role for complement in development of helicobacter-induced ... · activates complement through the...

INFECTION AND IMMUNITY, Dec. 2003, p. 7140–7148 Vol. 71, No. 120019-9567/03/$08.00�0 DOI: 10.1128/IAI.71.12.7140–7148.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Role for Complement in Development of Helicobacter-InducedGastritis in Interleukin-10-Deficient Mice

Hanan F. Ismail,1 Juan Zhang,1 Richard G. Lynch,2 Yi Wang,3 and Daniel J. Berg1*Department of Internal Medicine1 and Department of Pathology,2 University of Iowa College of Medicine,

Iowa City, Iowa 52242, and Alexion Pharmaceuticals, Inc., Cheshire, Connecticut 064103

Received 12 March 2003/Returned for modification 7 May 2003/Accepted 11 August 2003

The mechanisms by which the immune response can eradicate gastric Helicobacter infection are unknown.We hypothesized that Helicobacter-induced activation of the complement system could promote both inflam-mation and eradication of Helicobacter from the stomach. In vitro studies demonstrated that Helicobacter felisactivates complement in normal mouse serum but not in serum from Rag2�/� mice, indicating that H. felisactivates complement through the classical pathway. Next, we infected complement-depleted wild-type controland interleukin-10-deficient (IL-10�/�) mice with H. felis. Helicobacter infection of wild-type mice elicited amild, focal gastritis and did not alter serum complement levels. Infection of IL-10�/� mice with H. felis elicitedsevere gastritis. After the initial colonization, the IL-10�/� mice completely cleared Helicobacter from thestomach by day 8. In contrast to wild-type mice, H. felis-infected IL-10�/� mice had a marked increase in serumcomplement levels. Complement depletion of wild-type mice did not affect the intensity of gastric inflammationor the extent of Helicobacter colonization compared to that for the wild-type control mice. In contrast,complement depletion of Helicobacter-infected IL-10�/� mice decreased the severity of gastritis, decreased theHelicobacter-induced infiltration of neutrophils into the stomach, and delayed the clearance of bacteria. In vitrostudies of stimulated splenocytes and neutrophils from IL-10�/� mice produced a twofold increase in com-plement production compared to that for wild-type mice. Pretreatment with IL-10 inhibited this increase. Thesestudies identify a role for complement in the local immune response to gastric Helicobacter in IL-10�/� miceand suggest a role for IL-10 in the regulation of complement production.

Helicobacter pylori is a human gastric pathogen that cancause gastritis, peptic ulcer disease, and gastric cancer. Theorganism was first associated with peptic ulcer disease in 1984(25), and since that time there has been an exponential in-crease in our knowledge of the role of Helicobacter in disease.However, despite these advances in the understanding of thebiology of H. pylori, the mechanisms leading to eradication ofthis noninvasive bacterium remain poorly understood.

In recent years, evidence has accumulated to suggest that inboth human patients and animal models the host immuneresponse is an important determinant of the outcome of theinfection. H. pylori-infected individuals express proinflamma-tory cytokines in their gastric mucosa (49), and it has beenproposed that Helicobacter induces a Th1-type CD4� T-cellimmune response (12, 26). Indeed, T cells are required foreradication of gastric infection in animal models of Helicobac-ter infection (38). However, other components of the immuneresponse may have an important role in control of gastricHelicobacter infection. For example, Helicobacter infection alsoinduces a prominent neutrophilic infiltration (8), and it hasrecently been reported that depletion of neutrophils in ananimal model of Helicobacter infection resulted in delayedclearance of bacteria and a blunted immune response to Hel-icobacter (20).

Several lines of evidence suggest that the complement sys-tem may have an important role in H. pylori-induced gastritis.

H. pylori can activate complement in vitro (7). Moreover, com-plement is activated in H. pylori-positive gastritis (6). C3b,soluble terminal complement complexes, and C3b-opsonisedH. pylori have been found in the gastric mucus of H. pylori-infected individuals (6). However, the extent to which activa-tion of complement promotes Helicobacter-induced gastritisand clearance of gastric Helicobacter infection remains un-known.

Because of the difficulty of performing invasive studies inhumans, much of our understanding of the immune basis of H.pylori-related disease comes from studies with animal models.We have developed a murine model of Helicobacter felis gas-tritis in interleukin-10-deficient (IL-10�/�) mice that mimicsmany features of chronic H. pylori infection. Based on 16SrRNA sequence analysis, H. felis is genetically quite similar toH. pylori (33), and this organism has been demonstrated toefficiently colonize rodent gastric mucosa (10). H. felis infec-tion in mice with a targeted disruption of the IL-10 gene(IL-10�/� mice), a key antiinflammatory and immune regula-tory cytokine, results in severe inflammation with the develop-ment of Th1-type CD4� T cells (5). IL-10�/� mice infectedwith H. felis develop a chronic gastritis that simulates many ofthe features of human H. pylori infection, including preneo-plastic epithelial changes that occur within 4 weeks of infection(5), paralleling the epithelial changes which may take up to 1to 2 years in H. felis-infected wild-type (WT) mice (23) andwhich may also occur (over a period of decades) in patientschronically infected with H. pylori. Moreover, we have foundthat H. felis is rapidly cleared from IL-10�/� mice (20). There-fore, H. felis-infected IL-10�/� mice represent a useful modelfor study of the immune mechanisms that lead to Helicobacter-

* Corresponding author. Mailing address: Department of InternalMedicine, C32-GH, University of Iowa Hospitals, 200 Hawkins Drive,Iowa City, IA 52242. Phone: (319) 353-7800. Fax: (319) 353-8383.E-mail: [email protected].

