Local immunoglobulin G antibodies in the stomach may contribute to immunity against Helicobacter infection in mice

GASTROENTEROLOGY 1997;113:185–194

Local Immunoglobulin G Antibodies in the Stomach May Contribute to Immunity Against Helicobacter Infection in Mice RICHARD L. FERRERO, JEAN–MICHEL THIBERGE, and AGNES LABIGNE Unite´ de Pathoge´nie Bacte´rienne des Muqueuses, INSERM Unite´ 389, Institut Pasteur, Paris, France

Background & Aims: Orogastric immunization of mice with Helicobacter antigens, together with a mucosal adjuvant (cholera toxin), has been shown to confer immunity in the Helicobacter felis infection model. The aim of the study was to investigate the humoral immune responses associated with immunity and to compare these with responses in H. felis–infected mice. Methods: Enzyme-linked immunoassays were used to characterize the antibody-secreting cells and antibodies present at mucosal and systemic sites in mice. Animals were immunized orally with either whole-cell H. felis sonicates or Helicobacter pylori urease or heatshock proteins. Results: Infection of mice with H. felis preferentially induced the recruitment of plasma cells committed to immunoglobulin (Ig) A synthesis in salivary gland and gastric tissues. Antigen-specific IgA was the major antibody class detected in mucosal secretions recovered from these tissues. In contrast, immunization of mice against H. felis infection induced the proliferation of large numbers of IgG-secreting cells, as well as the synthesis of local IgG antibodies, in the gastric mucosa of the animals. Protection against H. felis infection occurred in the absence of gastric IgA responses in sonicate-immunized mice. Conclusions: It is proposed that locally synthesized specific IgG antibodies contribute to immunity against gastric Helicobacter infection.

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elicobacter pylori is a human pathogen that causes chronic gastritis1 and peptic ulceration2 and has been linked to gastric cancerigenesis.3 Infected individuals develop strong humoral and cellular immune responses to H. pylori but are unable to rid themselves of the infection.4 – 6 The detection of H. pylori specific antibodies, such as serum immunoglobulin (Ig) G or salivary IgA antibodies, can be used to accurately predict the presence of H. pylori infection in humans.4,5,7 Wyatt et al.8 reported the in vivo coating of H. pylori bacteria in gastric tissues by host Igs. Nevertheless, the role of such antibodies in the immune response was unclear. More recently, Thomas et al.9 observed that the development of H. pylori infection was delayed among infants who were breast-fed by H. pylori–positive mothers. It was concluded that the presence of specific IgA antibodies / 5e1e$$0027

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in maternal milk may protect infants from early acquisition of infection.9 Studies in murine infection models have shown that it is possible to modify the immune response to protect the host from gastric Helicobacter infection.10 – 17 Indeed, orogastric immunization of mice with antigen preparations, administered together with a mucosal adjuvant (usually cholera toxin), not only protected animals from gastric Helicobacter felis infection10 – 17 but could also clear an existent infection.18,19 The mechanism by which protection is mediated remains to be elucidated. Because passive immunization with monoclonal IgA was shown to prevent H. felis infection in mice,12,20 it was suggested that secretory IgA antibodies in mucosal secretions may mediate protection against gastric Helicobacter infection. Several investigators showed the presence of antigen-specific secretory IgA antibodies in the mucosal secretions of mice that had been immunized with Helicobacter antigens.12,15,21 Nevertheless, antibody measurements in these investigations were generally performed on sera collected after the mice had been challenged with H. felis inocula.15,21 Initial studies in our laboratory, using the enzymelinked immunospot (ELISPOT) technique, led to the observation that the mucosal tissues of mice that were infected with H. felis contained antibody-secreting cells (ASCs) that were mainly of the IgA type. Fox et al.22 also reported the presence of IgA/ cells in the mucosae of H. felis–infected animals. These observations seemed to militate against a role for IgA in immunity to H. felis infection. Therefore, a study was initiated to examine and directly compare the humoral immune responses of H. felis–infected mice with the responses of animals that had been immunized with Helicobacter antigens. For the latter, mice were immunized with known protective antigens such as sonicated whole-cell extracts of H. felis,10–14 Abbreviations used in this paper: ASC, antibody-secreting cell; ELISA, enzyme-linked immunosorbent assay; ELISPOT, enzyme-linked immunospot assay; HspA, heat-shock protein A; UreB, urease subunit B. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00

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recombinant H. pylori antigens derived either from the B subunit of H. pylori urease (UreB),13,14 or the GroES homologue of H. pylori (heat-shock protein A [HspA]).14 The mucosal immune responses in the gastric and salivary gland tissues of mice were analyzed for the presence of antibody-secreting cells in the tissues and for the presence of antigen-specific antibodies in mucosal secretions. From the data presented, we propose that locally synthesized IgG antibodies are a component of the immunity induced by orogastric immunization. Moreover, in contrast with other mucosal infections, IgA does not seem to mediate protection against gastric H. felis infection.

