Cellular Mechanism of Intraabdominal Abscess
Formation by Bacteroides fragilis
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J Immunol 1998; 160:5000-5006; ;
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References
Frank C. Gibson III, Andrew B. Onderdonk, Dennis L. Kasper and
Arthur O. Tzianabos
Cellular Mechanism of Intraabdominal Abscess Formation by
Bacteroides fragilis1
Frank C. Gibson III,2* Andrew B. Onderdonk,† Dennis L. Kasper,* and
Arthur O. Tzianabos*
D
espite the plethora of bacteria that cause human disease,
host responses to these organisms are comprised of three
pathologic mechanisms: tissue inflammation, granuloma, and abscess formation. Recent work has revealed a better
understanding of the mechanisms governing tissue inflammation
(1–3) and granuloma formation (4 – 6), while the processes underlying abscess formation remain ill defined. Clinically, intraabdominal abscesses are commonly formed following events that
lead to the perforation of the bowel and subsequent leakage of the
colonic contents into the abdomen. Although Bacteroides fragilis
is among the least prevalent anaerobic species in the intestinal
tract, it is responsible for the majority of all clinical cases of anaerobic sepsis and intraabdominal abscesses (7, 8). Studies investigating the virulence properties associated with this organism
have shown that the CPC3 of B. fragilis is responsible for abscess
formation (9 –14). The CPC is comprised of two distinctly charged
polysaccharides (PSA and PSB) coexpressed on the surface of this
organism. Positive and negative charged groups on PSA and PSB
mediate the biologic properties of these polymers (15, 16).
Abscess formation is a complex host response that involves
the recruitment and accumulation of neutrophils, fibrin deposition, and other incompletely defined processes. In experimental
models, abscesses develop following i.p. challenge with B. fra-
Channing Laboratory, Departments of *Medicine and †Pathology, Brigham and
Women’s Hospital and Harvard Medical School, Boston, MA 02115
Received for publication September 30, 1997. Accepted for publication January
13, 1998.
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
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was sponsored by National Institutes of Health Grants AI34073 and
AI39579.
2
Address correspondence and reprint requests to Dr. Frank C. Gibson III, Channing
Laboratory, Departments of Medicine and Pathology, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115. Email address: [email protected]
3
Abbreviations used in this paper: CPC, capsular polysaccharide complex; GBSTIII,
group B streptococcal type III capsular polysaccharide; MMC, murine mesothelial
cell; PGG, poly(1– 6)-b-glucotriosyl-(1–3)-b-glucopyranose; PMNL, polymorphonuclear leukocyte; pMo, murine peritoneal macrophage; PSA, polysaccharide A; PSB,
polysaccharide B.
Copyright © 1998 by The American Association of Immunologists
gilis or purified CPC, PSA, or PSB (15, 17–19). Early studies
(20) showed that encapsulated B. fragilis bound to the peritoneal walls of rats better than unencapsulated Bacteroides species, enabling B. fragilis to resist clearance from the peritoneal
cavity by the diaphragmatic lymph system (20). Several groups
have demonstrated that peritoneal mesothelium, a layer of cells
that constitutes a line of structural and immunologic defense in
the abdominal cavity, potentiates the deposition of fibrin (21),
and the production of an array of cytokines and cell adhesion
molecules (21–28), plays an important role in abdominal sepsis.
Despite the lack of information describing a role for peritoneal
mesothelium during the formation of intraabdominal abscesses,
it is likely that inflammatory cells interact with this physical
barrier during the migration from host tissues to the peritoneal
lumen. The processes governing accumulation of these cells in
the peritoneal cavity remain unclear (29); however, these mechanisms most likely parallel those elucidated for migration of
immune cells from the vasculature to a focus of infection: a
complex process regulated by cytokines, cell adhesion molecules, and cell activation (23, 30, 31).
Several studies have shown that the host immune response is
critical to abscess formation and that several cell types are important in the development of intraabdominal abscesses (9, 19,
32, 33). Intraperitoneal challenge of animals with B. fragilis is
followed by immune cell infiltration, with an initial influx of
lymphocytes into the peritoneal cavity and the appearance of
neutrophils and macrophages approximately 4 days postchallenge (9). Recent studies have shown that purified phagocytic
cells from mice or humans cultured in vitro with CPC produce
TNF-a, IL-1a, IL-8, and IL-10 (33). It has been suggested that
cytokines may be responsible for triggering the migration of
immune cells into the peritoneal cavity following contamination
with B. fragilis (33); however, the source and role of these
cytokines remain undefined.