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induced pathology and clearance of Helicobacter from thestomach.

The aim of this study was to evaluate the role of complementon the course of H. felis-induced gastritis in IL-10�/� mice. Weobserved that complement depletion decreased the ability ofIL-10�/� mice to clear H. felis infection. Moreover, comple-ment depletion significantly decreased the severity of H. felis-induced gastritis. These studies suggest an important role forcomplement in the immune and inflammatory responses toHelicobacter in IL-10�/� mice.

MATERIALS AND METHODS

Mice. Healthy 6-week-old IL-10�/� mice on a 129/SvEv background were usedfor this study (3). Helicobacter-free WT 129/SvEv mice were purchased fromTaconic Farms (Germantown, N.Y.). The mice were maintained in microisolatorcages under specific-pathogen-free conditions at the animal care facility at theUniversity of Iowa. All mice were maintained in accordance with the guidelinesof the University of Iowa Animal Care and Use Committee.

Bacteria. H. felis (ATCC 49179) was obtained from the American Type Cul-ture Collection (Rockville, Md.). The bacteria were grown as described previ-ously (28). Briefly, the bacteria were grown on brucella agar plates with tri-methoprim-vancomycin-polymyxin B (Remel, Lenexa, Kans.) under microaerophilicconditions at 37°C for 2 days. Confluent plates of bacteria were harvested, andthe number of organisms was determined by absorption at an optical density at450 nm (OD450), with one OD unit corresponding to 109 bacteria (28). Thebacteria were positively identified on the basis of morphology and the presenceof urease enzyme activity and via PCR amplification of the 16S rRNA gene byusing Helicobacter-specific primers (35).

Infection with H. felis. H. felis (1 � 108 bacteria in 100 �l of phosphate-bufferedsaline [PBS]) was instilled by gavage by using a 23-gauge feeding needle (Popperand Sons, New Hyde Park, N.Y). The mice received three inoculations over aperiod of 5 days, with 1 day separating each inoculation. The mice were made tofast overnight prior to the inoculations.

Complement depletion. To characterize the role of complement during gastricH. felis infection, mice were decomplemented by using cobra venom anticom-plementary protein (Naja Naja kaouthia; Sigma, St. Louis, Mo.), hereafter re-ferred to as CVF (cobra venom factor). WT mice received intraperitoneal in-jections of 0.8 �g of CVF/g of body weight every 48 h (44). IL-10�/� micereceived intraperitoneal injections of 3.0 �g of CVF/g of body weight every 24 h.To evaluate the efficiency of CVF in decomplementation of WT and IL-10�/�

mice, blood samples for assessment of C3 levels were obtained by tail veinbleeding prior to injection of CVF, 24 h after injection of CVF, and at thecompletion of the experiment (day 8). Each mouse served as its own control, andthe pretreatment value was set to 100%. Two days after the start of the treat-ment, the levels of C3 were decreased to 5 to 9% and remained at this level forthe duration of the experiment. Inoculation with H. felis began 1 day afterinitiation of CVF treatment. The mice were evaluated for colonization of H. felisand severity of inflammation 8 days after the initiation of infection.

C5 depletion. To characterize the effect of C5 depletion during gastric H. felisinfection, WT and IL-10�/� mice were treated with intraperitoneal injections ofBB5.1 (40 mg/kg every other day), a monoclonal antibody directed against C5(47). WT and IL-10�/� mice treated with the same dose and schedule of theisotype control antibody (135.8) served as the controls. Anti-C5 treatment began2 days before H. felis infection. Serum samples for determining C5 levels wereobtained before and after anti-C5 treatment, as well as 7 days after H. felisinfection. Sera were assessed for efficiency of C5 depletion using a hemolyticassay as described previously (46). Briefly, mouse serum samples were diluted to10% (vol/vol) with gelatin Veronal buffer 2� (Sigma) and added (50 �l/well) to96-well microtiter plates containing 50 �l of human C5-deficient serum (Quidel,San Diego, Calif.). The plates were incubated for 30 min at room temperature.Erythrocyte preparation and hemolytic assays were then performed as previouslydescribed (36). Each mouse served as its own control, and the pretreatment valuewas set to 100%. Anti-C5 treatment reduced the levels of C5 to 5% of thepretreatment control value for the duration of the experiment. Inoculation withH. felis began one day after initiation of CVF treatment. The mice were evalu-ated for colonization of H. felis and severity of inflammation 8 days after initi-ation of infection.

Complement activation assay. Normal nonimmune serum was obtained fromhealthy, uninfected 129/SvEv mice. Antibody-deficient nonimmune serum wasobtained from healthy 129/SvEv/Rag2�/� mice. Serum (90 �l) was incubated

with H. felis suspension (10 �l, approximately 3 � 106 CFU) or lipopolysaccha-ride (LPS) (10 �l; final concentration, 10 to 100 ng/ml) at 37°C. EDTA (pH 8.0)was added to a final concentration of 10 mmol/liter after 15, 30, or 60 min toterminate complement activation. Serum incubated with 10 mmol of EDTA/literwas used as the negative control. The mixtures were centrifuged at 14,000 � g for5 min, and the supernatants were stored at �80°C until assessment of C3 levelswas performed.

