Manifestations of the local gastric immune response in gnotobiotic piglets infected with Helicobacter pylori

Veterinary immunology ad immunopathoiogy

Veterinary Immunology

and lmmunopathology 52 (1996) 159-173

ELSEVIER

Manifestations of the local gastric immune response in gnotobiotic piglets infected with Helicobacter pylon! Steven Krakowka a**,Susan S. Ringler a, Katv Wendy B. Green a, Robert Leunk a Department

A. Eaton a,

of Veterinary Biosciences, CoIlege of Veterinary Medicine. The Ohio State University.

Colambus. OH 43210, USA b The Procter and Gamble Co.. Miami Valley Laboratories, P.O. Box 398707. Cincinnati. OH 45239. USA

Accepted 13 November

1995

Abstract Helicobacter pylori, a human gastric bacterial pathogen, was inoculated into gnotobiotic piglets and manifestations of the resultant gastric inflammation was analyzed by in situ immunochemistry and flow cytometric analysis of isolated lamina propria leukocytes (LPL) and peripheral blood leukocytes (PBL) recovered from infected and control piglets. Gastric mucosa tissue sections from uninfected control piglets were essentially negative for cluster differentiation- (CD-) positive leukocytes. Failure to isolate significant numbers of LPL from the gastric lamina propria confirmed this observation. A local and systemic immune response occurs in piglets after infection with H. pylori. This is manifest by the appearance of cells associated with a local immune response in gastric mucosa. In gastric tissue sections from H. pylori-infected piglets, CDCpositive leukocytes were sparse and closely associated with developing lymphoid follicles whereas the CDS-positive cellular phenotype was abundant. The latter formed a continuous band in the lamina propria just above the muscularis mucosa. Perivascular accumulations of lymphocytes in the outer muscular tunic(s) were strongly positive for expression of CD8 antigen. Class II-positive cells were prominent in CD8 lymphocytic infiltrates, developing follicles and vascular endothelia but were uniformly absent from gastric epithelia even in sites overlying areas of immunocyte proliferation and infiltration. Leukocytes possessing the monocyte and granulocyte markers were rare. Plasma cells

* Corresponding author at: Goss (614)-292-0231; fax: (614)-292.6473.

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containing IgA were common in the periphery of developing lymphoid follicles or distributed as discrete foci around individual gastric pits. Fewer numbers of IgG- and IgM-positive plasma cells were identified. When the LPL flow cytometry data were compared with the flow cytometry data obtained from PBL in these same II. pylori-infected piglets. leukocytes bearing the CD8 marker predominated in LPL whereas leukocytes bearing the CDCreactive and MHC class II markers predominated in PBL. Finally, local ELISA antibody responses were measured in mucosal explant culture supernatants and compared with in vivo antibody levels in sera, bile, and gastric juice. Antibody activity, specific for H. pylori. was detected in supematants and serum in all three isotypes in

actively infected piglets whereas gastric juice lacked antibodies. Gastric explants prepared from piglets in which infection had been successfully eradicated failed to produce local antibody into supematant fluids. These data support the concept mediated by local immunological events.

