Roto- [3H]PSA, and GBSTIII polysaccharide to MMCs. These Ags were for chamber (Bio-Rad, Hercules, CA) in 2% ampholytes (range 3–10) for weighed, diluted to a concentration of 1 mg/ml in DMEM without serum, 4to5hat12watts constant power. Focused fractions were collected, and and vortexed until completely dissolved; dilutions were then made with by guest on September 27, 2021 a sample of each fraction was subjected to immunoelectrophoresis and DMEM, and the Ags were added to MMCs. The amount of Ag bound to subsequent immunoprecipitation with high titer rabbit antiserum to B. fra- cells was evaluated by ELISA or liquid scintillation. gilis NCTC 9343 (34). Samples containing PSA were pooled and dialyzed In experiments designed to block the binding of B. fragilis to MMCs, against 1 M NaCl overnight, and then against distilled water for 2 days. bacteria were left untreated or treated with B. fragilis strain 9343-specific The purity of CPC and PSA was assessed by nuclear magnetic reso- capsular polysaccharide antiserum or irrelevant Ab for1hat37°C before nance spectroscopy, gas chromatography-mass spectrometry, immunoelec- addition of bacteria to MMC monolayers. Blocking of PSA binding to trophoresis (pH 7.3), UV spectroscopy (260 and 280 nm), and reducing MMCs was accomplished by adding various dilutions of a PSA-specific PAGE on gradient gels with subsequent silver staining, as described (34). mouse mAb (clone CE3) or nonimmune mouse control ascites (Sigma, St. The CPC and PSA used for these experiments were isolated from a single Louis, MO) to PSA (10 ␮g/ml) for1hat37°C. Untreated or Ab-treated B. extraction, tested for purity by the above methods, and stored dry at 4°C. fragilis or PSA was added to MMCs for 1 h, and binding was evaluated by Before use, each Ag was diluted to 1 mg/ml in pyrogen-free water and CFU/ml determinations or ELISA. tested for endotoxin by the Limulus amebocyte lysate assay (Cape Cod Competition experiments were performed to demonstrate specific bind- Associates, Woods Hole, MA). All Ags used in these studies tested free of ing of PSA to MMCs or pMo. To tritiated PSA (10 ␮g/ml), we added a endotoxin. 50-fold excess of native unmodified PSA (500 ␮g/ml). This polysaccharide The native and tritiated GBSTIII polysaccharides used in these exper- mixture was added to monolayers of cells in 24-well plates and cocultured iments were a kind gift from Dr. Lawrence Paoletti (Channing Laboratory). for1hat37°C. The cells were washed three times with DMEM and lysed Radiolabeling of PSA with 1 ml of sterile distilled water, and the lysates were collected and processed for liquid scintillation enumeration of 3H-radiolabeled PSA 3H-radiolabeled PSA was generated by oxidation and subsequent reduc- binding. tion. In brief, PSA was treated with sodium metaperiodate (0.01 M) to oxidize approximately 25% of the vicinal hydroxyl groups on the galacto- Quantitation of bacteria and Ag binding furanose of the PSA side chain to carbonyl groups. Ethylene glycol was added to stop oxidation, and the sample was dialyzed overnight against Colony counts. After incubation with bacteria, MMCs were washed with DMEM to remove unbound bacteria (with the efficiency of washing de- water. The newly generated carbonyl groups underwent reduction with ␮ tritiated sodium borohydride (DuPont NEN, Boston, MA) to form isotope- termined by plating of the final wash), and 100 l of sterile water was labeled hydroxymethyl groups on PSA. Excess unlabeled sodium borohy- added to the monolayers for 30 min to lyse MMCs. These lysates were dride was added to completely modify any remaining carbonyl groups, and subjected to cycles of vigorous aspiration and expulsion to disrupt cells and the modified Ag was dialyzed overnight against water lyophilized, and evenly disperse bacteria. The lysates were subjected to serial 10-fold di- stored dry at 4°C. We have demonstrated that this procedure does not alter lution in 1% peptone, plated onto Brucella blood agar plates, and incubated the biologic activity of the polymer. at 37°C in an anaerobic chamber. It was noted that the treatment of these bacteria in this manner did not affect organism viability. After 2 days, Isolation and culture of MMCs, murine peritoneal macrophage, colonies were enumerated. and PMNLs ELISA. MMCs cocultured with CPC were gently washed to remove ex- cess unbound Ag and were fixed with 2% formaldehyde in PBS (pH 7.2) MMCs. MMCs were isolated from the peritoneum of C57BL/6 mice by for 1 h. After fixation, monolayers were washed with PBS ϩ 0.05% enzymatic digestion and were cultivated in wells of collagen-coated culture Tween-20 (pH 7.2). High titer rabbit serum specific for B. fragilis was vessels (35). Briefly, omentum was harvested and digested with collage- added at a 1/2000 dilution in PBS (100 ␮l/well), and the monolayers were nase-dispase for 30 min at room temperature. Liberated cells were col- incubated for1hat37°C. Incubation was followed by three washes, after 5002 MECHANISMS OF INTRAABDOMINAL ABSCESS FORMATION

