Helicobacter pylori is the most common cause of chronic

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1 GASTROENTEROLOGY 2000;118: A Predominant Th1 Type of Immune Response Is Induced Early During Acute Helicobacter pylori Infection in Rhesus Macaques JOSEPH J. MATTAPALLIL,* SATYA DANDEKAR,* DON R. CANFIELD, and JAY V. SOLNICK* *Department of Internal Medicine, Division of Infectious Diseases, California Regional Primate Research Center, University of California Davis, Davis, California Background & Aims: The immune response of gastric T cells during acute Helicobacter pylori infection has not been previously characterized. The aim of this study was to delineate the phenotypic and functional responses of gastric T cells during acute H. pylori infection of rhesus macaques. Methods: Four monkeys were experimentally infected with H. pylori. Gastric biopsy specimens and peripheral blood samples were obtained 1 and 12 weeks after inoculation. Samples from 3 animals uninfected with H. pylori served as controls. The immunophenotypic changes and functional potential of CD4 and CD8 T cells in gastric mucosa and peripheral blood to produce cytokines (interleukin [IL]-2, IL-4, IL-13, interferon [IFN]-, MIP- 1, and tumor necrosis factor [TNF]- ) were determined at a single cell level using flow cytometry. Results: An increase in CD4 T cells occurred in the gastric mucosa during acute H. pylori infection as early as 1 week after infection. Acute infection was characterized by a predominantly T helper (Th)1 (IL-2 and IFN- ) and proinflammatory (TNF- and MIP-1 ) type of cytokine response and the absence of a Th2 type of response. Conclusions: A predominant Th1 type response was induced early during acute H. pylori infection and may contribute to the development of gastric disease. Helicobacter pylori is the most common cause of chronic active gastritis and peptic ulcer disease in humans and has been implicated in the development of gastric malignancy. 1 Much of our understanding of the pathogenesis of H. pylori is derived from studies of patients with chronic H. pylori infection. Understanding the early host-pathogen interactions may provide insights into the persistence and pathogenesis of H. pylori infection. However, because acute H. pylori infection rarely comes to clinical attention, these studies have been limited. The simian (Macaca mulatta) model provides an opportunity to study the pathogenesis of acute H. pylori infection in a host whose gastrointestinal immune system closely resembles that of humans. 2 4 Experimental infection of rhesus monkeys is characterized by histological gastritis, increased serum gastrin levels, and the presence of H. pylori specific immunoglobulin G, all features that mimic H. pylori infection in humans. 5 Some animals eventually develop gastric atrophy, the histological precursor to gastric adenocarcinoma. 5 These studies suggest that the rhesus macaque model is suitable for studies of H. pylori associated gastritis and the host immune responses that develop in gastric mucosa during acute infection. The gastric lamina propria harbors numerous lymphocytes called lamina propria lymphocytes (LPLs), which are an important component of the mucosa-associated immune defense. The cytokines produced by T cells in the gastric lamina propria may play a central role in the infiltration of neutrophils and mononuclear cells that is characteristic of H. pylori associated gastritis. Based on the pattern of cytokine expression, CD4 T cells and recently CD8 T cells have been classified as T helper (Th)1 and Th2 type of T cells. The Th1 type of cytokine expression (interleukin [IL]-2, interferon [IFN]-, and lymphotoxin) is associated with cellular immune responses generated against intracellular pathogens, whereas Th2 type of cytokine expression (IL-4, IL-5, IL-10, and IL-13) is primarily associated with extracellular pathogens and allergens and is characterized by an antibodymediated response. The qualitative and quantitative expression of various cytokines during H. pylori infection may have an effect on the persistence of infection and disease outcome. Previous studies have suggested that protection from H. pylori infection was associated with Th2 type of cytokine response, whereas chronic infection was associated with the predominance of a Th1 type of cytokine response Most studies have attempted to Abbreviations used in this paper: FITC, fluorescein isothiocyanate; IFN, interferon; IL, interleukin; LPL, lamina propria lymphocyte; MIP, macrophage inflammatory protein; PBMC, peripheral blood mononuclear cell; PE, phycoerythrin; TC, Tricolor; Th, T helper; TNF, tumor necrosis factor by the American Gastroenterological Association /00/$10.00