The prevalence of isolation of encapsulated B. fragilis from clinical cases of abscess formation led us to hypothesize that associated surface polysaccharides allow this organism to persist preferentially within the peritoneal cavity and initiate cellular events
that lead to the formation of this pathobiologic host response. In
0022-1767/98/$02.00
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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-a
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-a or
ICAM-1. Mice treated with TNF-a- or ICAM-1-specific Abs failed to develop intraabdominal abscesses following challenge with
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.
The Journal of Immunology
this work, we present data that demonstrate the preferential binding of B. fragilis, as well as purified surface polysaccharides from
this organism, to MMCs in vitro. These polysaccharides stimulate
TNF-a production by peritoneal macrophages that in turn elicited
the production of ICAM-1 by MMCs. ICAM-1 expression on
MMCs served as a functional ligand that supports increased
PMNL binding to these cells. mAb-blocking experiments in mice
demonstrated that both TNF-a and ICAM-1 expression are necessary for the development of intraabdominal abscesses in vivo.
These studies define cellular events critical for intraabdominal abscess formation by B. fragilis.
Materials and Methods
Cultivation and preparation of bacterial strains
Isolation of CPC; purification of PSA and group B
Streptococcus type III capsular polysaccharide
The CPC used in these studies was isolated from B. fragilis grown in
proteose-peptone yeast extract broth supplemented with hemin and menadione in a 20-L pH-controlled (pH 7.2) batch culture overnight at 37°C, as
described previously (34).
PSA was generated from pure CPC by isoelectric focusing with a Rotofor chamber (Bio-Rad, Hercules, CA) in 2% ampholytes (range 3–10) for
4 to 5 h at 12 watts constant power. Focused fractions were collected, and
a sample of each fraction was subjected to immunoelectrophoresis and
subsequent immunoprecipitation with high titer rabbit antiserum to B. fragilis NCTC 9343 (34). Samples containing PSA were pooled and dialyzed
against 1 M NaCl overnight, and then against distilled water for 2 days.
The purity of CPC and PSA was assessed by nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry, immunoelectrophoresis (pH 7.3), UV spectroscopy (260 and 280 nm), and reducing
PAGE on gradient gels with subsequent silver staining, as described (34).
The CPC and PSA used for these experiments were isolated from a single
extraction, tested for purity by the above methods, and stored dry at 4°C.
Before use, each Ag was diluted to 1 mg/ml in pyrogen-free water and
tested for endotoxin by the Limulus amebocyte lysate assay (Cape Cod
Associates, Woods Hole, MA). All Ags used in these studies tested free of
endotoxin.
The native and tritiated GBSTIII polysaccharides used in these experiments were a kind gift from Dr. Lawrence Paoletti (Channing Laboratory).
Radiolabeling of PSA
3
H-radiolabeled PSA was generated by oxidation and subsequent reduction. In brief, PSA was treated with sodium metaperiodate (0.01 M) to
oxidize approximately 25% of the vicinal hydroxyl groups on the galactofuranose of the PSA side chain to carbonyl groups. Ethylene glycol was
added to stop oxidation, and the sample was dialyzed overnight against
water. The newly generated carbonyl groups underwent reduction with
tritiated sodium borohydride (DuPont NEN, Boston, MA) to form isotopelabeled hydroxymethyl groups on PSA. Excess unlabeled sodium borohydride was added to completely modify any remaining carbonyl groups, and
the modified Ag was dialyzed overnight against water lyophilized, and
stored dry at 4°C. We have demonstrated that this procedure does not alter
the biologic activity of the polymer.