which 100 ␮l/well of a 1/4000 dilution of goat antiserum to rabbit IgG/ alkaline phosphatase conjugate (Biosource, Camarillo, CA) was added to monolayers and incubated for1hat37°C. The wells were washed, and 100 ␮lofp-nitrophenyl phosphate solution (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added for 15 min. The reaction was stopped, and plates were read with a microtiter plate reader at 405 nm. Liquid scintillation. After incubation for 1 h with tritiated polysaccha- rides, cells in either 24- or 96-well plates were washed with DMEM to remove unbound Ag, lysed with water in situ, and harvested onto glass fiber filters with a PHD cell harvester (Cambridge Technologies, Water- town, MA). The glass fiber disks were placed into glass vials with 2-ml liquid scintillation mixture, and cpm were measured with a liquid scintil- lation reader (Packard International, Downers Grove, IL). The sp. act. of tritiated PSA and GBSTIII were determined using a 10-␮g sample of each polysaccharide during each binding experiment, and cpm/ng dry weight of each polymer was calculated. Cytokine and ICAM-1 detection by ELISA MMCs were grown to confluency in 96-well collagen-coated cell culture FIGURE 1. Attachment of B. fragilis NCTC 9343 (B. fragilis), B. the- plates. Proinflammatory cytokines were detected from MMCs cultured taiotaomicron 491909 (B.theta), or B. distasonis 8503 (B. dist) to MMCs with CPC or PGG-glucan (10 ␮g/ml). Supernatants were collected 1, 4, 8, in vitro measured by viable bacterial colony counts. Bacteria were added at 4 and 24 h after stimulation, and levels of TNF-␣ and IL-1␣ were determined a multiplicity of infection of 1000 to 3 ϫ 10 MMCs in wells of 96-well Downloaded from with cytokine-specific ELISA kits (Endogen, Cambridge, MA). collagen-coated plates for1hat37°C. *p ϭ 0.0004, **p ϭ 0.0001 com- To detect ICAM-1 on the surface of MMCs, an in situ ELISA assay was pared with binding of B. fragilis NCTC 9343 to MMCs. developed. MMCs in 96-well plates were incubated for 18 h with 100 ␮l culture medium, 500 U/ml murine rTNF-␣ (R&D Systems, Cambridge, MA), CPC, or PGG-glucan (10 ␮g/ml), and supernatants from Ag-stimu- lated peritoneal macrophage. After stimulation, MMCs were washed to Statistical analyses remove Ags and fixed with 2% buffered formaldehyde. After fixation, cell All statistical analyses were performed with InStat statistical analysis soft- http://www.jimmunol.org/ ␮ monolayers were incubated with 100 l/well of a 1/500 dilution of rat ware (Graphpad Software, San Diego, CA) on an IBM Personal Computer anti-murine ICAM-1 mAb (clone YN/1.7.4; American Type Culture Col- AT. Results of in vitro data were calculated from three experiments, re- lection, Rockville, MD) for 1 h. The monolayers were washed and then corded as the mean Ϯ SD, and analyzed with the Kruskal-Wallis nonpara- incubated with 1/2000 dilution of goat antiserum specific for rat IgG/al- metric test. In vivo data were analyzed by the Fisher’s exact test. A p value kaline phosphatase (Sigma). Alkaline phosphatase substrate reagent of less than 0.05 was considered significant. (Kirkegaard & Perry Laboratories) was added to each well and developed for 1 h, and absorbance was read at 405 nm. Results Treatment of peritoneal macrophage supernatants with TNF-␣- Adherence of Bacteroides sp. to MMCs neutralizing Ab Initial experiments defined the binding kinetics of B. fragilis to by guest on September 27, 2021 Supernatant fluids from CPC-stimulated peritoneal macrophage were MMCs. B. fragilis was added to MMCs at various multiplicities of thawed and either added directly to monolayers of MMCs in 96-well plates infection ranging from 1 to 10,000. Bacterial viable counts indi- or treated with 10, 1, or 0.1 ␮g of goat neutralizing Ab to murine TNF-␣ (R&D Systems) or nonimmune goat IgG (Sigma) for1hat37°C before cated that a multiplicity of infection of 1000 saturated binding sites addition to MMCs. After the addition of these supernatants, MMCs were on MMCs (data not shown). Additional experiments compared the incubated for 18 h and then assayed for surface-expressed ICAM-1 by binding of B. fragilis with other Bacteroides species (Fig. 1). B. ELISA or in PMNL-binding experiments. fragilis (1.39 ϫ 106 CFU/ml) bound more avidly than B. thetaio- ϫ 5 ϭ PMNL/MMC cell adherence assays taomicron (2.35 10 CFU/ml; p 0.0004 vs B. fragilis)orB. distasonis (8.88 ϫ 104 CFU/ml; p ϭ 0.0001 vs B. fragilis). In We adapted the method for studying the binding of human PMNLs to similar experiments to characterize the attachment of B. fragilis to murine endothelium (36) and modified this for MMCs. Supernatant fluids from Ag-stimulated peritoneal macrophage were added to MMCs in 24- MMCs, bacteria were treated with either CPC-specific rabbit poly- well plates for 18 h, as previously described (this study). PMNLs were clonal Ab or irrelevant Ab before the addition to MMC monolay- added to MMC and allowed to adhere for 30 min. After binding, the co- ers. Irrelevant Ab-treated B. fragilis (1.28 ϫ 106 CFU/ml) bound cultures were washed to remove unbound PMNLs while taking care to to similar levels as untreated B. fragilis (1.15 ϫ 106 CFU/ml), maintain intact MMC monolayers. Additional experiments were performed while CPC-specific Ab treatment significantly reduced B. fragilis to characterize the mechanism of PMNL attachment to MMCs stimulated 5 with CPC-treated macrophage supernatant fluids. Following stimulation, attachment (1.21 ϫ 10 CFU/ml; p Ͻ 0.002 vs irrelevant Ab MMCs were treated with ICAM-1-specific mAb (100 ␮g/ml) or irrelevant treatment). Ab matched to isotype (clone IXB2; a kind gift from Dr. Gene Muller, Channing Laboratory, Brigham and Women’s Hospital) for 1 h before Characterization of CPC and PSA attachment to host peritoneal PMNL attachment. An observer blinded as to the treatments counted the cells number of PMNLs bound to MMCs per ϫ200 magnification field with an Olympus CK2 phase contrast microscope. Five random fields were counted MMCs. The binding of CPC to MMCs was measured by ELISA. per sample. With the addition of increasing doses of CPC (ranging from 10 ␮ In vivo Ab blocking of ICAM-1 and TNF-␣ ng/ml to 200 g/ml) in DMEM, saturation was achieved at a dose of 10 ␮g/ml (Fig. 2A). Maximal binding of this dose of CPC oc- A murine model of peritoneal abscess formation was adapted to assess the curred within 15 min. role of TNF-␣ and ICAM-1 during abscess formation (19). In brief, C57BL/6 mice received 100 ␮l i.p. injections of rat Ab to murine ICAM-1, To better define the binding characteristics of B. fragilis poly- goat Ab to murine TNF-␣, or sham mAb (clone IXB2) in PBS (1 mg/ml) saccharides to MMCs, we performed experiments with a compo- 24 and 4 h before implantation of an abscess-inducing inoculum of 100 ␮g nent polysaccharide of CPC, PSA. This repeating unit is readily CPC in the adjuvant sterile cecal contents. Mice received additional i.p. amenable to radiolabeling and was useful in quantifying polysac- injections of mAb 4, 24, 48, 72, and 96 h after challenge to down-regulate TNF-␣ or ICAM-1 in vivo (37). Six days after B. fragilis CPC challenge, charide binding and binding specificity to cells. Tritiated PSA (sp. an observer blinded as to the treatment, then graded for presence of i.p. act. 10.15 cpm/ng) bound to MMCs with a profile similar to that of abscesses in these animals. CPC (6% of the input polysaccharide or 1.997 pg/cell bound when The Journal of Immunology 5003