2 308 MATTAPALLIL ET AL. GASTROENTEROLOGY Vol. 118, No. 2 delineate the cytokine profiles using whole cell culture by enzyme-linked immunosorbent assay or by detecting the messages for cytokines in gastric mucosa These studies are limited by their inability to identify the phenotype and frequency of cytokine-producing cells in the gastric mucosa. Although the frequency and phenotype of IFN- and IL-4 producing T cells in patients chronically infected with H. pylori have been identified, 14 the cytokine responses of T cells during acute H. pylori infection have not been characterized. The main objective of this study was to characterize the phenotypic changes and cytokine expression in gastric lamina propria and peripheral blood T cells during acute H. pylori infection. We examined the frequency of CD4, CD4 CD8, and CD8 T cells in peripheral blood and gastric lamina propria of H. pylori infected and uninfected rhesus macaques using flow cytometry. The potential of T cells to produce IL-2, IL-4, IL-13, tumor necrosis factor (TNF)-, IFN-, and macrophage inflammatory protein (MIP)-1 was determined at a single cell level using flow cytometry. Our results showed that acute H. pylori infection was associated with increased prevalence of CD4 T cells and a predominantly Th1 (IL-2 and IFN- ) and proinflammatory (TNF- and MIP-1 ) cytokine response as early as 1 week after inoculation. This response was found to persist at 12 weeks after inoculation. This early Th1 response may contribute to the pathogenesis of H. pylori infection. Materials and Methods Animals Seven rhesus macaques (age, 3 6 years) used in this study were housed at the California Regional Primate Research Center according to protocols approved by the Association for the Assessment and Accreditation of Laboratory Animal Care. All monkeys were naturally infected with H. pylori before therapy as determined by culture of gastric biopsy samples. Histological examination of gastric biopsy specimens from 3 monkeys also showed evidence of H. heilmannii infection, an uncultivated Helicobacter species that commonly infects rhesus monkeys but that is associated with little or no inflammation. 2,15 To eliminate the natural Helicobacter infection, all monkeys were treated with omeprazole (0.3 mg/kg), clarithromycin (11 mg/kg), bismuth subsalicylate (20 mg/kg), and amoxicillin (14 mg/kg) twice daily for 14 days. Culture and histological examination of stomach biopsy specimens obtained after antibiotic treatment confirmed the absence of Helicobacter infection. Four monkeys were experimentally inoculated with H. pylori, and the remaining 3 monkeys served as uninfected controls. Experimental H. pylori Infection Four rhesus macaques were inoculated (10 weeks after antibiotic treatment) with H. pylori N246C3, a CagA-positive rhesus-derived strain (courtesy of C. Lee, OraVax, Cambridge, MA). Animals were fasted overnight and given cimetidine (10 mg/kg body wt) intramuscularly 1 hour before inoculation. Bacteria ( in 3.0 ml) were administered by orogastric inoculation, which was repeated on 3 alternate days. Before all inoculations, bacteria were examined by wet mount, Gram stain, and rapid urease assay with urea-indole medium 16 to confirm their identity as H. pylori, and by serial dilution plate counts to confirm quantitation of the inoculum. Sample Collection Peripheral blood samples and gastric biopsy specimens were obtained from the 4 rhesus macaques at 1 and 12 weeks after inoculation with H. pylori. Peripheral blood samples and gastric biopsy specimens were also obtained from the 3 uninfected control animals. Animals were fasted overnight and anesthetized by intramuscular administration of ketamine (10 mg/kg body wt) before endoscopy. Gastric biopsy specimens obtained from the antrum and the body were used for lymphocyte isolation and H. pylori detection. Histological Examination and H. pylori Culture Biopsy specimens were fixed in formalin, embedded in paraffin, and processed with Warthin Starry stain. The presence of acute and chronic gastritis was determined by the presence of polymorphonuclear leukocytes and mononuclear cell infiltration. H. pylori was cultured according to methods described previously. 