Materials and Methods Bacterial Strains, Media, and Growth H. pylori (85P) is a clinical isolate.23 H. felis (ATCC 49179) was originally isolated from cat gastric mucosa.24 Helicobacters were grown on a blood agar medium (Blood Agar Base No. 2; Oxoid, Basingstoke, England) supplemented with 10% horse blood (bioMe´rieux, Marcy L’Etoile, France), containing a Helicobacter-selective antibiotic mixture, and incubated under microaerobic conditions at 377C.13 Escherichia coli MC1061 cells were grown routinely at 377C in solid or liquid Luria medium.

Animals Four- to 6-week-old Swiss specific pathogen–free outbred mice (Centre d’Elevage R. Janvier, Le-Genest-St-Isle, France) were housed in polycarbonate cages in isolators and fed a commercial pellet diet with water ad libitum. These mice were previously shown to be free of the murine Helicobacter sp., Helicobacter muridarum.13 All animal experimentation was performed in accordance with institutional guidelines.

Recombinant antigens, consisting of H. pylori UreB or HspA, were produced in E. coli MC1061 cells, as previously described.13,14 The antigens were expressed as MalE fusion proteins (i.e., MalE-UreB and MalE-HspA) using the pMALc2 expression vector (New England BioLabs Inc., Beverly, MA). The fusion proteins were purified from culture lysates of recombinant E. coli cells by affinity and anion exchange chromatography. The purity of recombinant protein preparations was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and by immunoblotting. Protein concentrations were determined by the Bradford assay (Sigma Chemical Co., St. Louis, MO).

Sonicated Bacterial Extracts H. felis and H. pylori bacteria were harvested from culture plates in phosphate-buffered saline (PBS; pH 7.4). After centrifugation at 5000 rpm in a Sorvall RC-5 centrifuge (Sorvall, Norwalk, CT) for 10 minutes at 47C, the bacterial pellets

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Animal Colonization and Immunization Aliquots (100 mL) containing 104 H. felis bacteria prepared from a low-subculture stock suspension of H. felis were administered orogastrically to mice, as previously described.14 Mice were immunized orogastrically once per week, for 4 weeks, with antigen extracts (1 mg bacteria sonicates or 50 mg recombinant proteins) containing 5 mg purified cholera toxin (Sigma) that had been prepared in 100 mmol/L sodium bicarbonate.14 Mice were killed at the appropriate times by cervical dislocation, and the tissues were removed. H. felis colonization was confirmed using a biopsy urease test14 and by direct examination of mucus samples under phase contrast microscopy.24 For the former, portions of gastric antrum and body were placed on the surfaces of individual agar plates (1 cm by 1 cm) containing a modified Christensen’s urea medium, to which had been added an Helicobacter-selective antibiotic mixture.14 The plates were observed for up to 48 hours. The remaining stomach tissue was used for ELISPOT analyses. To correlate immune responses with protective immunity, groups of mice (n Å 25 and n Å 19) were immunized with either whole-cell sonicates of H. felis and cholera toxin or cholera toxin alone. Mice were challenged 2 weeks after immunization with 7 1 104 H. felis bacteria. Some of the sonicateimmunized mice (n Å 5) that were not challenged were killed 14 days after immunization. The remainder of the animals were killed approximately 3 days and 17 days after infection (equivalent to 17 and 31 days after immunization). Gastric immune responses were analyzed, and the levels of protection from gastric H. felis infection were determined using the biopsy urease test.

Extraction of Lymphocytes From Tissues

Production of Recombinant H. pylori Antigens

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were washed once and resuspended in PBS. Bacterial suspensions were sonicated and stored at 0207C until used.

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To recover lymphocytes from stomach tissues, glandular portions of the tissues were dissected into very small pieces and placed into 2.0 mL Jocklick’s medium (GIBCO BRL, Cergy Pontoise, France) supplemented with 5% (vol/vol) horse serum (GIBCO), 200 mmol/L L-glutamine, 10,000 IU/mL penicillin, and 10,000 mg/mL streptomycin (Jocklick’s complete medium; supplements from Seromed, Berlin, Germany) to which had been added 1.5 mg/mL dispase (Boehringer GmbH, Mannheim, Germany). The tissues were incubated with gentle agitation at 377C in six-well tissue culture plates (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) for 30 minutes. The digested tissues were then recovered on 70mm Falcon cell strainers (Becton Dickinson Labware) using 50-mL tubes as supports. The recovered tissues were washed in 2.0 mL Jocklick’s complete medium to collect single cells. After each digestion, single-cell suspensions corresponding to the same gastric tissue were pooled. The tissues were redigested three more times in 2.0 mL fresh Jocklick’s complete medium containing dispase, as above. The volumes of the single-cell suspensions were adjusted to 16 mL with Jocklick’s complete