Isolation and culture of MMCs, murine peritoneal macrophage,
and PMNLs
MMCs. MMCs were isolated from the peritoneum of C57BL/6 mice by
enzymatic digestion and were cultivated in wells of collagen-coated culture
vessels (35). Briefly, omentum was harvested and digested with collagenase-dispase for 30 min at room temperature. Liberated cells were col-
lected by centrifugation and washed extensively to remove enzyme. Cells
were grown in DMEM with 12% FCS supplemented with 2-hydrocortisone
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
used upon reaching confluency (approximately 2.5 3 105 and 3.3 3 104
cells/well, respectively, and .98% pure by morphology and immunofluorescent assay). Bacteroides sp. were added at various multiplicities of
infection, polysaccharide Ags were added at various concentrations, or
supernatant fluids from Ag-stimulated macrophage were added to these
cells. The number of MMCs per monolayer was determined for each assay.
Murine peritoneal macrophage. pMo were elicited in C57BL/6 mice by
peritoneal injection of thioglycolate broth. After 3 days, cells were harvested by lavage, washed, added to 24-well tissue culture plates at 1 3 106
cells/ml, and allowed to adhere to plastic for 2 h at 37°C. The monolayers
were washed to remove nonadherent cells and cultured (.90% macrophage) with 1 ml culture medium, or medium containing 10 mg/ml CPC,
CPC treated with 20 mg/ml polymyxin B, PGG-glucan (Alpha-Beta Technologies, Worcester, MA), or GBSTIII. After 24 h, supernatants were harvested, centrifuged at 1000 3 g for 20 min to remove cells, and stored
frozen at 280°C.
PMNLs. To isolate PMNLs, peripheral blood was taken from healthy human donors and layered over a bed of Mono-Poly resolving medium (ICN
Biomedicals, Palo Alto, CA). Following separation by centrifugation, the
PMNL fraction was collected (.95% pure), washed with ice-cold DMEM
to remove separation medium, resuspended in DMEM to 2 to 3 3 107
cells/ml, and maintained on ice (,30 min). A 1-ml sample of these cells
was warmed to 37°C and added to monolayers of MMCs in 24-well plates.
Bacteroides- and CPC-binding assays and blocking experiments
Monolayers of MMCs were cocultured with B. fragilis, B. thetaiotaomicron, or B. distasonis for 1 h at 37°C with 5% CO2. Monolayers were
washed extensively to remove unbound bacteria, and an equivalent volume
of sterile water was added to the monolayers. Following lysis, vigorous
aspiration-expulsion cycles were performed with a pipet to evenly distribute bacteria. The lysate was serially diluted in 1% peptone, plated on Brucella agar, and grown for 48 h for viable count (CFU/ml) determination.
Additional experiments involved the addition of B. fragilis CPC, PSA,
[3H]PSA, and GBSTIII polysaccharide to MMCs. These Ags were
weighed, diluted to a concentration of 1 mg/ml in DMEM without serum,
and vortexed until completely dissolved; dilutions were then made with
DMEM, and the Ags were added to MMCs. The amount of Ag bound to
cells was evaluated by ELISA or liquid scintillation.
In experiments designed to block the binding of B. fragilis to MMCs,
bacteria were left untreated or treated with B. fragilis strain 9343-specific
capsular polysaccharide antiserum or irrelevant Ab for 1 h at 37°C before
addition of bacteria to MMC monolayers. Blocking of PSA binding to
MMCs was accomplished by adding various dilutions of a PSA-specific
mouse mAb (clone CE3) or nonimmune mouse control ascites (Sigma, St.
Louis, MO) to PSA (10 mg/ml) for 1 h at 37°C. Untreated or Ab-treated B.
fragilis or PSA was added to MMCs for 1 h, and binding was evaluated by
CFU/ml determinations or ELISA.
Competition experiments were performed to demonstrate specific binding of PSA to MMCs or pMo. To tritiated PSA (10 mg/ml), we added a
50-fold excess of native unmodified PSA (500 mg/ml). This polysaccharide
mixture was added to monolayers of cells in 24-well plates and cocultured
for 1 h at 37°C. The cells were washed three times with DMEM and lysed
with 1 ml of sterile distilled water, and the lysates were collected and
processed for liquid scintillation enumeration of 3H-radiolabeled PSA
binding.