that the gastric

inflammation

observed

is

1. Introduction A bacterial infectious etiology is now established as the cause of type B gastritis in human beings (Blaser, 1992). The agent, Helicobucfer pylori. a gram negative microaerophilic bacterium. is widespread in the population. Duration and severity of infection are strongly linked to ulcerogenesis, mucosal atrophy and recently, gastric carcinoma (Buck. 1990; Madan et al., 1990; Blaser. 1992: Lee, 1994; O’Conner, 1994; Blaser and Parsonnet. 1994). In humans, the spectrum of microscopic inflammatory lesions attributable to this bacterium varies (Dixon et al., 1988; Dixon, 1994). Histologic lesions may include lymphocytic infiltrations and lymphoid follicle development alone (Stolte and Eidt, 1989; Bertram et al.. 1991; Dixon, 1994) and/or chronic active inflammation characterized by neutrophilic infiltrates, gland abscesses with attendant gastric epithelial degeneration, necrosis and mucosal atrophy (Wyatt et al., 1986; Stolte and Eidt, 1989; Buck, 1990; Bertram et al.. 1991; Blaser. 1992; Dixon. 1994; Lee, 1994). Previous studies have shown that the gnotobiotic piglet is susceptible to human strains of H. pylori and that the organism selectively colonizes the gastric microenvironment with subsequent development of lymphoplasmacytic gastritis (Krakowka et al.. 1987; Eaton et al.. 1989, Eaton et al., 1990, Eaton et al., 1991, Eaton et al., 1992; Eaton and Krakowka, 1992) and gastric ulcers (Krakowka et al., 1995). In infected piglets, the neutrophilic component is sparse (Krakowka et al.. 1987; Bertram et al., 1991) but is enhanced by parenteral immunization with formalin-killed bacteria prior to infection with the bacterium (Eaton and Krakowka. 1992). In order to gain insights into the mechanisms of induction of gastritis and gastric immunity. a series of studies were designed to determine the number, distribution and organization of inflammatory cell infiltrates in the gastric mucosa of H. pylori-infected piglets using monoclonal anlibodies to swine cluster differentiation (CD) antigens (Lunney, 1993). In addition. data regarding gastric mucosal immunoglobulin (Ig) production were sought.

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2. Materials and methods 2.1. Animals, microbiology and pathology A total of 31 gnotobiotic piglets from six litters were employed in this study. Details of derivation and husbandry conditions in gnotobiology are published (Krakowka et al., 1987). Piglets were maintained in isolation units and fed a sterile milk replacement diet throughout the study period of 15-31 days. Six piglets from one litter became monocontaminated with a yeast sp. and served as one set of H. pylori-negative controls; three piglets from one litter and four from another remained microbiologically sterile throughout the experiment and constituted the naive control germ-free piglet group. The remaining piglets (n = 18) were orally infected with 10’ colony forming units (cfu) of H. pyfori, strain 26695 (Krakowka et al., 1987; Eaton et al., 1989) at 3 days of age. At termination, ten-fold dilutions of gastric homogenate were used for quantitative determination (Eaton and Krakowka, 1992) of bacterial cfu. Formalin-fixed paraffin-embedded 6 micron sections of gastric tissue were stained with hematoxylin and eosin for histopathologic evaluation. 2.2. Antibody determinations Serum samples, bile and gastric contents were tested for isotype-specific antibodies to bacterial antigens by ELISA as described (Krakowka et al., 1987; Eaton et al., 1990, Eaton et al., 1992). In preliminary studies, selected sera were titrated for activity using constant antigen and constant secondary reagent(s) to establish the dilution of sample which gave linear OD,, values between 0 and 100% of a positive control serum for each Ig isotype. On the basis of these titrations (data not shown), all samples were prediluted 1: 10 before testing. Local mucosal production of bacterial antibodies was measured in supematant fluids collected from mucosal explant cultures (Sobala et al., 1991). For this, 2-4-mm diameter biopsies of cardia and antrum (three to six replicates of each per site in each piglet) were cultured in 96-well microtiter plates in RPM1 1640, 10% fetal calf serum (FCS) at 37°C 5% CO, for 4 days. Supematants were collected, replicates pooled and then frozen (- 20°C) until assayed. In all cases ELISA data is reported as corrected OD values less the OD values of the plate controls. 2.3. Antibodies to porcine antigens The following monoclonal antibodies and their isotype controls (VMRD, Pullman WA) to porcine leukocyte cluster differentiation (CD) antigens were used: CDClike (PT90A, IgG2a), CD8 (PT81B, IgG2b), MHC class II (MSA3, IgG2a), monocyte (PG130A, IgM) and granulocyte/monocyte (DH59B, IgGl). The monoclonal (PT9OA) used to detect CD4 marker on lymphocytes has recently been reported to react with myeloid cells as well (Blecha et al., 1994); others report that PT90A is specific for the CD4 determinant on lymphocytes (Pescovitz et al., 1994; Saalmueller et al., 1994). The uncertainty in the specificity of this monoclonal reagent renders designation of CD4positive cells as solely of lymphocyte lineage uncertain. Polyclonal goat and rabbit antisera to swine Igs (Kirkegaard and Perry, Gaithersburg, MD) were employed as secondary reagents in the ELISA and for immunocytochemical staining of plasma cells.