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medium. The tubes were then centrifuged at 1500 rpm (Jouan, Saint-Herblain, France) for 10 minutes at 167C. Cell pellets were resuspended in 0.5 mL RPMI medium (GIBCO) supplemented with 5% (vol/vol) horse serum, 200 mmol/L L-glutamine, 10,000 IU/mL penicillin, and 10,000 mg/mL streptomycin (RPMI complete medium). An aliquot of each of the cell suspensions was diluted appropriately in trypan blue (Seromed), and cell viability and density were assessed in a Malassez counting chamber (Optik Labor, PolyLabo Paul Block & Cie, Strasbourg, France). Salivary glands were dissected into very small pieces and then digested twice in RPMI complete medium containing 1 mg/mL collagenase type IV (Sigma), as described above. The digested tissues were then homogenized in Teflon tissue grinders. The volumes of the digested tissues were adjusted to 16 mL in RPMI complete medium, and these suspensions were layered gently onto 16 mL of a Ficoll-Paque solution (Pharmacia, Uppsala, Sweden), in a 50-mL centrifuge tube. The tubes were centrifuged at 1600 rpm (Jouan) for 30 minutes at 167C. The interphase, containing predominantly lymphocytes, was recovered according to the manufacturer’s instructions. The cells were diluted in 16 mL RPMI complete medium and centrifuged at 1800 rpm for 10 minutes. The cell pellets were again resuspended in RPMI complete medium and centrifuged at 1200 rpm for 10 minutes. Cell suspensions (2.0 mL) were prepared in RPMI complete medium. Cell viability and density were assessed as above. Spleens were placed in RPMI complete medium and homogenized directly using Teflon grinders. Lymphocytes were recovered from homogenates on Ficoll-Paque solution and were treated as described above.

Detection of ASCs in Murine Tissues The ELISPOT assay25 was used to detect ASCs in the gastric, salivary gland, and splenic tissues of mice. Briefly, 96well Multiscreen filtration plates (Millipore, Molsheim, France), containing nitrocellulose supports, were coated with H. felis or H. pylori sonicate preparations (50 mg of protein per well) and left overnight at 47C. After washing twice in PBS, the plates were saturated with RPMI complete medium (100 mL per well) and incubated at 377C for 1 hour. Aliquots (100 mL) of the lymphocyte cell suspensions, prepared in RPMI complete medium, were added to the wells. The cells were incubated at 377C in 10% CO2 for 4 hours or, alternatively, overnight. The plates were washed twice in PBS and then twice in PBS containing 0.05% (vol/vol) Tween 20 (PBSTween; Merck-Schuchardt, Hohenbrunn, Germany). Aliquots (100 mL) of biotinylated secondary antibodies to specific mouse Ig classes (Amersham, Les Ulis, France), which had been diluted 1:1000 in PBS-Tween buffer, were added to the wells, and the plates were left overnight at 47C. The plates were washed as above and then 100-mL aliquots of a streptavidinperoxidase (Amersham) solution, diluted 1:500 in PBS-Tween, were added to each well. The plates were incubated in the dark at room temperature for 1 hour. After three washes in PBS, 100 mL of substrate solution (containing 1.1 mmol/L 3-

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amino-9-ethylcarbazole dissolved in N,N-dimethylformamide [both from Sigma], 4.5 mmol/L H2O2, and 50 mmol/L sodium acetate; pH 5.0) was added to each well. The reaction was terminated by washing the plates with copius amounts of water. Immunospots were counted using a stereomicroscope (Olympus, Tokyo, Japan). The numbers of ASC (per 106 lymphocytes) represent the mean numbers of immunospots counted from triplicate or quadruplicate determinations.

Collection of Salivary and Gastric Lavage Samples To induce salivation, mice were given an intraperitoneal injection of 100 mg of pilocarpine (Sigma) before the collection of salivary and gastric lavage samples. Salivary secretions were aspirated from the oral cavities of mice using a pipetman and were immediately stored at 0207C. Gastric secretions were collected using a modification of Elson’s technique,26 developed for the collection of intestinal secretions. Briefly, mouse stomachs were washed in saline and then opened along the greater curvature to release the gastric contents directly into 2.7-mL aliquots of PBS in six-well tissue culture plates (Falcon). The tissues were washed in the PBS solutions. Solid matter present in the stomachs of mice at the time of sacrifice was gently dislodged into the PBS solution using a clean scalpel blade. To each sample of gastric contents was added 300 mL of 500 mmol/L ethylenediaminetetraacetic acid and 3 mL each of the protease inhibitors leupeptin (2 mmol/ L) and pepstatin (2 mmol/L) (Boehringer). The samples were transferred to 15-mL tubes, and the total volume of each was adjusted to 6.0 mL with PBS. After centrifugation at 650g for 10 minutes, the supernatants were transferred to 2.0-mL Eppendorf tubes. Twenty microliters of 100 mmol/L phenylmethylsulfonyl fluoride (Boehringer) was added to each of the tubes, which were then centrifuged at 15,000 rpm in a Sigma 2K15 centrifuge for 20 minutes at 47C. The supernatants corresponding to a single stomach were pooled before redistributing in 2.0-mL aliquots, to each of which was added 20 mL of 100 mmol/L phenylmethylsulfonyl fluoride. The supernatants were left at room temperature for 15 minutes. One hundred microliters of calf serum was added to the samples, and these were stored at 0207C.