Quantitation of bacteria and Ag binding
Colony counts. After incubation with bacteria, MMCs were washed with
DMEM to remove unbound bacteria (with the efficiency of washing determined by plating of the final wash), and 100 ml of sterile water was
added to the monolayers for 30 min to lyse MMCs. These lysates were
subjected to cycles of vigorous aspiration and expulsion to disrupt cells and
evenly disperse bacteria. The lysates were subjected to serial 10-fold dilution in 1% peptone, plated onto Brucella blood agar plates, and incubated
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,
colonies were enumerated.
ELISA. MMCs cocultured with CPC were gently washed to remove excess unbound Ag and were fixed with 2% formaldehyde in PBS (pH 7.2)
for 1 h. After fixation, monolayers were washed with PBS 1 0.05%
Tween-20 (pH 7.2). High titer rabbit serum specific for B. fragilis was
added at a 1/2000 dilution in PBS (100 ml/well), and the monolayers were
incubated for 1 h at 37°C. Incubation was followed by three washes, after
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B. fragilis NCTC 9343 was obtained from National Culture Type Collection, Rockville, MD. Bacteroides thetaiotaomicron strain 491909 and Bacteroides distasonis strain 8503 are clinical strains obtained from the Channing Laboratory’s (Brigham and Women’s Hospital, Boston, MA)
anaerobe stock culture collection, and were identified to the species level
by long chain fatty acid analysis and conventional biochemical reactions.
Each strain was passaged once on Brucella agar supplemented with 5%
defibrinated sheep’s blood and stored frozen at 280°C in peptone-yeast
extract broth. When needed, frozen aliquots were thawed, grown overnight
at 37°C in an anaerobic chamber. Bacterial growth was collected and resuspended in DMEM without serum to the desired multiplicity of infection
before bacterial binding experiments.
5001
5002
MECHANISMS OF INTRAABDOMINAL ABSCESS FORMATION
which 100 ml/well of a 1/4000 dilution of goat antiserum to rabbit IgG/
alkaline phosphatase conjugate (Biosource, Camarillo, CA) was added to
monolayers and incubated for 1 h at 37°C. The wells were washed, and 100
ml of p-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 polysaccharides, 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, Watertown, MA). The glass fiber disks were placed into glass vials with 2-ml
liquid scintillation mixture, and cpm were measured with a liquid scintillation reader (Packard International, Downers Grove, IL). The sp. act. of
tritiated PSA and GBSTIII were determined using a 10-mg 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
FIGURE 1. Attachment of B. fragilis NCTC 9343 (B. fragilis), B. thetaiotaomicron 491909 (B.theta), or B. distasonis 8503 (B. dist) to MMCs
in vitro measured by viable bacterial colony counts. Bacteria were added at
a multiplicity of infection of 1000 to 3 3 104 MMCs in wells of 96-well
collagen-coated plates for 1 h at 37°C. *p 5 0.0004, **p 5 0.0001 compared with binding of B. fragilis NCTC 9343 to MMCs.
Treatment of peritoneal macrophage supernatants with TNF-aneutralizing Ab
Adherence of Bacteroides sp. to MMCs
Supernatant fluids from CPC-stimulated peritoneal macrophage were
thawed and either added directly to monolayers of MMCs in 96-well plates
or treated with 10, 1, or 0.1 mg of goat neutralizing Ab to murine TNF-a
(R&D Systems) or nonimmune goat IgG (Sigma) for 1 h at 37°C before
addition to MMCs. After the addition of these supernatants, MMCs were
incubated for 18 h and then assayed for surface-expressed ICAM-1 by
ELISA or in PMNL-binding experiments.
PMNL/MMC cell adherence assays
We adapted the method for studying the binding of human PMNLs to
murine endothelium (36) and modified this for MMCs. Supernatant fluids
from Ag-stimulated peritoneal macrophage were added to MMCs in 24well plates for 18 h, as previously described (this study). PMNLs were
added to MMC and allowed to adhere for 30 min. After binding, the cocultures were washed to remove unbound PMNLs while taking care to
maintain intact MMC monolayers. Additional experiments were performed
to characterize the mechanism of PMNL attachment to MMCs stimulated
with CPC-treated macrophage supernatant fluids. Following stimulation,
MMCs were treated with ICAM-1-specific mAb (100 mg/ml) or irrelevant
Ab matched to isotype (clone IXB2; a kind gift from Dr. Gene Muller,
Channing Laboratory, Brigham and Women’s Hospital) for 1 h before
PMNL attachment. An observer blinded as to the treatments counted the
number of PMNLs bound to MMCs per 3200 magnification field with an
Olympus CK2 phase contrast microscope. Five random fields were counted
per sample.