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isolation

Leukocytes from 6 ml of heparinized peripheral blood (PBLI were purified by centrifugation on a ficoll-hypaque gradient (1.077 g ml-’ 1, 35 min, 600 X g. The interface was collected, washed twice in Ca’+ and Mg’+-free PBS, (CMF-PBS) and resuspended in RPM1 1640 supplemented with 10% FCS containing 1% glutamine and 1% antibiotics. Viability was determined by trypan blue dye exclusion and purity verified by microscopic examination of Wright Geimsa-stained cytospin preparations. Cells were incubated overnight at 37°C in 5% CO1 prior to staining with the CD monoclonals. The isolation of lamina propria leukocytes (LPL) from stomachs was achieved through modifications of several published techniques (Bull and Bookman, 1977; Gibson-D’Ambrosio et al., 1986; Wilson et al.. 1986a, Wilson et al., 1986b). After aseptic removal of the stomach and incision along the greater and lesser curvatures, the mucosa was scraped free of mucus, rinsed in three changes of cold Hank’s balanced salt solution (HBSS) and the external muscular layers were dissected and discarded. Excess mucus was again removed from the mucosa by gentle scraping, and the tissue was cut into 5-mm strips. To remove remaining adherent mucus, the tissue pieces were incubated for 25 min, 22°C in 10 ml of CMF-HBSS containing 1.O mM dithiothreitol (DTT). The tissue pieces were then rinsed in CMF-HBSS supplemented with 0.5% bovine serum albumin, 20 mM HEPES buffer, 1% glutamine, and 1% antibiotics (CMF-HBSS). To remove epitbelial cells, the tissue fragments were placed into 10 ml CMF-HBSS containing 0.75 mM EDTA and incubated for 20 min. 37°C with constant agitation. The tissue was then washed three times with lo-20 ml CMF-HBSS and this digestion and wash procedure was repeated twice. After the third wash, the tissue was transferred to RPM1 1640/10% FCS containing 0.5 mg ml- ’ collagenase (Type II), minced into l-2-mm pieces, and incubated for 2 h, 37°C with constant agitation. After collagenase digestion, methylcellulose was added to a final concentration of 0.1% and the cell slurry was transferred to a sterile Stomacher bag for mechanical isolation of LPL in a Stomacher Model 80 Laboratory Blender (Gibson-D’Ambrosio et al., 1986). The tissue was incubated for 10 min, 22°C with the paddle speed set at 150 to 160 strokes min- ’ The contents of the Stomacher bag were filtered through an 80-mesh filter and the filter rinsed with 10 ml RPM1 164O/ 10% FCS. The cell suspension was centrifuged at 200 X g for 10 min and the resultant cell pellet was resuspended in 10 ml of 43% (v/v) percoll ( 1.057 g 1- ’ ) and underlaid with 5 ml of 67% (v/v) percoll (1.088 g l- ’ ). The gradients were centrifuged at 600 X g. 30 min, 4°C. The cellular interface was collected, washed twice in CMF-PBS. and resuspended in 5 ml of RPM1 1640/10% FCS. Viability was assessed as above and the morphology was determined by examination of stained cytospins. 2.5. Flow cytomety Following overnight incubation at 37°C to permit cellular recovery, synthesis and re-expression of CD antigens, both PBL and LPL were again centrifuged through the percoll gradients to remove dead cells and debris, evaluated for viability and adjusted to