Detection of Antibody Isotypes in Secretions Antigen-specific antibodies in samples were detected by an enzyme-linked immunosorbent assay (ELISA).14,27 Briefly, 96-well Nunc Maxisorp plates (Nunc, Kamstrup, Denmark) were coated with sonicated extracts of H. felis or H. pylori (25 mg of protein per well), prepared in 0.1N carbonatebicarbonate buffer (pH 9.5). Antibody-containing samples to be analyzed were diluted in a PBS-Tween solution containing 0.5% (wt/vol) milk powder (Regilait Ecre´me´; Regilait, St. Martin-Belle-Roche, France), as described previously.27 Diluted gastric lavage samples (1:10–1:100) and saliva (1:20– 1:100) were added in 100-mL aliquots to coated microtiter wells. To allow for nonspecific antibody binding, samples were

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also added to uncoated wells. Bound antigen-specific Igs were detected with biotinylated goat anti-mouse antibodies (Amersham) and streptavidin-peroxidase conjugate (Amersham). Immune complexes were detected by reaction with a solution containing either o-phenylenediamine dihydrochloride (Sigma) or 2,2*-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (Sigma), and hydrogen peroxide. Optical density readings were read at either 405 or 405 and 492 nm, respectively, in an ELISA Multiskan RC plate reader (Labsystems, Helsinki, Finland). The readings for uncoated wells were subtracted from those of the respective test samples. Cutoff values for each antibody class and each antibody sample type (gastric wash vs. saliva) were determined from the mean optical density values { 2 SD for the corresponding samples from naive uninfected mice. Test samples with optical density readings greater than these cutoff values were considered to be positive for H. felis–specific antibodies. Total Igs were detected in a similar fashion as above except that the 96-well plates were coated with 100 ng of unconjugated goat antibodies raised against either mouse IgA (Harlan Sera-lab Ltd., Sussex, England) or mouse IgG (Valbiotech S. A., Paris, France), which had been prepared in 0.1N carbonate-bicarbonate buffer (pH 9.5). The ratio of specific to total Igs was determined using standard curves derived from purified murine polyclonal IgG and monoclonal IgA antibodies (both supplied by Sigma).

Statistical Analysis Data were analyzed using the Wilcoxon’s signed rank test or Fisher’s Exact Test (two-sided). P values were determined by the Statview 512/ computer software package (BrainPower, Calabasas, CA). Differences were considered significant for P values õ0.05.

Results H. felis Infection in Mice Stimulates Local IgA Synthesis The local immune responses of mice to Helicobacter antigens were examined by the ELISPOT and ELISA techniques. H. felis–infected mice had ASCs in their gastric and salivary gland tissues 2 and 8 weeks after infection. The majority of the cells in the gastric mucosa of these mice secreted antigen-specific antibodies of the IgA class; however, a small number of animals also had IgG-secreting cells at this site (Figure 1). The ASCs removed from the salivary glands of the infected mice secreted only IgA class antibodies (Figure 1). Measurements of antigen-specific and total antibodies in gastric and salivary secretions from H. felis–infected mice showed that a large proportion (9%–50%) of the IgA present in these secretions were directed against H. felis antigens (Figure 2). Within individual mice, a correlation was observed between the magnitude of anti– H. felis IgA responses in gastric secretions and that of responses in the saliva (Figure 2). Although there were / 5e1e$$0027

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Figure 1. Proliferation of ASCs in gastric (G) and salivary gland (SG) tissues from H. felis –infected mice. Mice were killed approximately (A ) 2 weeks and (B ) 8 weeks after infection (n Å 5 and n Å 7, respectively). Immunocytes that secreted H. felis –specific IgA (s) or IgG (h) antibodies were detected by the ELISPOT technique. The histograms correspond to the mean values for each group from the pooled results of two experiments. Antigen-specific IgA- or IgG-secreting cells were not present in the tissues of control uninfected mice (data not shown). P values for the numbers of IgA- vs. IgG-secreting cells in gastric and salivary tissues were (A ) P Å 0.275 and P Å 0.043, respectively, and (B ) P Å 0.028 and P Å 0.018, respectively.

approximately 10 times more total IgG than total IgA class antibodies present in the gastric secretions of the infected animals (data not shown), antigen-specific IgG antibodies represented a small proportion of the total IgG pool present (Figure 2A). H. felis–specific IgG antibodies were not detected in the saliva of infected animals (Figure 2B). WBS-Gastro