15 Biopsy specimens were placed in preweighed tubes containing 100 µl sterile 0.9% NaCl and immediately transported to the laboratory. Samples were homogenized, and serial dilutions were plated on selective Brucella agar with 5% fetal calf serum (GIBCO BRL, Gaithersburg, MD) containing TVP-A (5 mg/l trimethoprim, 10 mg/l vancomycin, 2.5 IU/L polymyxin B, and 4 mg/l amphotericin B; Sigma Chemical Co., St. Louis, MO). The plates were incubated in an atmosphere of 5% CO 2 for 10 days. H. pylori was identified using colony morphology (pinhead-sized translucent colonies), microscopy (gram-negative curved organisms), and biochemistry (oxidase, catalase, and urease positive). Isolation of Gastric LPLs and Peripheral Blood Mononuclear Cells LPLs were isolated according to previously published procedures with minor modifications. 17,18 Twelve 2 3-mg biopsy tissue samples were placed into cell isolation medium containing RPMI 1640 (GIBCO) supplemented with 100 U/mL penicillin (GIBCO), 100 U/mL streptomycin (GIBCO), 5% fetal calf serum (GIBCO), and 50 U/100 ml collagenase type II (Sigma) and subjected to rapid shaking at 37 C for 30 minutes. This procedure was repeated 3 times. Liberated cells were centrifuged and washed, and lymphocytes were isolated through a 35%/60% (vol/vol) isotonic discontinuous Percoll (Sigma) density gradient. Lymphocytes were found to band at the interface between the 35% and 60% gradients. Isolated

3 February 2000 IMMUNE RESPONSES IN ACUTE H. PYLORI INFECTION 309 lymphocytes were washed in Hank s balanced salt solution, and viability was assessed by trypan blue exclusion. More than 98% of the isolated mononuclear cells were viable as determined by trypan blue exclusion assay. The total yield of cells from all 12 biopsy specimens ranged from 1.7 to cells from treated animals before experimental infection and ranged from 2 to cells from infected animals. Approximately 23% 31% of the isolated cells from uninfected animals were found within the lymphocyte gate, whereas 36% 47% of isolated cells from H. pylori infected animals were found within the lymphocyte gate. Isolated cells were then used for flow-cytometric analysis. Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation. Peripheral blood was collected in tubes containing acid citrate dextrose and centrifuged at 500g for 20 minutes. The buffy coats were collected and mixed with an equal volume of 1 phosphate-buffered saline (PBS), layered over lymphocyte isolation medium (ICN, Aurora, OH), and centrifuged at 500g for 30 minutes. Mononuclear cells were found to band at the interface. Cells were washed in 1 PBS and used for flow-cytometric analysis. Antibodies Monoclonal antibodies to CD3 (Pharmingen, San Diego, CA), CD4 (OKT4; Ortho Diagnostic Systems Inc., Raritan, NJ), CD8 (Caltag Laboratories, San Francisco, CA), MIP-1 (Molecular Probes, Inc., Eugene, OR), and IL-2, IL-4, IL-13, TNF-, and IFN- (Pharmingen, San Diego, CA) were used in this study. Matched isotype controls for the cell surface antigens were obtained from Caltag Laboratories, and isotype control antibodies for intracellular cytokines were obtained from Pharmingen. Immunophenotyping of Gastric LPLs and PBMCs Freshly isolated cells were stained with anti-cd3 conjugated to fluorescein isothiocyanate (FITC), anti-cd4 conjugated to phycoerythrin (PE), and anti-cd8 conjugated to Tricolor (TC) for 30 minutes at 4 C. Cells were washed and suspended in PBS for analysis. Negative control samples were stained with matched isotype control antibodies. Flow-Cytometric Detection of Intracellular Cytokine Production Freshly isolated cells were stimulated with 10 ng/ml phorbol myristate acetate (Sigma), 500 ng/ml ionomycin (Calbiochem, La Jolla, CA), and 2 µmol/l monensin (Sigma) for 4 hours. Control samples were incubated with 2 µmol/l monensin only, to determine whether cells were producing cytokines in the absence of stimulation. After incubation, the cells were harvested and washed in cytoflow buffer (PBS with 1% bovine serum albumin) and labeled with fluorescent antibodies. To determine the capacity of gastric LPLs and PBMCs to produce cytokines, cells were subjected to 3-color flowcytometric analyses according to previously described methods. 