added at 10 ␮g/ml; Fig. 2B). PSA binding was inhibited with mAb CE3 (specific for PSA), as measured by ELISA (48% inhibition; p Ͻ 0.0001). Competitive binding experiments demonstrated spec- ificity of PSA binding to MMCs. The addition of 50-fold unlabeled PSA with tritiated PSA (10 ␮g/ml) to MMC monolayers signifi- cantly reduced the binding of tritiated PSA (Fig. 2B). Addition of 3H-radiolabeled GBSTIII (sp. act. 42.78 cpm/ng) to MMCs failed to bind appreciably irrespective of dose administered. Peritoneal macrophage. CPC and PSA of B. fragilis bound readily to pMo. The time-dependent binding of tritiated PSA to these cells is shown in Figure 2C. Saturation of binding sites on pMo occurred with a dose of 50 ␮g/ml (3% of the input Ag or 0.289 pg/cell; Fig. 2D) and occurred at 1 h following the addition of PSA (Fig. 2C). PSA bound specifically to pMo as 50-fold ex- cess unlabeled PSA significantly inhibited PSA attachment (Fig. 2D); furthermore, the addition of 50-fold excess GBSTIII poly- saccharide failed to inhibit PSA binding. GBSTIII polysaccharide did not bind appreciably to these cells. Downloaded from CPC stimulation of TNF-␣ and ICAM-1 Direct in vitro stimulation of MMCs with CPC failed to elicit detectable levels of the proinflammatory cytokines TNF-␣ or IL-1␣ from these cells, but resulted in a modest increase in sur- face-expressed ICAM-1 compared with untreated or PGG-glucan- treated cells ( p Ͻ 0.02 and p Ͻ 0.04, respectively; data not http://www.jimmunol.org/ shown). Additional experiments, in which culture supernatants from CPC-stimulated murine peritoneal macrophages were added to MMCs for 18 h, resulted in a potent ICAM-1 response ( p Ͻ 0.0001 vs medium supernatant transfer; Fig. 3) by these cells. This effect was dependent on the dose and time of CPC administration to the macrophages and was not elicited by PGG-glucan or GB- STIII. In addition, incubation of CPC with polymyxin B did not