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cated whole-cell extracts of H. felis, together with a mucosal adjuvant (cholera toxin), 2 weeks after immunization (Figure 3). Similarly, immunization with two known protective antigens, MalE-fused H. pylori HspA or UreB polypeptides,10 induced the recruitment of H. pylori– specific IgG-secreting cells to the salivary glands of mice (Figure 4). Because immunization with these recombinant H. pylori antigens seemed to induce only low numbers of antigen-specific ASC in the gastric tissues of the animals,14 it was not possible to accurately quantitate the numbers of such cells in the gastric tissues of the animals. The presence of IgG-secreting cells in the stomachs of sonicate-immunized animals correlated with the detection of H. felis–specific IgG antibodies (representing between 3%–9% of total IgG) in gastric lavage samples (Figure 2B). Despite the presence of significant quantities of nonspecific IgG antibodies in the salivary secretions of sonicate-immunized mice (data not shown) and the proliferation of specific IgG-secreting cells in salivary gland tissues, H. felis–specific IgG antibodies were not detected in these secretions (Figure 2B). In addition, the

Figure 2. Antibody responses in the (A ) gastric and (B ) salivary secretions of mice. The levels of H. felis –specific IgA (circles) and IgG (squares) were measured in mice infected for 8 weeks with H. felis (● and j; numbers 1–9) and in mice that had been orogastrically immunized with sonicated H. felis extracts and cholera toxin (s and h; numbers 10–16). Crosses represent samples that were not tested. Samples from control uninfected mice (n Å 3) were negative for H. felis –specific antibodies (data not shown). Specific antibody levels are presented as a function of the total Ig detected (in micrograms of protein, 1001).

Mucosal Immunization With Protective Antigens Induces Local IgG Synthesis IgG was the major antibody class secreted by ASCs recovered from the gastric and salivary tissues of mice that had been orogastrically immunized with soni/ 5e1e$$0027

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Figure 3. Proliferation of ASCs in gastric (G) and salivary gland (SG) tissues from mice that had been orogastrically immunized with wholecell H. felis sonicates. Mice (n Å 8 and n Å 14, respectively) were killed approximately 2 weeks after the last immunization. Immunocytes that secreted H. felis –specific IgA (s) or IgG (h) antibodies were detected by the ELISPOT technique. The histograms correspond to the mean values for each group from the pooled results of three experiments. Antigen-specific IgA- or IgG-secreting cells were not present in the tissues of control uninfected mice (data not shown). Significant differences were found between the numbers of IgA- vs. IgGsecreting cells in gastric and salivary gland tissues (P Å 0.047 and P Å 0.002, respectively).

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Figure 4. Proliferation of ASCs in salivary gland tissues from mice that had been orogastrically immunized with either recombinant H. pylori (A ) HspA or (B ) UreB antigens and cholera toxin (n Å 9 and n Å 6, respectively). Mice were killed 2 weeks after the final immunization. Immunocytes that secreted H. felis –specific IgA (s) or IgG (h) antibodies are shown. The results from two experiments are presented in B. The histograms correspond to the mean values for the results in each group. Antigen-specific IgA- or IgG-secreting cells were not present in the tissues of control uninfected mice (data not shown). Significant differences were found between the numbers of IgA- vs. IgG-secreting cells in the tissues of HspA- and UreB-immunized mice (P Å 0.012 and P Å 0.028, respectively).

immunocytes (138 { 80; n Å 5) were present in the stomachs of the animals. Assessment of H. felis colonization in the mice showed 100% protection for sonicate-immunized mice, both at 3 days (n Å 10) and 17 days (n Å 10) after the challenge. In comparison, 44% (4 of 9) and 10% (1 of 9), respectively, of mice that had been immunized with cholera toxin alone were not infected by H. felis at these time points (P Å 0.022 and P Å 0.0001, respectively). The relatively mediocre level of infection among the cholera toxin-immunized mice 3 days after the challenge was attributed to a temporary suppression of the infection below the level of detection. It is possible that nonspecific immune mechanisms induced by cholera toxin13 were responsible for this temporary suppression of H. felis infection. Consistent with this, Marchetti et al.16 reported

salivary and gastric secretions of immunized mice did not contain H. felis–specific IgA class antibodies (Figure 2). Gastric Mucosal Responses and Protective Immunity Mice that had been immunized orogastrically with H. felis sonicate preparations and cholera toxin all developed strong H. felis–specific IgG antibody responses in their gastric tissues both before and at 3 and 17 days after the challenge with H. felis bacteria (Figure 5). The levels of these specific IgG antibodies, as a proportion of total IgG antibodies detected in gastric lavage samples, varied from 1.3% (for mouse no. 6; Figure 5) to 77% (for mouse no. 18; Figure 5). Furthermore, the presence of antigen-specific IgG antibodies in gastric secretions correlated with the detection of specific IgG-secreting ASCs in the stomachs of immunized/uninfected mice (276 { 99; n Å 4) and in those of immunized/infected animals, at 3 days (247 { 308; n Å 4) and 17 days (159 { 131, n Å 5) after infection. In contrast, neither H. felis–specific IgA antibodies nor specific IgA-secreting ASCs were detected before or immediately after H. felis infection. However, 17 days after the challenge, H. felis– specific gastric IgA antibodies and antigen-specific IgA / 5e1e$$0027