Assessment of C3 levels in serum. A sandwich enzyme-linked immunosorbentassay (ELISA) was used to quantify C3 in mouse sera (13). Briefly, 96-wellmicrotiter plates were coated with immunoglobulin G anti-mouse C3 (1:3000)(Cappel), washed with PBS, and incubated for 2 h at 37°C with serial dilutions ofmouse sera. The plates were washed with PBS containing 0.05% Tween 80 andthen incubated with affinity-purified peroxidase-labeled goat anti-mouse C3 di-luted 1:500 in PBS (Cappel). After incubation with the peroxidase substrate(o-phenylenediamine dihydrochloride; Sigma) the plates were read at OD492. Atitration of pooled normal mouse serum obtained from uninfected WT 129/SvEvmice was used to generate a standard curve. One unit of C3 was defined as theamount of C3 contained in a 1/256,000 dilution of the standard pooled mouseserum (11, 18).

Gastric colonization by H. felis. To assess the effect of decomplementation ongastric colonization of H. felis, WT and IL-10�/� mice were inoculated with H.felis as described above. Decomplementation with CVF was initiated 1 day priorto inoculation with H. felis and continued throughout the duration of the exper-iment. The day of the first inoculation of H. felis is referred to as day 1. Stomachswere assessed for colonization with H. felis on day 8. Each in vivo experimenttesting the effect of complement depletion on IL-10�/� mice consisted of six WTmice (as a positive control for Helicobacter infection), six IL-10�/� control mice,and six CVF-treated IL-10�/� mice. To test the effect of CVF on Helicobacterinfection in WT mice, CVF was administered to six WT mice and the results werecompared with those for six WT control mice.

The presence of H. felis was determined on histologic sections stained by usinga modified Steiner method (Sigma). Eight longitudinal cross sections of thestomach from each WT or IL-10�/� mouse were examined. The number ofinfected glands and the number of bacteria in each gland were counted in eachsection to determine the number of bacteria per section. The data are presentedas the mean number of bacteria for all mice in each group, with the value fromthe section with the highest number of bacteria being used. In WT mice, gastriccolonization was uniform and bacterial counts did not vary significantly betweensections. With IL-10�/� control (non-CVF-treated) mice, bacteria were notpresent in any of the multiple stomach cross sections. In a previous study (20),PCR analysis of stomach DNA from infected WT and IL-10�/� mice (day 8postinoculation) for the 16S rRNA gene with Helicobacter-specific primers (35)and reisolation cultures of Helicobacter validated the quantification obtainedwith the histochemical method.

Histologic analysis. Stomachs from WT and IL-10�/� mice were fixed andflattened in 95% ethanol, routinely processed, sectioned at 6 �m, and stainedwith hematoxylin and eosin (H&E) for light microscopic examination. For eachstomach, eight longitudinal sections extending from the gastroesophageal junc-tion to the duodenum were examined. Sections were examined by the samepathologist (R. G. Lynch) without knowledge of the identity of the samples.Because lesions were multifocal and of variable severity, the grade given to anysection of stomach took into account the number of lesions as well as theirseverity. Scores from 0 to 6 were based on the following criteria: grade 0, nochange from normal tissue; grade 1, unifocal mild cellular infiltration in thelamina propria, usually located in the distal stomach or at the junction of thesquamous and glandular epithelium; grade 2, few multifocal lesions of moderateinflammatory cell infiltrates in the lamina propria; grade 3, lesions over a largearea of the mucosa, more frequent than for grade 2; grade 4, more severe lesionsthan for grade 3 over most of the section; grade 5, moderate inflammation, ofteninvolving the submucosa but rarely transmural, with inflammatory cells consistingof a mixture of mononuclear cells and neutrophils and with moderate epithelialmetaplasia; grade 6, diffuse and severe inflammation, including transmural in-flammation, with inflammatory cells consisting of a mixture of mononuclear cellsand neutrophils and with epithelial metaplasia and ulcerations.

Quantification of neutrophil percentage in peripheral blood. Peripheral bloodsmears were prepared before CVF administration, 24 h after CVF administra-tion, and at the completion of the experiment (day 8). The smears were stainedwith Wright-Giemsa, and 100-cell differentials were performed to determine thepercentage of circulating neutrophils.

Quantification of neutrophils in stomach. A myeloperoxidase assay was usedto quantify the degree of neutrophil infiltration in the stomachs of the H.felis-infected WT and IL-10�/� mice. The glandular portion of the stomach wasweighed and subsequently homogenized in a solution of PBS with 0.5% hexa-

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decyltrimethylammonium bromide. The samples were freeze-thawed three timesand centrifuged at 10,000 � g for 20 min. The supernatants were diluted 1:2 in50 mM NaPO4 buffer, and 20 �l of sample was added to 180 �l of O-dianisidineHCl (0.2 mg/ml in NaPO4 buffer) with or without 0.0006% H2O2. The plateswere read at OD450. The values were multiplied by 2.655 � 10�4 to calculate theIU/sample (40) and were normalized to the weight of the tissue.