17,18 The cells were stained with a monoclonal antibody to CD4-TC or CD8-TC. After washing in PBS, cells were fixed (Cell Perm & Fix Kit; Caltag) for 15 minutes at room temperature in the dark, washed, permeabilized (Cell Perm & Fix Kit), and stained with either anti-human IL-2 FITC and anti-human MIP-1 PE, or anti-human IFN- FITC and anti-human IL-4 PE, or anti-human TNF- FITC and antihuman IL-13 PE for 15 minutes at room temperature in the dark. Negative controls included samples stained with matched isotype control antibodies. Figure 1. Gastric inflammation observed in H. pylori infection of rhesus macaques. Histological examination of gastric mucosa from (A) uninfected animal (B) 1 week after H. pylori infection showing plasma cells, scattered neutrophils, and H. pylori organisms closely apposed to the epithelial surface of gastric pits. (C) Twelve weeks after infection; H. pylori accompanied by extensive inflammation. Bar 100 µm (A C).

4 310 MATTAPALLIL ET AL. GASTROENTEROLOGY Vol. 118, No. 2 Cells prepared for both immunophenotypic analysis and intracellular detection of cytokines were analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). For immunophenotypic analysis, events were collected in list mode after simultaneously gating on lymphocytes (based on their forward and light scatter characteristics) and CD3 T cells (based on FL1). For the intracellular detection of cytokines, 2000 events were collected by simultaneously gating on lymphocytes (based on their forward and light scatter characteristics) and CD4 or CD8 T cells (based on FL3). Collected data were analyzed using the Cell Quest software (Becton Dickinson). The frequency of T cells producing IL-2, IL-4, IL-13, IFN-, MIP-1, and TNF- was determined as a percentage of gated CD4 and CD8 T cells. No constitutive expression of IL-2, IL-4, IL-13, IFN-, MIP-1, and TNF- was observed in the absence of mitogenic stimulation. Results Experimental H. pylori Infection Leads to Persistent Infection in Rhesus Macaques All 4 animals experimentally inoculated with H. pylori were found to be infected 1 and 12 weeks after inoculation by histology and culture. The mean colonyforming units per gram of gastric tissue were at 1 week after inoculation and at 12 weeks after inoculation. Infection was associated with histological gastritis characterized by plasma cells and scattered neutrophils predominantly in the gastric antrum (Figure 1). H. pylori organisms were found in close proximity to the epithelial surfaces at 1 week after inoculation and persisted in gastric mucosa at 12 weeks after inoculation. Prevalence of CD4 T Cells in Gastric Lamina Propria Increased During Acute H. pylori Infection The frequency of CD3-gated CD4, CD4 CD8, and CD8 T cells in gastric lamina propria and peripheral blood was determined before and after H. pylori infection using flow cytometry (Figure 2). The frequency of CD4 T cells increased in the gastric lamina propria immediately after infection. The increase was detected as early as 1 week after inoculation and remained elevated at 12 weeks after inoculation. A coincident decrease was observed in the frequency of CD8 T cells in the gastric lamina propria. No major changes were observed in the frequency of CD4 CD8 double-positive T cells in the gastric lamina propria. Relative proportions of CD4, CD8, and CD4 CD8 double-positive T cells in peripheral blood did not differ after H. pylori infection. Figure 2. Acute H. pylori infection led to an increased prevalence of CD3 CD4 T cells in the gastric mucosa. Freshly isolated cells from the gastric mucosa and peripheral blood from uninfected controls and at 1 and 12 weeks after infection were stained for CD3, CD4, and CD8 and analyzed by 3-color flow cytometry. Bars represent mean values.