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1.0 to 3.0X lo6 live cells ml-‘. Cellular suspensions were then stained with diluted (1: 100 to 1: 1000) monoclonal or isotype control antibodies followed by fluorosceinated flow cytometry grade antiglobulin secondary reagents. Dual labelled (CDClike:CDS)positive cells were determined by staining cell suspensions with both monoclonals followed by staining with fluorescein- and phycoerythrin-conjugated secondary reagents. Prior to cytometric evaluation, single cell suspensions were created by fine needle aspiration of cell pellets through a 26-g needle. The stained cells were fixed in freshly prepared 1.0% paraformaldehyde in CMF-PBS and processed through an EPICS 753 Flow Cytometer (Coulter Electronics Corp., Kendall, FL). 2.4. Immunocytochemishy Sections of gastric and lymphoid tissue from infected gnotobiotic piglets were examined for the distribution of selected CD markers and for Ig-producing plasma cells by immunocytochemistry. The enzyme digestion method used to detect Ig-bearing cells in formalin-fixed tissues (Axthelm and Krakowka, 1986; Cork et al., 1991) was employed to identify plasma cells in gastric tissues. For localization of leukocyte CD antigens, unfixed sections of gastric cardia and antrum were collected, embedded in OCT compound, snap-frozen in liquid nitrogen and stored at -7O’C. Six micron sections were cut on a cryostat and mounted on glass microslides, air dried and fixed in acetone for 10 min. Sections were treated for 5 min, 22°C with l.O-mM DTT, blocked with normal horse serum (lo%, v/v) and then stained with the appropriate monoclonal or isotype control for 2 h, 22°C. After extensive washing, the appropriate secondary biotinylated horse anti-murine Ig (diluted 1:3) was added and incubated for 30 min, 22°C. This was followed by ABC (Vectastain) for 60 min, 22°C. Bound horseradish peroxidase reaction product was visualized with a DAB substrate kit (Kirkegaard and Perry Inc., Gaithersberg, MD) on sections counterstained with hematoxylin.

3. Results 3.1. Parameters of infection All piglets inoculated with H. pylori became infected and developed histologically evident lymphoplasmacytic gastritis typical of this disease (Krakowka et al., 1987; Eaton et al., 1989, Eaton et al., 1990, Eaton et al., 1991; Bertram et al., 1991). Colonization levels achieved at termination ranged from 106-* cfu g- ’ (Akopyants et al., 1995) values consistent with previous studies in piglets and comparable to results in humans (Blaser and Parsonnet, 1994). Uninfected control gnotobiotes were housed in separate isolation units and remained culture-negative for H. pylori throughout the experiments. Table 1 (top panel) summarizes ELISA antibody data (as group mean values) from four infected and four uninfected control gnotobiotic piglets. Terminal immune sera contained readily detectable quantities of H. pylon’-specific lgs whereas uninfected control piglet sera (presented in parentheses) did not. Bile from infected piglets was rich in IgG and IgA, but lacked H. pylori-specific IgM; no antibodies were present in bile

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Table I Group mean anti-t/. pyhri-specific Ig isotype-specific ELISA antibody determinations from H. pylori-infected piglets (Group A) and their respective litter-mate controls (parentheses). Also presented is a second group of H. pylori-infected piglets (Group B) successfully treated and cleared of bacterial infection with a proprietary antimicrobial along with their litter-mate infected and untreated controls (parentheses) Location of Sample(s)

Helicobucter

pyhri-specific

IgG

Group A: H. pylori-infectedpig1et.s. Gastric mucosal explants: Cardia 0.48 Antrum 0.27 Gastric Contents 0.02 Bile 0. I2 Serum 0.77 Group B: H. pylori-infectedpiglets Gastric mucosal explants: Cardia 0.08 Antrum 0.02 Serum 0.16

immunoglobulins IgM

(Ig) IgA

PID ” 28 (n = 4) b (0.04) c (0.03) (0.04) (0.03) (0.03)

0. IS 0.15 0.00 0.01 0.52

(0.04) (0.01) (0.01) (0.00) (0.2 I)

treated successfully with an antimicrobial. (0.33) (0.26) (0.79)

0.07 (0.16) 0.02 (0.07) 0.42 (0.46)

0.5 I 0.46 0.0 I 0.17 0. I6

(0.03) (0.02) (0.02) (0.02) (0.04)

PID 21 (n = 3) 0. IO (0.65) 0.04 (0.44) 0.15 (0.17)

a PID. post infection day. b Data expressed as the group corrected mean OD,,, nm values (e.g. less plate control values) of triplicate determinations from four infected piglets, (Group A) and three infected piglets (Group B). ’ Group corrected mean OD,, nm litter-mate control values (e.g. less plate control values) of triplicate determinations. For Group A, controls consisted of four uninfected control piglets. For Group B, controls consisted of three H. pyiori-infected piglets not treated with a proprietary antimicrobial agent.