affect ICAM-1 expression. Based on our previous data in which by guest on September 27, 2021 CPC was shown to elicit a potent TNF-␣ response from murine peritoneal macrophages (33), we hypothesized that this cytokine was responsible for ICAM-1 expression by MMCs. Therefore, CPC-stimulated macrophage supernatants were treated with neu- tralizing Ab specific for murine TNF-␣ (1 ␮g/ml) before addition of the supernatants to MMCs. This treatment significantly reduced the level of ICAM-1 expressed by MMCs ( p ϭ 0.0022 vs non- immune goat IgG; Fig. 3).

PMNL binding to MMCs To assess the biologic function of ICAM-1 expression by MMCs, a PMNL-binding assay was performed. In this assay, human PMNLs were added to MMC monolayers following culture with supernatants from pMo stimulated with medium, PGG-glucan, GBSTIII, or CPC. In these experiments, direct stimulation of MMCs with TNF-␣ for 18 h resulted in enhanced PMNL binding

FIGURE 2. Binding kinetics of B. fragilis capsular polysaccharides to host cells. A, Dose-dependent binding of CPC to MMCs measured by ELISA. Ag was added to 3 ϫ 104 MMCs for1hat37°C. B, Dose- dependent binding of [3H]PSA and specificity of PSA binding to MMCs. Ag was added directly to MMCs for1hat37°C. Addition of 50-fold excess unlabeled PSA with 3H-labeled PSA prevented binding of the la- beled Ag (*p Ͻ 0.001 vs 10 ␮g/ml PSA dose). C, Time-dependent binding of 50 ␮g/ml [3H]PSA to pMo. Ag was added to 1 ϫ 106 pMofor1hat 37°C. D, Dose-dependent binding of [3H]PSA to pMo. Ag was added to 1 ϫ 106 pMofor1hat37°C. Addition of 50-fold excess unlabeled PSA with 3H-labeled PSA prevented binding of the labeled Ag (*p Ͻ 0.002 vs 50 ␮g/ml PSA dose). 5004 MECHANISMS OF INTRAABDOMINAL ABSCESS FORMATION