In vivo Ab blocking of ICAM-1 and TNF-a
A murine model of peritoneal abscess formation was adapted to assess the
role of TNF-a and ICAM-1 during abscess formation (19). In brief,
C57BL/6 mice received 100 ml i.p. injections of rat Ab to murine ICAM-1,
goat Ab to murine TNF-a, or sham mAb (clone IXB2) in PBS (1 mg/ml)
24 and 4 h before implantation of an abscess-inducing inoculum of 100 mg
CPC in the adjuvant sterile cecal contents. Mice received additional i.p.
injections of mAb 4, 24, 48, 72, and 96 h after challenge to down-regulate
TNF-a or ICAM-1 in vivo (37). Six days after B. fragilis CPC challenge,
an observer blinded as to the treatment, then graded for presence of i.p.
abscesses in these animals.
Statistical analyses
All statistical analyses were performed with InStat statistical analysis software (Graphpad Software, San Diego, CA) on an IBM Personal Computer
AT. Results of in vitro data were calculated from three experiments, recorded as the mean 6 SD, and analyzed with the Kruskal-Wallis nonparametric test. In vivo data were analyzed by the Fisher’s exact test. A p value
of less than 0.05 was considered significant.
Results
Initial experiments defined the binding kinetics of B. fragilis to
MMCs. B. fragilis was added to MMCs at various multiplicities of
infection ranging from 1 to 10,000. Bacterial viable counts indicated that a multiplicity of infection of 1000 saturated binding sites
on MMCs (data not shown). Additional experiments compared the
binding of B. fragilis with other Bacteroides species (Fig. 1). B.
fragilis (1.39 3 106 CFU/ml) bound more avidly than B. thetaiotaomicron (2.35 3 105 CFU/ml; p 5 0.0004 vs B. fragilis) or B.
distasonis (8.88 3 104 CFU/ml; p 5 0.0001 vs B. fragilis). In
similar experiments to characterize the attachment of B. fragilis to
MMCs, bacteria were treated with either CPC-specific rabbit polyclonal Ab or irrelevant Ab before the addition to MMC monolayers. Irrelevant Ab-treated B. fragilis (1.28 3 106 CFU/ml) bound
to similar levels as untreated B. fragilis (1.15 3 106 CFU/ml),
while CPC-specific Ab treatment significantly reduced B. fragilis
attachment (1.21 3 105 CFU/ml; p , 0.002 vs irrelevant Ab
treatment).
Characterization of CPC and PSA attachment to host peritoneal
cells
MMCs. The binding of CPC to MMCs was measured by ELISA.
With the addition of increasing doses of CPC (ranging from 10
ng/ml to 200 mg/ml) in DMEM, saturation was achieved at a dose
of 10 mg/ml (Fig. 2A). Maximal binding of this dose of CPC occurred within 15 min.
To better define the binding characteristics of B. fragilis polysaccharides to MMCs, we performed experiments with a component polysaccharide of CPC, PSA. This repeating unit is readily
amenable to radiolabeling and was useful in quantifying polysaccharide binding and binding specificity to cells. Tritiated PSA (sp.
act. 10.15 cpm/ng) bound to MMCs with a profile similar to that of
CPC (6% of the input polysaccharide or 1.997 pg/cell bound when
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MMCs were grown to confluency in 96-well collagen-coated cell culture
plates. Proinflammatory cytokines were detected from MMCs cultured
with CPC or PGG-glucan (10 mg/ml). Supernatants were collected 1, 4, 8,
and 24 h after stimulation, and levels of TNF-a and IL-1a were determined
with cytokine-specific ELISA kits (Endogen, Cambridge, MA).