collected from control animals. Gastric juice from both infected and control piglets lacked detectible antibodies. However, substantial quantities of Ig of all three isotypes were detected in explant culture supematants prepared from infected but not control piglets. In a second series of explant experiments (Table 1, bottom panel), biopsies from two groups of infected piglets were established as above. All had been infected with H. pylori 23 days previously. Piglets of Group B were successfully treated (e.g. H. pylori was not recovered from gastric homogenates at termination) with a proprietary drug product beginning at 10 days post infection. Explant biopsies prepared from the stomachs of treated piglets produced virtually no antibody when compared with untreated actively infected litter-mate controls (corresponding group mean values in parentheses); levels of IgM and IgA in terminal sera were equivalent in both groups whereas sera from culture-negative piglets demonstrated reduced levels of H. pylorispecific IgG. 3.2. Flow cytometry of isolated PBL and LPL Monoclonal reagents were titrated on PBL isolated from several gnotobiotic piglets and then applied to equivalent numbers of PBL isolated from control and infected piglets (Table 2). In swine PBL, the CDCpositive cellular phenotype predominated and

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Table 2 Plow cytometric determination of selected swine leukocyte cluster differentiation (CD) monoclonal antibody markers for isolated porcine peripheral blood leukocytes (PBL) and isolated gastric lamina propria leukocytes (LPL) from H. pylori-infected and uninfected control gnotobiotic piglets Peripheral blood leukocytes (PBL)

Swine CD leukocyte marker

Controls Germ-free (n=3)

CD4 Pl90A a CD8 PT8lB CD4CD8 dual-labelled MHC-Class II MSA-3 Monocyte PG 130A Granulocyte/monocyte

DH59B

48.3 b 15.9 2.2 12.5 _c -

Contaminated (n=3) 27.4 13.4 1.9 9.1

Gastric lamina propria leukocytes (LPL) H. pylori

Controls

Infected

Germ-free

(n=.5)

(n=3)

30.9 13.0 0.8 11.9 29.7 1.1

4.5 42.5 1.9 28.7 -

H. pylori Contammated (n= 3) 3.6 46.2 2.8 33.2

Infected (n=6) 3.1 18.6 3.1 33.7 5.2 6.3

a This monoclonal, designated as CD4 specific (VMRD, Inc., Pullman, WA) reacts with porcine myeloid cells (Blecha et al., 1994) and lymphocytes (Pescovitz et al., 1994; Saalmueller et al., 1994). b The data are expressed as the group mean percent positive staining cells as determined by flow cytometric analysis. c -, not done.

no obvious difference attributable to infection with Z-Z.pylori was seen between the infected and control groups in the percent distribution of the various labelled leukocyte phenotype(s). Lamina propria leukocytes were readily isolated from gastric mucosa of H. pylori-infected piglets and sufficient numbers (range of 9.8-28.3 X lo6 leukocytes per one-half or whole stomach) were recovered to permit complete flow cytometric analysis. In contrast, only a few (range of 0.2-0.8 X 106) LPL were isolated from uninfected control stomachs. Similarly, very few (1.0-4.4 X 106) LPL were recovered from the gastric mucosa of the yeast-contaminated control piglets. For both infected and control groups, the viability of isolated LPL was high (> 95%) immediately after isolation and 24 h later. In the LPL recovered from uninfected control piglets, the CD8-positive cell population accounted for 30-65% of the total and were approximately ten-fold more abundant than were the CDcreactive cells (Table 2). The next most abundant cell type did not stain with T cell reagents used and were likely of null or B-cell origin. When compared with PBL, LPL were enriched for cells expressing MHC class II-reactive mononuclear cells (B cells and/or monocytes) and the CD8 phenotype and were relatively devoid of cells reacting with the CD4 reagent and the reagents for the monocyte/granulocyte phenotypes. Granulocytes (neutrophils) were uncommon in LPL and accounted for only 6.3 to 8.2% of the cells recovered from infected gastric tissue. Even though many more LPL were recovered from infected vs. control piglets, the only consistent feature of LPL recovered from H. pylori-infected piglets was the relative decrease in the percent CD8 cells (18.6%) compared with control values (42.5 to 46.2%). The importance of this