FIGURE 3. ICAM-1 expression on MMCs following supernatant trans- FIGURE 4. PMNL binding to macrophage supernatant-stimulated Downloaded from fer from polysaccharide-stimulated peritoneal macrophage. Monolayers of MMCs correlates with enhanced levels of ICAM-1 on MMCs and is me- MMCs were stimulated with culture medium, murine rTNF-␣ (TNF-␣; 500 diated by TNF-␣ and ICAM-1. MMCs were directly stimulated with me- 1 U/ml), or TNF-␣ treated with TNF-␣-neutralizing Ab (Ab ,1␮g/ml) or dium or murine rTNF-␣ (rmTNF-␣; 500 U/ml) to produce ICAM-1. Su- stimulated with supernatants from peritoneal macrophages cocultured with pernatants collected from peritoneal macrophage cocultured with medium, DMEM (medium), PGG-glucan, GBSTIII, or CPC (10 ␮g/ml). In similar PGG-glucan, GBSTIII, or CPC (10 ␮g/ml) were added to MMCs for 18 h. experiments, CPC-stimulated peritoneal macrophage supernatants were In similar wells, CPC-stimulated macrophage supernatants were treated treated with TNF-␣-neutralizing Ab or nonimmune goat IgG (IRR-Ab, 1 with TNF-␣-neutralizing Ab or nonimmune goat IgG (IRR1) for 1 h before http://www.jimmunol.org/ ␮g/ml) for 1 h before addition to monolayers of MMCs. After 18 h, sur- addition to monolayers of MMCs or following stimulation with ICAM-1- face-expressed ICAM-1 was measured by ELISA. *p ϭ 0.0022 when com- blocking Ab or nonimmune rat IgG (IRR2). After these treatments, PMNLs pared with ICAM-1 stimulation by supernatant fluids from CPC-treated were added to MMCs, and cell attachment was measured. *p Ͻ 0.002 when peritoneal macrophages; **p ϭ 0.0022 when compared with ICAM-1 stim- compared with PMNL binding to MMCs stimulated with supernatant fluids ulation by supernatant fluids from medium-treated peritoneal macrophage. from CPC-treated peritoneal macrophage; **p Ͻ 0.002 when compared with PMNL binding to MMCs stimulated with supernatant fluids from medium-treated peritoneal macrophage. ( p Ͻ 0.002 vs medium control; Fig. 4). MMCs treated with su- pernatant fluids from CPC-stimulated macrophages supported in- by guest on September 27, 2021 creased PMNL binding compared with supernatants from PGG- mesothelium, and that CPC is the primary attachment factor. To glucan- or GBSTIII-treated macrophages ( p Ͻ 0.002 and p Ͻ evaluate the role of B. fragilis in initiating intraabdominal ab- 0.002, respectively, vs CPC stimulation; Fig. 4). Treatment of scesses, we developed an in vitro system to study the interactions CPC-stimulated supernatant fluids with murine TNF-␣-neutraliz- of this organism or the purified polysaccharides from its surface ing Ab significantly reduced PMNL binding to MMCs ( p Ͻ 0.002 with the first cell boundary likely to be encountered in the perito- vs irrelevant Ab treatment; Fig. 4). Furthermore, PMNL binding to neal cavity: peritoneal mesothelium. MMCs was inhibited by treatment of monolayers with ICAM-1- B. fragilis adhered more avidly to MMCs than either B. dista- specific mAb ( p Ͻ 0.002 vs irrelevant Ab treatment; Fig. 4). sonis or B. thetaiotaomicron. This result suggested that the CPC In vivo role of TNF-␣ and ICAM-1 in abscess formation functions as an attachment factor. Previous studies have shown that B. thetaiotaomicron has only a thin capsule layer (41), while The role of TNF-␣ and ICAM-1 in the development of intraab- B. distasonis lacks a capsule. This difference most likely explains dominal abscesses was studied in a murine model of peritoneal why B. thetaiotaomicron binds less avidly than B. fragilis but more sepsis. Mice received i.p. injections of TNF-␣-neutralizing Ab, avidly than the unencapsulated B. distasonis. Although little is ICAM-1-specific mAb, or sham Ab (100 ␮g/injection) 24 and 4 h known about the capsular polysaccharide of B. thetaiotaomicron, before challenge, and 2, 24, 48, 72, and 96 h after B. fragilis CPC its binding capacity is interesting since B. thetaiotaomicron is the challenge. Treatment with these mAbs significantly reduced the second most frequently isolated Bacteroides species in human development of abdominal abscesses following CPC challenge, disease. while treatment with a sham Ab did not affect abscess formation The finding that CPC adhered to different cell types (MMCs and ( p Ͻ 0.0005 for TNF-␣, and p Ͻ 0.0005 for ICAM-1 vs irrelevant pMo) was not surprising, as surface-expressed polysaccharides Ab treatment; Table I). from Actinobacillus actinomycetem comitans and Staphylococcus aureus type 5 and 8 bind to a variety of host cells (42–44). Fur- Discussion thermore, recent studies have shown that binding of microbial The binding of bacteria to host cells is, in many cases, critical to polysaccharides to host cells is important for eliciting proinflam- the progression of bacterial infections, including those of the peri- matory cytokines (43–45). Previous work by our group has dem- toneal cavity (22, 25, 38–40). B. fragilis, an encapsulated organ- onstrated that the CPC of B. fragilis elicits potent TNF-␣, IL-1␣, ism that is the primary cause of intraabdominal abscesses and IL-8, and IL-10 response from phagocytic cells of human or mu- Gram-negative anaerobic bacteremia, binds to the abdominal wall rine origin (33). In the present study, we were unable to detect the of rats more readily than do other unencapsulated Bacteroides or- proinflammatory cytokines TNF-␣ or IL-1␣ from MMCs cocul- ganisms (20). We hypothesized that encapsulated B. fragilis or- tured with CPC. Although other cytokines may be produced from ganisms resist clearance from the peritoneal cavity by adhering to CPC-stimulated MMCs, we limited our current studies to these The Journal of Immunology 5005