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Figure 5. Antibody responses in the gastric secretions of sonicateimmunized mice before and after H. felis challenge. The levels of H. felis –specific (A ) IgA and (B ) IgG were determined in mice that had been immunized with H. felis sonicates and cholera toxin. Mice were left for 2 weeks after the final immunization and killed as follows: I, before H. felis challenge (n Å 5); II, 3 days after H. felis challenge (n Å 10); and III, 17 days after H. felis challenge (n Å 10). H. felis – specific antibody readings of gastric samples from individual mice were determined in triplicate and compared with a reference value corresponding to the mean optical density { 2 SD for gastric samples from control uninfected mice (n Å 4; not shown). Specific antibody levels are presented as a function of the total Ig detected (in micrograms of protein, 1001).

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that prior immunization with the heat-labile toxin from E. coli was sufficient to induce nonspecific immunity against H. pylori infection in 25% of mice. Systemic Immune Responses in Colonized and Immunized Mice The spleens of mice that had been infected with H. felis for 8 weeks contained large numbers of H. felis– specific IgA- and IgG-secreting cells (Figure 6). The IgG immunocytes recovered from these animals secreted antibodies of the IgG1, IgG2a, and IgG2b isotypes in similar proportions (Figure 7). In contrast with infected mice, sonicate-immunized animals had splenic ASCs that were exclusively committed to the synthesis of specific IgG class antibodies (Figure 6). Furthermore, isotype analyses of the ASCs recovered from immunized mice (at 2 weeks after immunization) showed that the anti–H. felis antibodies secreted by these cells were mainly of the IgG1 subclass and, to a lesser extent, of the IgG2b isotype (Figure 7). To determine whether these responses were influenced by the nature of the stimulatory antigen administered, IgG isotype analyses were performed on the ASCs recovered from the spleens of mice that had been immunized with MalE-fused H. pylori UreB. Nevertheless, it seemed that IgG1 was again the dominant subclass secreted by these cells (Figure 7).

Figure 7. Proliferation of ASCs in the spleens of mice (n Å 6 per group) that had been infected for 8 weeks with H. felis (L) or orogastrically immunized either with sonicated H. felis extracts (s) or recombinant H. pylori UreB antigen (n) and cholera toxin. Immunocytes were scored according to the type of Helicobacter-specific IgG isotype (IgG1, IgG2a, or IgG2b) that was secreted. Antigen-specific IgA- or IgG-secreting cells were not present in the tissues of control uninfected mice (data not shown). Significant differences were found between the numbers of IgG1- and IgG2a-secreting cells in sonicate-immunized mice (P Å 0.028) and in animals that had been immunized with H. pylori UreB antigen (P Å 0.043).

Discussion

Figure 6. Proliferation of ASCs in the spleens of mice that had been (A ) infected for 8 weeks with H. felis or (B ) orogastrically immunized with sonicated H. felis extracts and cholera toxin. Immunocytes that secreted H. felis –specific IgA (s) and IgG (h) antibodies are shown. The histograms correspond to the mean values for the results in each group (n Å 6 mice per group), pooled from two experiments. Antigenspecific IgA- or IgG-secreting cells were not present in the tissues of control uninfected mice (data not shown). P values for the numbers of IgA- vs. IgG-secreting cells were P Å 0.689 and P Å 0.028, respectively.

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The local and systemic immune responses of mice infected with H. felis have been compared with those of animals that were immunized with known protective antigens. We found that orogastric immunization of mice, either with whole-cell sonicated Helicobacter extracts or with recombinant H. pylori antigens, induced the recruitment of large numbers of specific IgG-secreting cells in the mucosal tissues of the animals (Figures 3 and 4). In addition, antigen-specific IgG antibodies were present in gastric lavage samples from sonicateimmunized mice both before and after challenge with H. felis bacteria (Figures 2 and 5). The significant level of protection against H. felis infection, observed among sonicate-immunized mice at 3 days after the challenge, occurred in the absence of detectable H. felis–specific IgA antibodies or immunocytes in the gastric mucosa (Figure 5). On the basis of the above findings, it is proposed that gastric IgG antibodies induced by mucosal immunization are an important component of effector immune responses involved in protection against H. felis infection. WBS-Gastro