To detect ICAM-1 on the surface of MMCs, an in situ ELISA assay was
developed. MMCs in 96-well plates were incubated for 18 h with 100 ml
culture medium, 500 U/ml murine rTNF-a (R&D Systems, Cambridge,
MA), CPC, or PGG-glucan (10 mg/ml), and supernatants from Ag-stimulated peritoneal macrophage. After stimulation, MMCs were washed to
remove Ags and fixed with 2% buffered formaldehyde. After fixation, cell
monolayers were incubated with 100 ml/well of a 1/500 dilution of rat
anti-murine ICAM-1 mAb (clone YN/1.7.4; American Type Culture Collection, Rockville, MD) for 1 h. The monolayers were washed and then
incubated with 1/2000 dilution of goat antiserum specific for rat IgG/alkaline phosphatase (Sigma). Alkaline phosphatase substrate reagent
(Kirkegaard & Perry Laboratories) was added to each well and developed
for 1 h, and absorbance was read at 405 nm.
The Journal of Immunology
5003
added at 10 mg/ml; Fig. 2B). PSA binding was inhibited with mAb
CE3 (specific for PSA), as measured by ELISA (48% inhibition;
p , 0.0001). Competitive binding experiments demonstrated specificity of PSA binding to MMCs. The addition of 50-fold unlabeled
PSA with tritiated PSA (10 mg/ml) to MMC monolayers significantly reduced the binding of tritiated PSA (Fig. 2B). Addition of
3
H-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 mg/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 excess unlabeled PSA significantly inhibited PSA attachment (Fig.
2D); furthermore, the addition of 50-fold excess GBSTIII polysaccharide failed to inhibit PSA binding. GBSTIII polysaccharide
did not bind appreciably to these cells.
Direct in vitro stimulation of MMCs with CPC failed to elicit
detectable levels of the proinflammatory cytokines TNF-a or
IL-1a from these cells, but resulted in a modest increase in surface-expressed ICAM-1 compared with untreated or PGG-glucantreated cells ( p , 0.02 and p , 0.04, respectively; data not
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 ,
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 GBSTIII. In addition, incubation of CPC with polymyxin B did not
affect ICAM-1 expression. Based on our previous data in which
CPC was shown to elicit a potent TNF-a 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 neutralizing Ab specific for murine TNF-a (1 mg/ml) before addition
of the supernatants to MMCs. This treatment significantly reduced
the level of ICAM-1 expressed by MMCs ( p 5 0.0022 vs nonimmune 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-a 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 3 104 MMCs for 1 h at 37°C. B, Dosedependent binding of [3H]PSA and specificity of PSA binding to MMCs.
Ag was added directly to MMCs for 1 h at 37°C. Addition of 50-fold
excess unlabeled PSA with 3H-labeled PSA prevented binding of the labeled Ag (*p , 0.001 vs 10 mg/ml PSA dose). C, Time-dependent binding
of 50 mg/ml [3H]PSA to pMo. Ag was added to 1 3 106 pMo for 1 h at
37°C. D, Dose-dependent binding of [3H]PSA to pMo. Ag was added to
1 3 106 pMo for 1 h at 37°C. Addition of 50-fold excess unlabeled PSA
with 3H-labeled PSA prevented binding of the labeled Ag (*p , 0.002 vs
50 mg/ml PSA dose).
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CPC stimulation of TNF-a and ICAM-1
5004
MECHANISMS OF INTRAABDOMINAL ABSCESS FORMATION
( p , 0.002 vs medium control; Fig. 4). MMCs treated with supernatant fluids from CPC-stimulated macrophages supported increased PMNL binding compared with supernatants from PGGglucan- or GBSTIII-treated macrophages ( p , 0.002 and p ,
0.002, respectively, vs CPC stimulation; Fig. 4). Treatment of
CPC-stimulated supernatant fluids with murine TNF-a-neutralizing Ab significantly reduced PMNL binding to MMCs ( p , 0.002
vs irrelevant Ab treatment; Fig. 4). Furthermore, PMNL binding to
MMCs was inhibited by treatment of monolayers with ICAM-1specific mAb ( p , 0.002 vs irrelevant Ab treatment; Fig. 4).