Table I. Role of TNF-␣ and ICAM-1 in a murine model of abscess formation to B. fragilis CPCa

Treatmentb Challenge Animals Challenged Positive Abscesses pc

TNF-␣ B. fragilis CPC ϩ SCCd 16 5 0.0005 ICAM-1 B. fragilis CPC ϩ SCC 13 3 0.0005 Sham Ab B. fragilis CPC ϩ SCC 10 10 — Saline ϩ SCC 10 0 ND — B. fragilis CPC ϩ SCC 10 9 ND

a Mice challenged with 100 ␮g of CPC mixed with sterile cecal contents by i.p. injection. b Mice received Ab (100 ␮g/dose) 24 and 4 h prechallenge and 2, 24, 48, 72, and 96 h postchallenge. c Compared with sham Ab (IX2b). d SCC, sterile cecal contents. cytokines since they are major inflammatory stimuli following mi- In summary, this work demonstrates that the CPC of B. fragilis crobial contamination in the peritoneal cavity (27, 46) and are interacts with the host immune system in a number of ways to prominent in the induction of cell adhesion molecules on cell sur- coordinate a cellular response leading to abscess formation. We are faces (36, 47). currently investigating the chemotactic properties of CPC and PSA Based on our previous observations that B. fragilis and CPC that may be responsible for the recruitment of PMNLs to the peri- Downloaded from promote rapid infiltration of lymphocytes, neutrophils, and mac- toneal cavity and the possible contribution of these factors to ab- rophages into the peritoneal cavity of animals following i.p. chal- scess formation associated with intraabdominal sepsis. lenge, we hypothesized that cell adhesion molecules such as ICAM-1 might play a role in the extravasation and localization of References these cells to the peritoneum (47, 48). Direct stimulation of MMCs 1. Lorant, D. E., M. K. Topham, R. E. Whatlley, R. P. McEver, T. M. McIntyre, with CPC produced higher levels of surface-expressed ICAM-1 S. M. Prescott, and G. A. Zimmerman. 1993. Inflammatory role of P-selectin. than cells in medium alone, or PGG-glucan-treated cells, although J. Clin. Invest. 92:559. http://www.jimmunol.org/ this increase was modest. Additional experiments showed that 2. Marrack, P., and J. Kappler. 1994. Subversion of the immune system by patho- gens. Cell 76:323. transfer of culture supernatants from CPC-stimulated peritoneal 3. Gunn, M. D., N. A. Nelken, X. Liao, and L. T. Williams. 1997. Monocyte che- macrophages elicited a maximal ICAM-1 response from MMCs. moattractant protein-1 is sufficient for the chemotaxis of monocytes and lympho- Since we have shown previously that murine peritoneal macro- cytes in transgenic mice but requires an additional stimulus for inflammatory activation. J. Immunol. 158:376. phages cultured with the CPC produced TNF-␣, we believed that 4. Lukacs, N. W., S. W. Chensue, R. M. Strieter, K. Warmington, and S. L. Kunkel. this macrophage-derived cytokine played a major role in up-reg- 1994. Inflammatory granuloma formation is mediated by tumor necrosis factor- ␣-inducible intercellular adhesion molecule-1. J. Immunol. 152:5883. ulating the expression of ICAM-1 on MMCs and is critical to the 5. Wynn, T. A., A. W. Cheever, D. Jankovich, R. W. Poindexter, P. Casper,

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