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modest percent reduction is balanced by the fact that at least 35fold more total cells of the CD8 phenotype were recovered from infected gastric mucosae than were recovered from control tissues. 3.3. Immunocytochemistry For clarity, the tissue distribution and structural arrangement of gastric inflammation, either as unorganized diffuse or focal cellular infiltrates or organized lymphoid aggregates and mucosa-associated follicles are described for each CD marker. Each summary description represents the results of four to six section replicates of cardia and antrum from four to six different infected piglets. The paucity of identifiable leukocytes in the uninfected controls precluded meaningful immunocytochemical evaluation of them. 3.3.1. CD4 cell distribution CD4-positive leukocytes were sparsely distributed in gastric lamina propria. They were most abundant in the outer mantle region of developing mucosal lymphoid follicles; occasional cells stained with the CD4 reagent used were identified in the lamina propria, closely apposed to epithelial basement membranes or crypt areas of the gastric pits. A few CD4-positive cells were also detected in the perivascular infiltrates in the outer muscular tunics. The rare intraepithelial leukocytes (IELI seen were not stained with this monoclonal antibody. 3.32. CD8-positive T cell distribution In contrast to CD6positive leukocytes, the CD8-reactive T cells were abundant and strongly stained in both the lamina propria and the large vessels of the external muscular tunics of H. pylori-infected piglets. These formed a discrete and almost continuous band of immunopositive-staining cells in the deepest layer of the lamina propria, subadjacent to the muscularis (Fig. 1). The numerous and well-developed mucosal lymphoid follicles contained few CD8-positive cells. Occasional CD8-positive cells were present in the lamina propria between gastric pits and in subepithelial/epithelial locations consistent with the location of IEL (Fig. 1). Virtually every perivascular leukocyte in the outer muscular layers was strongly positive for CD8 antigen. This had the effect of outlining the vascular system with intense immunoreactive staining product (not shown). 3.3.3. MHC class II-positive leukocyte distribution Strong immunoreactivity with MSA-3 (MHC II) was noted in lymphoid follicles. The stromal network (presumably dendritic cells) underlying the outer mantle and central core regions of the lymphoid follicles were also MHC II-positive. In extrafollicular (diffuse) cellular infiltrates, the distribution of MHC II-reactive cells in the lamina propria largely mirrored the distribution of CD8 cells described above except for the deepest layer of cells subadjacent to the muscularis. In tissue sections, unidentified cells. most likely capillary vascular endothelia. were also strongly reactive with this monoclonal. This had the effect of emphasizing the vasculature in both the lamina propria and gastric mucosa. At least one-half of the cells surrounding the vessels as well as vascular endothelia in the muscularis externa were also positive for this antigen. In all instances,

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Fig. 1. Distribution of CDI-positive leukocytes (arrows) in the gastric lamina propria gnotobiotic piglet. Note that the positive staining cells form an almost continuous propria of the stomach. In addition, rare staining product is discernible in cells possibly within the gastric epithelium as intraepithelial leukocytes. Section stained and counterstained with hematoxylin ( X 175).

gastric epithelia, II-negative.

even over prominent

3.4. Monocyte/macrophage

accumulations

167

of an H. pylori-infected layer in the deep lamina below the epithelium or with monoclonal PT81B

of leukocytes,

were MHC class

distribution

Monocytes were stained with two monoclonal antibodies: PG130A specific for swine monocytes and DH59B, a monocyte/granulocyte swine leukocyte CD marker. PG130A stained only a small population (< 5%) of cells. These cells were scattered singly throughout the lamina propria, but were most prominent in the regions subadjacent to gastric pits. Similarly, developing lymphoid follicles were sparsely stained, if at all, and then only in the interior portions of the follicles. The granulocyte/monocyte monoclonal (DH59B) stained only an occasional cell in the lamina propria and follicles; the numbers of immunopositive cells, as with PGl30A, were low. 3.5. Plasma cell distribution Formalin-fixed replicates stained with isotype-specific reagents for swine Ig revealed numerous Ig-positive plasma cells in the gastric lamina propria. lgM-positive plasma

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Fig. 2. A cluster of IgA-positive plasma cells adjacent to a developing mucosal lymphoid follicle and blood vessels in the lamina propria. Formalin-fixed paraffin-embedded section stained with polyclonal anti-IgA (Axthelm and Krakowka, 1986) and counterstained with hematoxylin (X 750).

cells were infrequent and largely restricted to areas adjacent to B cell origin lymphoid follicles. In contrast, both single cells and clusters of cells which stained for either cytoplasmic IgG and IgA (Fig. 2) were closely apposed to the vasculature of the lamina propria and were widely distributed in regions of inflammation throughout the stomach.