In vivo role of TNF-a and ICAM-1 in abscess formation
The role of TNF-a and ICAM-1 in the development of intraabdominal abscesses was studied in a murine model of peritoneal
sepsis. Mice received i.p. injections of TNF-a-neutralizing Ab,
ICAM-1-specific mAb, or sham Ab (100 mg/injection) 24 and 4 h
before challenge, and 2, 24, 48, 72, and 96 h after B. fragilis CPC
challenge. Treatment with these mAbs significantly reduced the
development of abdominal abscesses following CPC challenge,
while treatment with a sham Ab did not affect abscess formation
( p , 0.0005 for TNF-a, and p , 0.0005 for ICAM-1 vs irrelevant
Ab treatment; Table I).
Discussion
The binding of bacteria to host cells is, in many cases, critical to
the progression of bacterial infections, including those of the peritoneal cavity (22, 25, 38 – 40). B. fragilis, an encapsulated organism that is the primary cause of intraabdominal abscesses and
Gram-negative anaerobic bacteremia, binds to the abdominal wall
of rats more readily than do other unencapsulated Bacteroides organisms (20). We hypothesized that encapsulated B. fragilis organisms resist clearance from the peritoneal cavity by adhering to
FIGURE 4. PMNL binding to macrophage supernatant-stimulated
MMCs correlates with enhanced levels of ICAM-1 on MMCs and is mediated by TNF-a and ICAM-1. MMCs were directly stimulated with medium or murine rTNF-a (rmTNF-a; 500 U/ml) to produce ICAM-1. Supernatants collected from peritoneal macrophage cocultured with medium,
PGG-glucan, GBSTIII, or CPC (10 mg/ml) were added to MMCs for 18 h.
In similar wells, CPC-stimulated macrophage supernatants were treated
with TNF-a-neutralizing Ab or nonimmune goat IgG (IRR1) for 1 h before
addition to monolayers of MMCs or following stimulation with ICAM-1blocking Ab or nonimmune rat IgG (IRR2). After these treatments, PMNLs
were added to MMCs, and cell attachment was measured. *p , 0.002 when
compared with PMNL binding to MMCs stimulated with supernatant fluids
from CPC-treated peritoneal macrophage; **p , 0.002 when compared
with PMNL binding to MMCs stimulated with supernatant fluids from
medium-treated peritoneal macrophage.
mesothelium, and that CPC is the primary attachment factor. To
evaluate the role of B. fragilis in initiating intraabdominal abscesses, we developed an in vitro system to study the interactions
of this organism or the purified polysaccharides from its surface
with the first cell boundary likely to be encountered in the peritoneal cavity: peritoneal mesothelium.
B. fragilis adhered more avidly to MMCs than either B. distasonis or B. thetaiotaomicron. This result suggested that the CPC
functions as an attachment factor. Previous studies have shown
that B. thetaiotaomicron has only a thin capsule layer (41), while
B. distasonis lacks a capsule. This difference most likely explains
why B. thetaiotaomicron binds less avidly than B. fragilis but more
avidly than the unencapsulated B. distasonis. Although little is
known about the capsular polysaccharide of B. thetaiotaomicron,
its binding capacity is interesting since B. thetaiotaomicron is the
second most frequently isolated Bacteroides species in human
disease.
The finding that CPC adhered to different cell types (MMCs and
pMo) was not surprising, as surface-expressed polysaccharides
from Actinobacillus actinomycetem comitans and Staphylococcus
aureus type 5 and 8 bind to a variety of host cells (42– 44). Furthermore, recent studies have shown that binding of microbial
polysaccharides to host cells is important for eliciting proinflammatory cytokines (43– 45). Previous work by our group has demonstrated that the CPC of B. fragilis elicits potent TNF-a, IL-1a,
IL-8, and IL-10 response from phagocytic cells of human or murine origin (33). In the present study, we were unable to detect the
proinflammatory cytokines TNF-a or IL-1a from MMCs cocultured with CPC. Although other cytokines may be produced from
CPC-stimulated MMCs, we limited our current studies to these
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FIGURE 3. ICAM-1 expression on MMCs following supernatant transfer from polysaccharide-stimulated peritoneal macrophage. Monolayers of
MMCs were stimulated with culture medium, murine rTNF-a (TNF-a; 500
U/ml), or TNF-a treated with TNF-a-neutralizing Ab (Ab1, 1 mg/ml) or
stimulated with supernatants from peritoneal macrophages cocultured with
DMEM (medium), PGG-glucan, GBSTIII, or CPC (10 mg/ml). In similar
experiments, CPC-stimulated peritoneal macrophage supernatants were
treated with TNF-a-neutralizing Ab or nonimmune goat IgG (IRR-Ab, 1
mg/ml) for 1 h before addition to monolayers of MMCs. After 18 h, surface-expressed ICAM-1 was measured by ELISA. *p 5 0.0022 when compared with ICAM-1 stimulation by supernatant fluids from CPC-treated
peritoneal macrophages; **p 5 0.0022 when compared with ICAM-1 stimulation by supernatant fluids from medium-treated peritoneal macrophage.