5 February 2000 IMMUNE RESPONSES IN ACUTE H. PYLORI INFECTION 311 percentage of gated CD4 and CD8 T cells, showing the absence of IL-4 production (Figure 5). The predominant Th1 type of cytokine response displayed by both CD4 and CD8 T cells from gastric lamina propria and peripheral blood at 1 week after inoculation was found to persist at 12 weeks after inoculation. To determine the phenotype of IL-2 and IFN- producing T cells, analysis gates were set to include only IL-2 and IFN- positive cells. The majority of IFN- producing gastric T cells were CD8 ( 65%), whereas the majority of IL-2 producing T cells were CD4 ( 56%), both before and after H. pylori infection (data not shown). A similar trend was observed in peripheral blood, in which more than 62% of the IFN- producing T cells were CD8 and more than 78% of the IL-2 producing T cells were CD4 (data not shown). Figure 3. Acute H. pylor infection led to a predominantly proinflammatory and Th1 type of response in the gastric mucosa. The potential of gastric lamina propria CD4 and CD8 T cells to produce IL-2, IL-4, IL-13, and IFN- was determined after short-term in vitro stimulation. Isolated cells were stained for cell surface expression of CD4 or CD8, fixed, permeabilized, and stained for intracellular production of IL-2, IL-4, IL-13, and IFN-. The frequency of cytokine-producing T cells was determined as a percentage of gated CD4 and CD8 T cells. Bars represent mean values. Acute H. pylori Infection Induced a Predominantly Th1 Type of Cytokine Response in Both Gastric and Peripheral Blood CD4 and CD8 T Cells To determine whether acute H. pylori infection induced a Th1 (IL-2 and IFN- ) or Th2 (IL-4 and IL-13) type of cytokine response in gastric and peripheral T cells, we measured the potential of CD4 and CD8 T cells to produce IL-2, IL-4, IL-13, and IFN- at 1 and 12 weeks after inoculation using flow cytometry. Both CD4 and CD8 T cells in gastric mucosa (Figure 3) and in peripheral blood (Figure 4) had a Th1 type of cytokine profile at 1 week after inoculation. No change was observed in the frequency of IL-4 and IL-13 producing CD4 and CD8 T cells after H. pylori infection (Figures 3 and 4). A representative dot plot is presented as a Figure 4. Peripheral blood T cells displayed a predominantly proinflammatory and Th1 type of response during acute H. pylori infection. The potential of peripheral blood CD4 and CD8 T cells to produce IL-2, IL-4, IL-13, and IFN- was determined after short-term in vitro stimulation. Isolated cells were stained for cell surface expression of CD4 or CD8, fixed, permeabilized, and stained for intracellular production of IL-2, IL-4, IL-13, and IFN-. The frequency of cytokineproducing T cells was determined as a percentage of gated CD4 and CD8 T cells. Bars represent mean values.