4. Discussion In this study, the phenotypic distribution of gastric inflammatory cells and selected parameters of gastric mucosal humoral immune responses to H. pylori in young infected gnotobiotic swine are described. The experiments were designed to determine the distribution, numbers, anatomical arrangement and cellular phenotype(s) of gastric

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immune and inflammatory cell infiltrates and to correlate these findings to those in H. pylon’-naive piglets and also to comparable data in other species. A second objective was to gain preliminary insight into the gastric mucosal expression of humoral immunity to this gastric bacterial pathogen. An important and often unappreciated feature of experiments conducted in germ-free or microbe-defined (gnotobiotic) conditions such as these described here is the opportunity to study the ‘pure’ infectious disease process without the complicating factors of intercurrent subclinical infections and the confounding effects of commensal microbiota and their products. Previously, we reported that gastric (as well as intestinal) lamina propria of gnotobiotic swine are devoid of visually detectible leukocytes in the lamina propria (Krakowka et al., 1987). Here we confirm this observation by demonstrating that very few leukocytes could be recovered from stomachs of microbiologically sterile individuals and that only a few cells were obtained from piglets contaminated with a commensal yeast sp., an organism which lacks gastric specificity. For both control groups, the numbers of stained cells were too low to determine the distribution of isolated cells in the tissue sections. Thus, the leukocytes recovered from H. pylori-infected lamina propria were, in fact, attracted to that location as a direct consequence of gastric colonization with H. pylon’. Both the flow cytometric evaluations and the in situ immunocytochemical data demonstrated that the most of the invading immune cells (sum of CD4 and CD8 values) in the gastric lamina propria of H. pylon’-infected piglets were likely of T cell origin. Similar conclusions have also been reached in humans (Engstrand et al., 1989; Blaser, 1992; Nielsen and Andersen, 1993). However, in both infected humans and mice colonized with a related Helicobacfer sp., (Fox et al., 1993), CDCpositive cells predominate in the gastric lamina propria and most of the detectible CD8 cells were in intraepithelial locations (Papadimitriou et al., 1988). The H. pylori-infected gnotobiotic piglet differs substantially from these species in that the bulk of the recovered LPL are of the CD8 phenotype and very few intraepithelial leukocytes were observed. The distribution of leukocytes in the stomach more closely resembles that recently described for the distribution of intestinal leukocyte population(s) in conventional swine (VegaLopez et al., 1993). In pigs, like humans, CDClike leukocytes were closely associated with developing lymphoid (B-cell) follicles, a location consistent with their putative helper function (Jonjic et al., 1987). In contrast, a distinct region of the submucosa, directly adjacent to the muscularis mucosa was strongly positive for the CD&positive cells. These cells formed a more or less continuous and distinct layer along the lamina propria above the muscularis. A similar distribution of this cell type has been described in other segments of the porcine gut (Vega-Lopez et al., 1993). Perhaps the location of these cells constitute an immunoprotective barrier to inappropriate absorption of unwanted antigens into the circulation. The vessels in the outer muscular layers were also strongly outlined by perivascular infiltrates of CD8-positive cells, a finding which suggests that some proportion of the suppressor/cytotoxic phenotype differentiate at extragastric sites and are attracted to the stomach by continuous antigenic stimulation. Flow cytometric analysis of isolated LPL confirmed immunohistologic impressions in that the percent of T lymphocytes bearing the CD8 marker predominated. The presence of accessory cells expressing class II and monocyte markers is