Isolation and culture of peritoneal neutrophils. Peritoneal neutrophils wereelicited in WT and IL-10�/� mice by an intraperitoneal injection (2 ml) of freshlyprepared 2% glycogen (Sigma) in sterile isotonic saline (2). Four hours later, themice were euthanized and the peritoneal cavity was irrigated three times withPBS. The collected peritoneal exudates were centrifuged at 1500 � g for 7 min.Cytospin slides were prepared and stained with Wright-Geimsa staining. Thecells were 88 to 91% neutrophils. Viability was 92 to 95%, as assessed by trypanblue staining. Prior to cell culture, red blood cells were lysed by using a red bloodcell lysis buffer (8.26 g of NH4Cl/liter, 37 mg of EDTA-2Na/liter, and 1.0 g ofKHCO3/liter). Isolated peritoneal neutrophils from uninfected WT or IL-10�/�

mice were cultured at 3 � 106 cells/ml in RPMI 1640 supplemented with 10%fetal calf serum (complement-deactivated)–2 mM L-glutamine–0.05 mM 2-mer-captoethanol–100 U of penicillin/ml–100 U of streptomycin/ml in 48-well tissueculture plates (Costar, Corning, N.Y.). The cells were incubated in mediumalone or in medium supplemented with 12-O-tetradecanoylphorbol-13-acetate(TPA). In some experiments, IL-10�/� neutrophils were pretreated with 20 ng ofrecombinant IL-10 (rIL-10)/ml for 4 h before being cultured and activated withTPA. Supernatants from triplicate cultures were harvested after 20 h and storedat �80°C before being analyzed for C3 levels.

Spleen cell culture. Spleen cells from IL-10�/� control or H. felis-infected WTmice were cultured at 5 � 106 cells/ml in RPMI 1640 supplemented with 10%fetal calf serum–2 mM L-glutamine–0.05 mM 2-mercaptoethanol–100 U of pen-icillin/ml–100 U of streptomycin/ml in 12-well tissue culture plates (Costar). Thecells were incubated in medium alone or in medium supplemented with H. felissonicate at 1.0 �g of protein/ml. Supernatants from triplicate cultures wereharvested after 48 h and stored at �80°C before being analyzed for C3 levels.

Statistics. The data are expressed as means � standard deviations (SDs).Significant differences between experimental groups were evaluated by the non-parametric Mann-Whitney U test. A P value of �0.05 was considered statisticallysignificant.

RESULTS

H. felis fixes complement in vitro. It has previously beenreported that IL-10�/� mice rapidly clear gastric Helicobacterinfection, whereas in WT mice the gastric burden of Helico-

FIG. 1. Activation of complement in vitro by H. felis. Serum fromcontrol WT or Rag2�/� mice was incubated with H. felis suspension orLPS for various lengths of time, and C3 levels were subsequentlymeasured by ELISA at various time points. The results are expressedas OD492 � SD and represent the mean values of the results for atriplicate of a given sample. LPS (positive control), rapidly activatedcomplement; NMS, normal mouse serum; RMS, Rag2�/� mouse se-rum. An asterisk (�) indicates a P value of �0.001 compared to theresults for the control, and a number sign (#) indicates a P value of�0.001 compared to the results for normal mouse serum incubatedwith H. felis. The results are representative of three independent ex-periments.

FIG. 2. Levels of complement in serum in control and H. felis-infected WT and IL-10�/� mice. C3 levels were determined 8 daysafter inoculation with H. felis. Levels of C3 in serum are expressed asOD492 in a dilution series of serum. The results are expressed as means� SDs and represent the mean values of the results for 10 mice withina given group. The results are representative of 10 independent ex-periments.

FIG. 3. Effect of decomplementation on clearance of H. felis infec-tion in WT and IL-10�/� mice. WT mice received intraperitonealinjections of 0.8 �g of CVF/g of body weight every 48 h, and IL-10�/�

mice received 3.0 �g of CVF/g every 24 h. The control mice wereinjected with vehicle. Anti-C5 was given as intraperitoneal injections of40 mg/kg every 48 h. CVF and anti-C5 injections began 2 days prior toinoculation with H. felis and continued for the duration of the exper-iment. One week after inoculation, the mice were sacrificed for anal-ysis of H. felis colonization. The results are expressed as the means �SDs of the results for 4 to 6 mice within a given group. An asterisk (�)indicates a P value of �0.001 compared to the results for WT, and anumber sign (#) indicates a P value of �0.001 compared to the resultsfor untreated IL-10�/� mice. The results are representative of threeindependent experiments.

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bacter increases with time (20). As it has previously been re-ported that H. pylori is capable of fixing complement (7), wehypothesized that complement may play a role in eradicationof gastric Helicobacter. In order to assess the mechanisms un-derlying Helicobacter eradication in IL-10�/� mice, we first

tested whether H. felis would fix complement (Fig. 1). Incuba-tion of H. felis with normal (uninfected) mouse serum resultedin a rapid decline of C3 levels, indicating that H. felis, like H.pylori (7), can fix complement. In contrast, when H. felis wasincubated with serum from Rag2�/� mice (which have no

FIG. 4. H. felis colonization and histopathology of H. felis-infected WT and IL-10�/� mice. Gastric sections from WT and IL-10�/� mice werestained by using a modified Steiner method for the detection of H. felis or with H&E for light microscopic examination. Histologic evaluation wasperformed 8 days after inoculation with H. felis. (A) Steiner stain of H. felis-infected WT mouse (20� magnification). Note the dense colonizationof the gastric glands. (B) H&E stain of H. felis-infected WT mouse (20� magnification). Note the minimal inflammatory response. (C) Steiner stainof H. felis-infected IL-10�/� mouse (20� magnification). Note the absence of Helicobacter. (D) H&E stain of H. felis-infected IL-10�/� mouse (20�magnification). The lamina propria is heavily infiltrated with both mononuclear cells and neutrophils. (E) Steiner stain of H. felis-infectedCVF-treated IL-10�/� mouse (20� magnification). Note the colonization of the gastric glands with Helicobacter. (F) H&E stain of H. felis-infectedCVF-treated IL-10�/� mouse (20� magnification). Note the absence of cellular infiltrate in the lamina propria.