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It has previously been suggested that protection in the H. felis mouse model is mediated by the induction of local sIgA production.12,15 This conclusion reposed primarily on the following lines of evidence: first, that orogastric immunization of mice with antigens, together with a mucosal adjuvant, resulted in the synthesis of Helicobacter-specific IgA antibodies in the salivary,15 intestinal,15,21 or biliary secretions11 of the animals; and, second, the finding that passive immunization of mice with monoclonal IgA antibodies, directed against the UreB subunit of H. felis urease, protected the animals from H. felis infection.12,20 In the light of the present findings reported, it is perhaps necessary to reappraise the evidence in support of a role for secretory IgA in protection against gastric H. felis infection. IgA antibodies have been detected in the sera and secretions of mice immunized against H. felis infection; however, the interpretation of the data has been confounded by the fact that antibody determinations were often performed after infection of the animals with H. felis.12,15,21 For example, in mice that were orally immunized with whole cell sonicates, little or no anti–H. felis IgA antibodies were present in fecal extracts taken from the mice, yet antibodies could be detected when the immunized animals were also subjected to H. felis challenge.21 In contrast, immunization with recombinant H. pylori urease holoenzyme alone was sufficient to induce detectable antiurease IgA responses in the feces of the animals.21 This finding was confirmed by the detection of mild infiltrations of IgA/- and IgM/-staining cells in the gastric tissues of the mice.21 In the present study, gastric IgA antibody responses were observed in the stomachs of sonicate-immunized mice at 17 days after the challenge (Figure 5), suggesting that local IgA responses develop as a response to antigenic stimulation after bacterial challenge. This, however, does not preclude gastric IgA or other effector mechanisms from playing a role in protection after immunization because it has been shown that after challenge with H. felis, a transient infection occurs. This could perhaps be cleared by gastric IgG and/ or gastric IgA antibodies that begin to increase coincident with clearance of the infection. The results of passive immunization experiments have been another key element supporting the involvement of secretory IgA in the protection against mucosal H. felis infection. Recently, however, Blanchard et al.20 reported that an IgG monoclonal antibody was also able to passively protect mice against H. felis infection. In common with known protective IgA monoclonal antibodies, the IgG molecule recognized a portion of the UreB subunit of H. felis urease,20 although the efficacy of the IgG antibody may be attributed to the experimental / 5e1e$$0027

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conditions used20 or to the fact that the murine stomach is less hostile than its human counterpart. An equally valid interpretation might be that the IgG antibodies are able to neutralize H. felis bacteria in the inoculum and consequently prevent gastric Helicobacter infection. The induction of strong IgG antibody responses at mucosal sites have been described for various pathogenic agents28 – 31; the local production of antigen-specific IgA and IgG antibodies secreted into cervico-vaginal fluids of women with genital herpes simplex virus type 2 or Chlamydia trachomatis led to the observation that both antibody classes are important in controlling the magnitude and duration of these diseases.28,29 In addition, it was reported that the presence of circulating antilipopolysaccharide IgG antibodies, in the absence of seric or mucosal IgA, was able to protect mice against gastrointestinal infection with Pseudomonas aeruginosa.30 From the current results, it seems that locally synthesized IgG antibodies in the gastric mucosa mediate protection against H. felis infection. Nevertheless, as H. felis–specific IgG antibodies were present in the gastric secretions of both immunized and infected animals (Figure 2), qualitative differences between the Igs, such as antibody isotype or epitope specificity, might account for the apparent differences in anti-H. felis activity of the respective antibodies. Investigation of these differences should permit a greater understanding of the mechanism by which mucosal IgG antibodies mediate immunity to gastric Helicobacter infection. H. felis infection induces strong IgA responses in mice, characterized by the presence of IgA-secreting cells in the gastric mucosa and the production of mucosal and serum IgA antibodies (Figures 1, 2, 5, and 6). Similarly, infection with H. pylori in humans has been described to result in the production of mucosal IgA antibodies.7,8,32 It remains unclear why local IgA antibodies are unable to eradicate Helicobacter bacteria from the gastric mucosa. Several possible explanations for this observation are that (1) the antibodies do not bind to the bacteria and hence are unable to promote immune exclusion; (2) the antibodies per se are defective; or (3) the bacteria produce proteases that degrade IgA antibodies. There are no data showing the existence of IgA proteases in H. felis, but interesting data relevant to the other suggestions have been provided by studies of H. pylori–infected individuals. In the first instance, Darwin et al.33 reported that H. pylori bacteria present in gastric brushings from H. pylori–infected individuals were not coated in IgA or IgG antibodies and were poorly recognized by serum antibodies. The lack of reactivity of the bacteria was attributed to the poor immunogenicity of surface membrane antigens expressed by H. pylori bacteria in vivo.33 In the second WBS-Gastro