6 312 MATTAPALLIL ET AL. GASTROENTEROLOGY Vol. 118, No. 2 Figure 5. Flow-cytometric analysis revealed that gastric CD4 and CD8 T cells were primed to produce IFN- but not IL-4 during acute H. pylori infection. The potential of gastric CD4 and CD8 T cells to produce IL-4 and IFN- was determined after short-term in vitro stimulation. Isolated cells were stained for cell surface expression of CD4 or CD8, fixed, permeabilized, and stained for intracellular production of IL-4 and IFN-. The frequency of IL-4 and IFN- producing T cells is shown as a percentage of gated CD4 and CD8 T cells. P.I., post infection. Acute H. pylori Infection Increased the Frequency of MIP-1 Producing Gastric Lamina Propria T Cells Because MIP-1 may play an important role in infiltration of mononuclear cells into gastric mucosa, the potential of CD4 and CD8 T cells from gastric mucosa and peripheral blood to produce MIP-1 was examined after H. pylori infection and compared with uninfected controls. The frequency of both CD4 and CD8 T cells producing MIP-1 increased dramatically in gastric lamina propria at 1 week after inoculation and remained high at 12 weeks after inoculation compared with uninfected controls (Figure 6). A similar trend was observed in peripheral blood (Figure 6). To determine the phenotype of MIP-1 producing T cells, analysis gates were set to include only MIP-1 positive cells. The CD8 T cells were found to be the major source of MIP-1 in both gastric lamina propria (50% 76%) and peripheral blood (52% 80%) after H. pylori infection (data not shown). A representative dot plot (Figure 7A) shows that among the gastric lymphocytes, most of the MIP-1 producing cells before and after H. pylori infection were CD8 T cells. Acute H. pylori Infection Leads to an Increased Frequency of TNF- Producing T Cells in Gastric Lamina Propria Because TNF- is an inflammatory cytokine associated with gastritis, we examined the potential of CD4 and CD8 T cells from gastric mucosa and peripheral blood to produce TNF- after H. pylori infection and compared them with uninfected controls. The frequency of TNF- producing CD4 and CD8 T cells increased in the gastric lamina propria at 1 week after inoculation and remained high at 12 weeks after inoculation (Figure 6). A similar trend was observed in peripheral blood T cells after H. pylori infection (Figure 6). To determine the phenotype of TNF- producing T cells, analysis gates were set to include only TNF- positive cells. Interestingly, the CD4 T cells were the major producers of TNF- in gastric lamina propria (54% 62%), whereas CD8 T cells were the major producers of TNF- in peripheral blood ( 50%) after H. pylori infection (data not shown). A representative dot plot (Figure 7B) shows that among the gastric lymphocytes, most of the TNF- producing cells before and after H. pylori infection were CD4 T cells. Discussion Numerous studies have shown a predominantly Th1 type of cytokine response in the gastric mucosa of patients chronically infected with H. pylori, 14,19,20 but no information is available on the phenotype and frequency of cytokine-producing cells in the gastric mucosa and in peripheral blood during acute H. pylori infection. Using the simian model, our results showed that the gastric mucosa of acute H. pylori infected animals was characterized by an expansion of CD4 T cells and by an increased

7 February 2000 IMMUNE RESPONSES IN ACUTE H. PYLORI INFECTION 313 Figure 6. Acute H. pylor infection led to a predominantly proinflammatory type of immune response. The potential of gastric lamina propria and peripheral blood CD4 and CD8 T cells to produce TNF- and MIP-1 was determined after short-term in vitro stimulation. Isolated cells were stained for cell surface expression of CD4 or CD8, fixed, permeabilized, and stained for intracellular production of TNF- and MIP-1. The frequency of TNF- and MIP-1 producing T cells was determined as a percentage of gated CD4 and CD8 T cells. Bars represent mean values. frequency of both CD4 and CD8 T cells producing IL-2, IFN-, TNF-, and MIP-1. In contrast, few CD4 and CD8 T cells were primed to produce IL-4 and IL-13 during acute