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compatible with an infectious disease process in the stomach. Similarly, class II antigenic product(s) were detected within organized follicular structures and this distribution is again consistent with the presence of macrophages and B cells. In addition to staining LPL. the class II monoclonal used also stained vascular endothelia. However, porcine gastric epithelia, like porcine enterocytes of the lower bowel (Vega-Lopez et al., 1993) were negative for this antigen. This finding differs from that described in both mice (Fox et al., 1993) and humans (Papadimitriou et al., 1988; Engstrand et al., 1989) in which both enterocytes and gastric epithelia are MHC-positive and hence theoretically capable of antigen presentation (Kaiserlain and Vidal, 1993). Neutrophilic infiltrates, the oft-cited histological hallmark of human H. pylori gastritis (Dixon et al., 1988; Buck, 1990; Madan et al., 1990; Sobala et al., 1991; Blaser, 1992; Dixon, 1994) were uncommon in either isolated porcine LPL or in stained sections. In humans, bacterial chemotactic factors (Blaser. 1992; Nielsen and Andersen. 1992; Mai et al., 1992; Moutiala et al., 1992), and/or inflammatory mediators are thought to be responsible for neutrophilic gastritis. Data presented here and elsewhere (Eaton and Krakowka, 1992), indicate that this belief incompletely explains the development of the neutrophilic component of gastritis in vivo. For example, locally absorbed bacterial urease (Blaser, 1992) has been proposed as the pivotal in vivo chemotaxin; the absence of neutrophilic inflammation in piglets infected with urease-producing bacteria for 4 weeks indicate that absorption of urease alone is insufficient to produce local neutrophilia. Finally, we have previously shown that neutrophilic gastritis and lymphoplasmacytic inflammation are enhanced by parenteral immunization of piglets prior to infection (Eaton and Krakowka. 1992); convalescent sera from immunized animals contained unusually high levels of H. pylori-specific IgG. These data are consistent with the concept that immune IgG potentiates gastric inflammation and may even determine the histologic form(s) seen (Valnes et al., 1986; Brandtzaeg et al., 1987) and also with the consequences of a vigorous systemic humoral immune response. Mucosal production of anti-H. pylori Ig further illustrates the antigen-dependent nature of local gastric immune and inflammatory responses. The ELISA data demonstrate that substantial local production of Ig occurs in infected gastric mucosae and these findings were corroborated by concurrent demonstration of plasma cells in gastric tissues. Moreover, there was a positive correlation between bacterial infection and local Ig production. Piglets rendered microbe-negative by treatment rapidly cease producing local antibody, as determined by in vitro gastric mucosa explant culture data. Thus, local mucosal antibody production may well be an early, sensitive and specific indicator of active bacterial infection in piglets and possibly human beings as well. The lack of detectible antibody in gastric contents from infected piglets was unexpected. Mucosal explants prepared from these same piglets, produced Ig of all three isotypes in vitro. In humans, gastritis may be accompanied by nonspecific leakage of Ig, especially IgG into the gastric lumen (Brandtzaeg et al., 1987). Lack of lumenal antibody may be biologically important and could explain why the gastric mucosal antibody response is apparently incapable of clearing the stomach of this bacterium. An inhibitory substance may be present in gastric juice which prevents detection of biologically active Ig. Perhaps the acidic microenvironment denatures free Ig as it is released into the stomach or effects binding affinity for antigen(s) thereby rendering it

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biologically inactive? Recently completed studies have shown that porcine gastric epithelia produce secretory component and gastric contents, even when diluted to pH 7.0 immediately, are still antibody-negative (Green et al., unpublished observations, 1994). Finally, lack of antibody in gastric juice may be due to the fact that secreted IgA or IgG which leaks into the lumen as a consequence of infection all bind the bacteria or bacterial products in vivo, leaving little if any free Ig available for detection by ELISA. The gnotobiotic piglet model of human gastric disease has proven value in assessment of experimental antimicrobial drug therapies and bacterial virulence factor studies (Eaton et al., 1989, Eaton et al., 1991, Eaton et al., 1992). It is likely that additional insight(s) into the disease process will be gained by further immunological studies in this animal model.

Acknowledgements This research was supported by Grants DK3950 and R29DK45340, National Institutes of Health (NIH), Public Health Health Service (PHS) and a grant from the Procter and Gamble Co. The authors thank Judy Younger, Michelle Trumble and Bryan Kessler for technical assistance and Dr. Michael Murtaugh for concise, insightful and helpful critique of this manuscript.

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