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instance, Birkholz et al.34 found that IgA molecules in the gastric juice of H. pylori–infected persons were infrequently coupled to secretory component despite the presence of normal levels of secretory component in the sera. As the uncoupled molecules of IgA are likely to be susceptible to gastric acidity, local IgA would probably be ineffective in eradicating H. pylori from the stomach.34 Disturbances in IgA structure and/or function have also been documented in chronic diseases, such as in inflammatory bowel disease and in periodontal disease.35 Further investigations on the nature of the local immune responses induced by Helicobacter infection should clarify the relative contributions of bacterial and host factors to chronic disease. The current interest in developing oral vaccines against mucosal pathogens is founded on the concept of a common mucosal immune system, in which lymphocytes activated in the gut, for example, can induce immunity in the intestine and in other mucosal tissues.36 From the evidence of the present study and previous data,14,15,21,22 it seems that the stomach is part of the mucosal immune system. However, the inductive sites that need to be stimulated to produce effector responses in the stomach have not been elucidated. Other investigators reported that immunization via the oral route was able to induce protective immunity in the stomach, suggesting that induction of the immune response might occur in the orobuccal cavity itself.15,21 In the present study, orogastric immunization yielded an increased synthesis of specific IgG class antibodies in the gastric mucosa of mice, but not in the saliva (Figure 2). On the other hand, H. felis infection induced the synthesis of antigen-specific antibodies in the gastric tissue and, at a distant mucosal site, the saliva (Figure 2). Similarly, Fox et al.37 recently reported the presence of elevated anti– H. pylori IgA antibody levels in the salivary and gastric secretions of H. pylori–infected cats. These results indicate that the inductive sites involved in the uptake and processing of antigens introduced into the body by orogastric immunization may be different to the site(s) stimulated by an infection. In addition, it seems that the measurement of antibodies in mucosal secretions from extragastric sites may not truly reflect the induction of immunity in the stomach. The activation of splenic immunocytes devoted to systemic antigen-specific IgG1 antibody synthesis in immunized animals (Figure 7) was consistent with previous data14 suggesting that protective immunity to H. felis infection might be associated with a T helper 2 type response. Because H. pylori infection in humans seems to trigger the production of cytokines normally associated with a T helper 1 response,38 – 40 one might speculate that / 5e1e$$0027

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the switching of the immune system toward a predominantly T helper 2 type response is more likely to be associated with immunity to Helicobacter infection. Further investigations using the H. felis infection model, together with techniques permitting the analysis of cytokines synthesized in vivo, will permit this hypothesis to be tested.

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31. Walker MJ, Rohde M, Timmis KN, Guzma´n CA. Specific lung mucosal and systemic immune responses after oral immunization of mice with Salmonella typhimurium aroA, Salmonella typhi Ty21a, and invasive Escherichia coli expressing recombinant pertussis toxin S1 subunit. Infect Immun 1992;60:4260–4268. 32. Luzza F, Imeneo M, Maletta M, Monteleone G, Doldo P, Biancone L, Pallone F. Isotypic analysis of specific antibody response in serum, saliva, gastric, and rectal homogenates of Helicobacter pylori-infected patients. FEMS Immunol Med Microbiol 1995;10: 285–288. 33. Darwin PE, Sztein MB, Zheng Q-X, James SP, Fantry GT. Immune evasion by Helicobacter pylori: gastric spiral bacteria lack surface immunoglobulin deposition and reactivity with homologous antibodies. Helicobacter 1996;1:20–27. 34. Birkholz S, Knipp U, von Blohn G, Stallmach A, Schneider T, Zeitz M. Local antibodies to Helicobacter pylori antigens (abstr). Gut 1995;37:A32. 35. Kilian M, Russell MW. Function of mucosal immunoglobulins. In: Ogra PL, Mestecky J, Lamm ME, Strober W, McGhee JR, Bienenstock J. Handbook of mucosal immunology. Volume 1. San Diego, CA: Academic, 1994:127–137. 36. McGhee JR, Kiyono H. New perspectives in vaccine development: mucosal immunity to infections. Infect Agents Dis 1993;2:55– 73. 37. Fox JG, Perkins S, Yan L, Shen Z, Attardo L, Pappo J. Local immune response in Helicobacter pylori-infected cats and identification of H. pylori in saliva, gastric fluid, and faeces. Immunology 1996;88:400–406. 38. Ha¨berle H, Kubin M, Trinchieri G, Luthra R, Gourley WK, Garofalo R, Crowe SE, Reyes VE, Graham DY, Karrtunen R, Ernst PB. The regulation of activated helper T cells in the gastric mucosa during infection with H. pylori (abstr). Gut 1995;37(S1):A50. 39. Crabtree JE, Peichl P, Wyatt JI, Stachl U, Lindley IJD. Gastric interleukin 8 and IgA IL-8 autoantibodies in Helicobacter pylori infection. Scand J Immunol 1993;37:65–70. 40. Crowe SE, Alvarez L, Dytoc M, Hunt RH, Muller M, Sherman P, Patel J, Jin Y, Ernst PB. Expression of interleukin 8 and CD54 by human gastric epithelium after Helicobacter pylori infection in vitro. Gastroenterology 1995;108:65–74. Received May 10, 1996. Accepted March 18, 1997. Address requests for reprints to: Richard L. Ferrero, Ph.D., Unite´ de Pathoge´nie Bacte´rienne des Muqueuses, Institut Pasteur, 28 Rue du Dr Roux, Paris 75724, France. e-mail: [email protected]; fax: (33) 1-40613640. Supported by grants from OraVax Inc., Boston, Massachusetts, and Pasteur-Me´rieux Connaught (PMC), Lyon, France. The authors thank Dr. Marie-Jose´ Quentin-Millet and Dr. Bruno Guy (PMC, France) for introducing us to the ELISPOT technique and for helpful discussions.

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