H. pylori infection. These findings showed that acute H. pylori infection induced a predominantly proinflammatory and Th1 type of cytokine response as early as 1 week after inoculation, which persisted at 12 weeks after inoculation with no apparent shift to a Th2 type of response. The Th1 cytokine response was evident in peripheral blood but at a lower magnitude. Together, these results suggest that the expansion of CD4 T cells and Th1 type of cytokine response observed during chronic H. pylori infection of humans 6 10,14,21,22 likely occurred very early during acute infection. However, prior incidence of natural H. pylori infection in the monkeys used in our study may have predisposed both gastric and peripheral blood T cells to produce a stronger Th1 response. Future studies with specific pathogen (H. pylori) free rhesus monkeys 15 will be required to examine the cytokine responses in animals with no prior exposure to H. pylori. The emergence of a Th1-driven cell-mediated immune response during early acute H. pylori infection was not sufficient to resolve the infection by 12 weeks after inoculation. The persistence of bacteria may actually contribute to an up-regulation of the inflammatory cascade. Consistent with this hypothesis, our results showed that the frequency of gastric T cells primed to produce IL-2, IFN-, TNF-, and MIP-1 remained high at 12 weeks after inoculation. These cytokines have been shown to play a role in the activation of the inflammatory cascade by activating macrophages and polymorphonuclear leukocytes, which would release the reactive oxygen radicals that could damage the gastric mucosa. Further, MIP-1 may contribute to tissue damage by stimulating the release of cytokines such as TNF-, IL-1, and IL-6, which perpetuate the inflammatory response. Mohammadi et al. 23 have shown that Th1-mediated immune response characterized by an increased production of IFN- led to mucosal damage in the H. felis infected mouse model. The gastric inflammation was significantly reduced when IFN- neutralization antibodies were administered in vivo. Further, studies using the mouse model have shown that severe gastric inflammation was observed in mice that generated a strong Th1 type response, 23 whereas only mild gastritis was observed in mice generating a weak Th1 response. 24 IFN- and TNF- have also been shown to alter the barrier function of cultured epithelial cells 25,26 and thus may contribute to the pathogenesis. Although acute H. pylori infection stimulated cytokine production in both CD4 and CD8 T cells, differences were observed in the phenotype of the cytokineproducing cells. The CD8 T cells were the major producers of IFN- and MIP-1 in both peripheral blood and the gastric mucosa. On the other hand, CD4 T cells were the primary producers of IL-2 and TNF- in both gastric mucosa and peripheral blood. Previous studies 14 have shown that gastric CD8 T cells are the primary producers of IFN- in chronically H. pylori infected subjects. Our results have shown that among the gastric lymphocytes, CD8 T cells were the primary producers of MIP-1 and CD4 T cells were the primary producers of TNF- in H. pylori infected rhesus macaques. The differential cytokine responses of these T cells combined with the altered phenotypic profile of gastric T cells after H. pylori infection may have implications for the pathogenesis of H. pylori. The production of IL-2 by gastric CD4 T cells could contribute to the increased prevalence of

8 314 MATTAPALLIL ET AL. GASTROENTEROLOGY Vol. 118, No. 2 Figure 7. Flow-cytometric analysis of gastric lymphocytes revealed that (A) CD8 T cells were the primary producers of MIP-1 and (B) CD4 T cells were the primary producers of TNF-. The potential of gastric CD4 T cells to produce TNF- and gastric CD8 T cells to produce MIP-1 was determined after short-term in vitro stimulation. Isolated cells were stained for cell surface expression of CD4 or CD8, fixed, permeabilized, and stained for intracellular production of TNF- and MIP-1. The frequency of TNF- and MIP-1 producing lymphocytes is shown as a percentage of gated lymphocytes. P.I., post infection. CD4 T cells in the