The Journal of Immunology
5005
Table I. Role of TNF-a and ICAM-1 in a murine model of abscess formation to B. fragilis CPCa
Treatmentb
TNF-a
ICAM-1
Sham Ab
—
—
Challenge
B. fragilis CPC
B. fragilis CPC
B. fragilis CPC
Saline 1 SCC
B. fragilis CPC
Animals Challenged
Positive Abscesses
pc
16
13
10
10
10
5
3
10
0
9
0.0005
0.0005
1 SCCd
1 SCC
1 SCC
1 SCC
ND
ND
Mice challenged with 100 mg of CPC mixed with sterile cecal contents by i.p. injection.
Mice received Ab (100 mg/dose) 24 and 4 h prechallenge and 2, 24, 48, 72, and 96 h postchallenge.
Compared with sham Ab (IX2b).
d
SCC, sterile cecal contents.
a
b
c
In summary, this work demonstrates that the CPC of B. fragilis
interacts with the host immune system in a number of ways to
coordinate a cellular response leading to abscess formation. We are
currently investigating the chemotactic properties of CPC and PSA
that may be responsible for the recruitment of PMNLs to the peritoneal cavity and the possible contribution of these factors to abscess formation associated with intraabdominal sepsis.
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cytokines since they are major inflammatory stimuli following microbial contamination in the peritoneal cavity (27, 46) and are
prominent in the induction of cell adhesion molecules on cell surfaces (36, 47).
Based on our previous observations that B. fragilis and CPC
promote rapid infiltration of lymphocytes, neutrophils, and macrophages into the peritoneal cavity of animals following i.p. challenge, we hypothesized that cell adhesion molecules such as
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Since we have shown previously that murine peritoneal macrophages cultured with the CPC produced TNF-a, we believed that
this macrophage-derived cytokine played a major role in up-regulating the expression of ICAM-1 on MMCs and is critical to the
development of intraabdominal abscesses. Treatment of pMo supernatants from CPC-stimulated macrophages with TNF-a-neutralizing Ab significantly reduced the ICAM-1 response, indicating
that TNF-a is a major factor eliciting expression of this cell adhesion molecule on MMCs. Taken together with our demonstration of CPC and PSA binding to pMo, these data suggest that
following challenge, peritoneal macrophages recognize B. fragilis
capsular polysaccharide, either bound to mesothelium or in the
peritoneal cavity, and secrete TNF-a, which in turn activates a
potent inflammatory response leading to ICAM-1 expression on
MMCs. The binding of human PMNLs to MMCs cultured with
supernatants from CPC-stimulated macrophages confirmed the importance of TNF-a and ICAM-1 in the localization of these cells
to mesothelial tissue.
The ability of TNF-a- and ICAM-1-specific Abs to significantly
reduce abscess formation in the mouse model confirmed the biologic importance of these immune mediators in the formation of
this host response. We propose that the binding of B. fragilis to
MMCs serves two roles: 1) localization of the organism on the
mesothelial surface to form a nidus of infection in the peritoneal
cavity; and 2) stimulation of ICAM-1 expression to provide a ligand for infiltrating PMNLs. These two factors most likely form
the first stages of intraabdominal abscess formation in the infected
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However, it appears that TNF-a produced by resident or infiltrating phagocytes in response to B. fragilis CPC plays the major role
in up-regulating ICAM-1 expression. This latter response leads to
the accumulation of PMNLs within the abdominal cavity, the hallmark of abscess formation.
5006
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