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serum antibodies and thus cannot activate the classical path-way), C3 levels did not differ from those of the control mice.This finding demonstrates that H. felis can activate comple-ment through the classical pathway (Fig. 1), although a specificanti-Helicobacter antibody does not appear to be required.

Serum complement levels in H. felis-infected IL-10�/� andWT mice. To further assess the role of complement in H.felis-infected mice, we tested whether gastric H. felis infectionwould alter the levels of complement in serum in WT andIL-10�/� mice. Infection of WT mice with H. felis did notsignificantly change levels of C3 in serum (Fig. 2). In contrast,levels of C3 in serum were significantly higher in IL-10�/� miceafter 1 week of H. felis infection than in uninfected IL-10�/�

control mice (Fig. 2).Effects of decomplementation on clearance of H. felis infec-

tion in WT and IL-10�/� mice. We next assessed the extent towhich decomplementation would alter the ability of IL-10�/�

mice to clear the gastric Helicobacter infection. Bacterial col-onization in both WT and IL-10�/� mice with or without CVF-mediated decomplementation was determined. After 8 days ofH. felis infection, all H. felis-infected WT mice were denselycolonized (Fig. 3A and 4A). Complement depletion of WTmice did not alter the level of gastric H. felis colonization (Fig.3A). IL-10�/� control mice were able to completely clear thegastric Helicobacter infection by day 8 (Fig. 3B and 4C). How-ever, CVF-mediated decomplementation of IL-10�/� mice re-sulted in persistent H. felis infection, with a level of H. feliscolonization that was approximately 70% that of WT mice atthe same time point (Fig. 3B and 4E). In one experiment,complement depletion was extended to 14 days. Complementdepletion of WT mice for 14 days had no effect on colonizationby H. felis. In contrast, IL-10�/� mice depleted of complementfor 14 days had persistent infection with H. felis at a level thatwas approximately 51% that of WT mice at the same timepoint (data not shown). We also assessed the effect of de-complementation mediated by anti-C5 treatment on H. felisinfection in WT and IL-10�/� mice. Depletion of complementby anti-C5 treatment had no effect on H. felis infection in WTmice at the time point assessed (Fig. 3A). In contrast, IL-10�/�

mice depleted of complement by anti-C5 treatment had per-sistent infections that were approximately 80% of the level ofinfection in WT stomach at the same time point (Fig. 3B).

Effect of decomplementation on H. felis-induced gastritis inWT and IL-10�/� mice. Since decomplementation resulted inpersistent H. felis infection in IL-10�/� mice, we next assessedthe effect of decomplementation on the development of Heli-cobacter-induced gastritis. As has been reported in previousstudies (5), infection of IL-10�/� mice with Helicobacter resultsin rapid development of severe gastric inflammation. After 8days of infection, H. felis-infected IL-10�/� stomachs devel-oped pathological lesions that consisted of diffuse inflamma-tion with intensive cellular infiltration (Fig. 4D and 5B). Incontrast, infected WT stomachs showed either no inflamma-tion or a very mild degree of inflammation that was unifocal innature and distal in location (Fig. 4B and 5A). Decomplemen-tation did not result in any alteration to the minimal gastritisthat develops in H. felis-infected WT mice. In contrast, de-complementation of IL-10�/� mice resulted in a 45% decreasein the severity of H. felis-induced pathology (Fig. 4F and 5B).Similarly, decomplementation mediated by anti-C5 treatment

in H. felis-infected IL-10�/� mice resulted in a 30% decrease inthe severity of H. felis-induced pathology (Fig. 5B).

Effect of decomplementation on the neutrophil response inH. felis-infected IL-10�/� mice. As it has previously been foundthat depletion of neutrophils delayed Helicobacter clearanceand significantly reduced Helicobacter-induced gastritis in IL-10�/� mice (20), we assessed the effect of decomplementationon the neutrophil response in H. felis-infected IL-10�/� mice.CVF treatment of (control) noninfected IL-10�/� mice did notinduce a significant increase in the percentage of circulatingneutrophils (data not shown). In contrast, CVF treatment ofinfected IL-10�/� mice resulted in a threefold increase in thepercentage of circulating neutrophils (Fig. 6A). We then as-sessed the effect of decomplementation on the level of neutro-phil infiltration in the stomachs of H. felis-infected IL-10�/�

mice. Despite the increased percentage of circulating neutro-phils in the CVF-treated mice, decomplementation resulted ina 45% decrease in neutrophil infiltration in the stomachs of theH. felis-infected IL-10�/� mice compared to that for the H.felis-infected IL-10�/� control mice (Fig. 6B).

Complement levels in spleen cell cultures from control andH. felis-infected WT and IL-10�/�mice. We next assessed thelevel of complement production induced in spleen cell culturesof H. felis-infected WT and IL-10�/� mice. Splenocytes fromcontrol and H. felis-infected WT and IL-10�/� mice were cul-tured with and without sonicated H. felis antigen, and culturesupernatants were evaluated for the concentration of C3. Al-though splenocytes from naïve IL-10�/� mice produced a levelof C3 that was 60% of that produced by splenocytes from naïveWT mice (Fig. 7A, B), when stimulated with H. felis antigen,splenocytes from naïve IL-10�/� mice produced a twofold-higher level of C3 than that produced by splenocytes fromnaïve WT mice (Fig. 7A and B). Stimulated splenocytes fromH. felis-infected IL-10�/� mice produced a 10-fold-higher levelof C3 than that produced by splenocytes of the unstimulatednaïve IL-10�/� mice; this level was also twofold higher thanthat produced by stimulated splenocytes of H. felis-infectedWT mice.

Complement secretion by naïve WT and IL-10�/� peritonealneutrophils. Since neutrophils are important mediators of theimmune response to H. felis in IL-10�/� mice (20) and are alsoan important source of complement in inflammation (30, 31),we evaluated the levels of complement secretion by neutro-phils isolated from the peritoneal exudates of both naïve IL-10�/� and WT mice. Neutrophils from WT and IL-10�/� micewere cultured with and without TPA stimulation, and culturesupernatants were evaluated for the concentration of C3 20 hlater. Interestingly, neutrophils from naïve IL-10�/� mice pro-duced 50% of the level of complement secreted by neutrophilsfrom naïve WT mice. However, TPA stimulation induced anapproximately fivefold increase in complement secretion fromneutrophils of IL-10�/� mice but not from those of WT mice(Fig. 8A and B). When neutrophils from IL-10�/� mice werepretreated with rIL-10, TPA stimulation did not induce a sig-nificant increase in complement level (Fig. 8A and B).

DISCUSSION

We have used H. felis-infected WT and IL-10-deficient miceto further define the role of complement in the immune re-



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sponse to gastric Helicobacter infection. Our studies show thatH. felis is capable of fixing complement in vitro. We found thatinfection of IL-10�/� mice with H. felis resulted in a largeincrease in the levels of complement in serum in IL-10�/�

mice. Depletion of complement did not alter the level of gas-tric colonization or inflammation in WT mice. In contrast,complement depletion of IL-10�/� mice resulted in persis-tence of the gastric Helicobacter infection and a significant

reduction in Helicobacter-induced gastric inflammation, as wellas reduced neutrophil infiltration of the stomach. Helicobacter-stimulated production of complement was significantly in-creased in spleen cells from H. felis-infected IL-10-deficientmice compared to that for infected WT mice, and stimulation

FIG. 5. Effect of complement depletion on H. felis-induced gastritisin WT and IL-10�/� mice. Histopathologic evaluation was performed8 days after inoculation with H. felis. The average pathological score isindicated for each group. An asterisk (�) indicates a P value of �0.001compared to the results for the WT, and a number sign (#) indicatesa P value of �0.001 compared to the results for the IL-10�/� controlmice. The results are representative of three independent experiments.

FIG. 6. (A) Effect of CVF-mediated decomplementation on thepercentage of circulating neutrophils in control and H. felis-infectedIL-10�/� mice. The percentage of circulating neutrophils was deter-mined at day 8 of H. felis infection or after 8 days of CVF treatment.Smears were stained with Wright-Giemsa, and 100-cell differentialswere performed to determine the percentage of circulating neutro-phils. (B) Effect of decomplementation on the level of neutrophilinfiltration of the stomachs of H. felis-infected IL-10�/� mice, as mea-sured by myeloperoxidase activity. An asterisk (�) indicates a P valueof �0.001 compared to the results for the IL-10�/� control mice, anda number sign (#) indicates a P value of �0.001 compared to theresults for H. felis-infected non-CVF-treated IL-10�/� mice. The re-sults are representative of two independent experiments. MPO, my-eloperoxidase activity.

FIG. 7. C3 production by splenocytes from WT and IL-10�/� mice.(A) Spleen cells were cultured at 5 � 106 cells/ml in complete mediumor medium supplemented with H. felis antigen as described in Mate-rials and Methods. After 48 h of culture, supernatants were harvestedand analyzed for C3 level by ELISA. Data at each time point areexpressed as means � SDs of the results of observations of 10 mice pergroup. An asterisk (�) indicates a P value of �0.001 compared to theresults for the WT control mice, a number sign (#) indicates a P valueof �0.0001 compared to the results for the IL-10�/� control mice, anda plus sign (�) indicates a P value of �0.001 compared to the resultsfor Ag- stimulated H. felis-infected WT mice. The results are repre-sentative of four independent experiments.

FIG. 8. C3 secretion by peritoneal neutrophils from WT and IL-10�/� mice. Glycogen-elicited neutrophils from uninfected WT orIL-10�/� mice were cultured in control medium or medium supple-mented with TPA. Supernatants from triplicate cultures were har-vested after 20 h. C3 levels in the samples were evaluated by ELISA.In some experiments, IL-10�/� neutrophils were pretreated withrIL-10 before culture and activation with TPA. C3 levels are plotted asOD492. Results are expressed as means � SDs and represent the meanvalue of the results for four mice within a given group. An asterisk (�)indicates a P value of �0.001 compared to the results for WT neutro-phils, and a number sign (#) indicates a P value of �0.001 comparedto the results for the IL-10�/� control neutrophils. The results arerepresentative of three independent experiments.



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of neutrophils from IL-10�/� mice resulted in higher levels ofC3 secretion than for WT mice. Taken together, these resultssuggest that complement is a central mediator of Helicobacter-induced inflammation and clearance of gastric Helicobacter inIL-10�/� mice.

Several lines of evidence have suggested a role for comple-ment in the immune-inflammatory response to gastric Helico-bacter infection. The complement system is a key mediator ofthe immune response to multiple extracellular organisms, in-cluding, for example, pneumococci (42), Haemophilus influen-zae (29), Candida (24), and Leishmania major (39). Moreover,evidence of complement activation has been demonstrated ingastric biopsies from individuals infected with H. pylori (6).The complement products were present only in those areasassociated with Helicobacter, suggesting that H. pylori inducedlocal activation of complement. In the present study, we foundthat H. felis is capable of complement fixation in the presenceof normal serum antibodies but not in Rag2�/� serum, whichdoes not have antibodies. These results show that H. felis, justlike H. pylori (7), activates complement through the classicalpathway in the absence of a specific antibody to Helicobacter.The mechanism for this phenomenon is not known but couldbe secondary to activation of complement via low levels ofantiendotoxin antibody. Alternatively, the activation of com-plement may be secondary to antibodies to conserved proteins,such as heat shock proteins. Indeed, we have detected antibodyto HSP-70 from H. felis in uninfected mice (data not shown).The finding that H. felis can activate complement suggestedthat complement may have a role in the immune response togastric Helicobacter infection.

The potential role of the complement cascade in the devel-opment of H. felis-induced gastritis was studied by depletingcomplement levels in WT and IL-10�/� mice by using anti-complementary CVF. CVF is a peptide fragment of cobra C3that is capable of activating the alternative complement path-way, but unlike C3b, it is not cleaved by factors H and I (44).As a result, it continues to activate the alternative pathway inan unregulated way, leading to the depletion of several pro-teins that are necessary for complement function. Persistenttreatment with CVF results in severe depletion of C3, factor B,and C5 through C9 and, subsequently, total abolishment ofcomplement activity (17). Complement depletion of WT micedid not affect Helicobacter clearance or development of theminimal gastritis seen in these mice at the time point tested. Atthis early time point, WT mice have minimal inflammation inresponse to Helicobacter. With time (months), WT mice candevelop significant gastric inflammation and clear the organ-ism. It is possible that complement plays a more important roleat this later time point. In contrast to the results for WT mice,complement depletion of IL-10�/� mice resulted in signifi-cantly less gastritis in H. felis-infected IL-10�/� mice. Impor-tantly, this finding was associated with a decreased ability toclear H. felis in the decomplemented IL-10�/� mice, as highnumbers of H. felis were present in CVF-treated IL-10�/� micewhereas control IL-10�/� mice effectively cleared the Helico-bacter. Although the overall pathology grading of anti-C5-treated H. felis-infected IL-10�/� mice demonstrated a 40%decrease in the severity of gastritis, when the cardia, body, andpylorus of the stomach were evaluated separately for pathol-ogy, we found that the bodies of the stomachs of all anti-C5-

treated mice were completely free of pathology (grade 0), andthe number of H. felis organisms was comparable to that forWT mice (data not shown). These data suggest that comple-ment activation plays a role in the eradication of Helicobacterand the development of gastritis in Helicobacter-infected IL-10�/� mice.

To control for the possibility that CVF might have inducedanother mechanism that altered the course of H. felis infectionin IL-10�/� mice, we also depleted complement in H. felis-infected IL-10�/� mice by using a monoclonal antibody spe-cific for the C5 component of complement. This antibodyblocks the cleavage of C5 and thus prevents the generation ofthe potent proinflammatory factors C5a and C5b-9 (47). An-ti-C5 treatment induced a similar effect to that of CVF, as theanti-C5-treated IL-10�/� mice did not clear the H. felis infec-tion and had a reduced level of gastritis. Overall, absence ofcomplement prevented clearance of gastric Helicobacter anddecreased Helicobacter-induced inflammation in IL-10�/�

mice.There are multiple mechanisms by which complement acti-

vation may affect the immune and inflammatory responses togastric Helicobacter infection. In this study, we demonstratethat H. felis can fix complement in the presence of antibody;thus, H. felis clearance in IL-10�/� mice may be due to directcomplement lysis. Several lines of evidence suggest that eva-sion of complement is important for maintenance of chronicgastric Helicobacter infection. Both H. pylori and H. felis pro-duce the urease enzyme, and the ammonia produced by thisenzyme can directly inhibit complement activation (37). Inaddition, H. pylori is known to survive complement lysis bybinding of GPI-anchored protectin (CD59) (34). The en-hanced clearance of H. felis from IL-10�/� mice may be sec-ondary to direct bacterial lysis due to the increased levels ofcomplement induced by infection in these mice. However,complement proteins have pleiotropic effects on the immunesystem and may have indirectly enhanced gastritis and Helico-bacter eradication through effects on the immune response toHelicobacter.

Depletion of complement may have impaired Helicobacterclearance in IL-10�/� mice by interfering with homing of theneutrophils into the infected stomach. Complement is a majorcomponent of innate immunity and is involved in early protec-tive immune responses against pathogens that occur before theinduction of acquired T-cell and B-cell immunity (14). Com-plement is an important regulator of neutrophil function. Forexample, C3a (1) and C5a (16) promote chemotaxis of neu-trophils toward the site of inflammation; as would be expected,homing and/or extravasation of neutrophils is significantly de-creased in CVF-treated rats and mice (43). For example, CVFtreatment was reported to reduce accumulation of neutrophilsat the site of lung injury (15). Neutrophils are a prominentcomponent of infiltration in the H. pylori-infected stomachs. Aprevious study found that depletion of neutrophils in IL-10�/�

mice delayed clearance of Helicobacter and significantly re-duced Helicobacter-induced gastritis (20). Therefore, we hy-pothesized that decomplementation of IL-10�/� mice couldimpair migration and extravasation of neutrophils into thestomach, resulting in decreased inflammation and impairedclearance of Helicobacter. Indeed, decomplemented IL-10�/�

mice that were infected with H. felis showed a significant in-



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crease in neutrophil percentages in peripheral blood, but themyeloperoxidase levels in the stomach showed a significantdecrease in neutrophil infiltration. These data suggest that inthe absence of complement, emigration of neutrophils into thestomach is diminished, which may lead to a decrease in theability of mice to clear the infection and to diminished inflam-mation and pathology.

Absence of complement proteins might indirectly affect theacquired immune system. In the absence of complement, theremay be less targeting of microbial antigen to antigen-present-ing cells, such as dendritic cells, impairing T-cell activation asa result of impaired antigen presentation. The role of comple-ment in direct regulation of T-cell responses is not completelyworked out. A negative regulatory role for complement incellular immunity was suggested recently by demonstratingthat the cross-linking of membrane cofactor protein (CD46)led to suppression of IL-12 production (21). However, murineand human T cells also express complement receptors, and ithas been proposed that activated complement functions tofacilitate recruitment of T cells to areas of inflammation (22,27, 41, 48). The impaired recruitment of T cells into the stom-ach may have contributed to the decreased clearance of H. felisin the decomplemented IL-10�/� mice.

Interestingly, epithelial pathology was not connected to theabsolute number of gastric Helicobacter. It is well establishedthat Helicobacter produces bacterial products (e.g., VacA cy-totoxin) that have cytotoxic effects on the epithelium (32).However, in this study (and in other animal models of Helico-bacter infection) (12), the development of gastric pathology isclearly related to the immune response rather than to thepresence of Helicobacter. Interestingly, in our study, the IL-10�/� control mice cleared the Helicobacter infection within 1week and developed significant gastritis. In contrast, the com-plement-depleted IL-10�/� mice continued to harbor the bac-terium yet had less histologic damage, despite having increasednumbers of gastric Helicobacter. The decrease in pathologymay have been due to the decreased infiltration of neutrophils.We have found that neutrophil depletion reduced gastritis inH. felis-infected IL-10�/� mice (20). Neutrophils have beenpostulated to be important mediators of the gastric pathologyseen in H. pylori infection, due to their production of tissue-damaging degradative enzymes and reactive oxygen species(45).

Of note, we uncovered a previously unappreciated relation-ship between IL-10 and the complement system. We foundthat in vitro stimulation of spleen cells from IL-10�/� miceresulted in a twofold increase in C3 production compared tothat for stimulated WT spleen cells. Neutrophils have beenidentified as a source of C3 at the site of inflammation. Withina few hours after stimulation, neutrophils are able to releasestored C3 (30, 31). In this study, we have shown that TPA-stimulated neutrophils from naïve IL-10�/� mice produce two-fold more C3 in culture than that produced by stimulated WTneutrophils. This finding implies that in the absence of IL-10,neutrophils might store higher levels of their C3 to be secretedin response to stimulation. Interestingly, IL-10 was able toblock the TPA-induced secretion, suggesting a role for IL-10 inthe regulation of complement secretion.

Absence of IL-10 may lead to increased complement pro-duction due to increased levels of inflammatory cytokines (9).

Multiple studies have demonstrated that inflammatory cyto-kines, such as tumor necrosis factor alpha, IL-1, IL-6, andgamma interferon, can induce increased production of com-plement from several cell types (19). It has previously beendemonstrated that LPS stimulation in the absence of IL-10results in marked increases in inflammatory cytokine produc-tion (e.g., tumor necrosis factor alpha, IL-1, IL-6, and gammainterferon) (4), and we hypothesize that increased inflamma-tory cytokine production in IL-10-deficient mice may contrib-ute to an increase in the levels of complement in serum. Cor-responding to the increased ability of IL-10�/� mice toproduce complement, we found that IL-10�/� mice required asixfold higher dose of CVF to deplete complement than didWT mice.

In summary, our studies indicate that the complement sys-tem has a pivotal role in the regulation of the immune andinflammatory response to gastric Helicobacter in IL-10�/�

mice. We found that Helicobacter infection of IL-10�/� miceresulted in a marked increase in the levels of complement inserum. Moreover, depletion of complement resulted in de-creased gastritis and a decreased ability of IL-10�/� mice toclear gastric Helicobacter infection. These phenomena werecorrelated with a decrease in neutrophil infiltration into thegastric mucosa. Taken together, these data suggest that com-plement activation with subsequent induction of neutrophilinfiltration may play a key role in the development of gastritisand eradication of gastric Helicobacter infection in IL-10�/�

mice.

ACKNOWLEDGMENT

This work was supported by Public Health Service grant R29 CA-76129 from the National Cancer Institute (D.J.B).

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