gastric mucosa. Further, the increased production of TNF- by gastric CD4 T cells may be significant because this, combined with an increased prevalence of CD4 T cells in the gastric mucosa during acute H. pylori infection, could contribute to perpetuation of the inflammatory response. In conclusion, our studies showed that acute H. pylori infection of rhesus macaques leads to an alteration in the phenotypic profile of T cells in the gastric mucosa that is accompanied by a predominantly Th1 type of cytokine response early in infection. The persistence of infection at 12 weeks after inoculation suggests a failure of the Th1-mediated host immune responses to resolve infection. Immunologic abnormalities in acute infection may predispose the host to chronic or persistent H. pylori infection and associated disease. References 1. Dunn BE, Cohen H, Blaser MJ. Helicobacter pylori. Clin Microbiol Rev 1997;10: Dubois A, Tarnawski A, Newell DG, Fiala N, Dabros W, Stachura J, Krivan H, Heman-Ackah LM. Gastric injury and invasion of parietal cells by spiral bacteria in rhesus monkeys. Are gastritis and hyperchlorhydria infectious diseases? Gastroenterology 1991; 100: Dubois A, Fiala N, Heman-Ackah LM, Drazek ES, Tarnawski A, Fishbein WN, Perez-Perez GI, Blaser MJ. Natural gastric infection with Helicobacter pylori in monkeys: a model for spiral bacteria infection in humans. Gastroenterology 1994;106: Dubois A. Animal models of Helicobacter infection. Lab Anim Sci 1998;48: Dubois A, Berg DE, Incecik ET, Fiala N, Heman-Ackah LM, Perez-Perez GI, Blaser MJ. Transient and persistent experimental infection of nonhuman primates with Helicobacter pylori: implications for human disease. Infect Immun 1996;64: Blanchard TG, Czinn SJ, Maurer R, Thomas WD, Soman G, Nedrud JG. Urease-specific monoclonal antibodies prevent Helicobacter felis infection in mice. Infect Immun 1995;63: Blanchard TG, Czinn SJ, Nedrud JG. Host response and vaccine development to Helicobacter pylori infection. Curr Top Microbiol Immunol 1999;241: Corthesy-Theulaz I, Porta N, Glauser M, Saraga E, Vaney AC, Haas R, Kraehenbuhl JP, Blum AL, Michetti P. Oral immunization with Helicobacter pylori urease B subunit as a treatment against Helicobacter infection in mice. Gastroenterology 1995;109: Doidge C, Crust I, Lee A, Buck F, Hazell S, Manne U. Therapeutic immunisation against Helicobacter infection. Lancet 1994;343: Lee CK, Weltzin R, Thomas WD Jr, Kleanthous H, Ermak TH, Soman G, Hill JE, Ackerman SK, Monath TP. Oral immunization with recombinant Helicobacter pylori urease induces secretory IgA antibodies and protects mice from challenge with Helicobacter felis. J Infect Dis 1995;172: Yamaoka Y, Kita M, Kodama T, Sawai N, Kashima K, Imanishi J. Expression of cytokine mrna in gastric mucosa with Helicobacter pylor infection. Scand J Gastroenterol 1995;30: Yamaoka Y, Kita M, Kodama T, Sawai N, Kashima K, Imanishi J. Induction of various cytokines and development of severe mucosal inflammation by caga gene positive Helicobacter pylori strains. Gut 1997;41: Yamaoka Y, Kita M, Kodama T, Sawai N, Tanahashi T, Kashima K,

9 February 2000 IMMUNE RESPONSES IN ACUTE H. PYLORI INFECTION 315 Imanishi J. Chemokines in the gastric mucosa in Helicobacter pylor infection. Gut 1998;42: Bamford KB, Fan X, Crowe SE, Leary JF, Gourley WK, Luthra GK, Brooks EG, Graham DY, Reyes VE, Ernst PB. Lymphocytes in the human gastric mucosa during Helicobacter pylori have a T helper cell 1 phenotype. Gastroenterology 1998;114: Solnick JV, Canfield DR, Yang S, Parsonnet J. Rhesus monkey (Macaca mulatta) model of Helicobacter pylori: noninvasive detection and derivation of specific pathogen-free monkeys. Lab Anim Sci 1999;49: Labigne A, Cussac V, Courcoux P. Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity. J Bacteriol 1991;173: Mattapallil JJ, Smit-McBride Z, McChesney M, Dandekar S. Intestinal intraepithelial lymphocytes are primed for gamma interferon and MIP-1 expression and display antiviral cytotoxic activity despite severe CD4( ) T-cell depletion in primary simian immunodeficiency virus infection. J Virol 1998;72: Mattapallil JJ, Smit-McBride Z, Dailey P, Dandekar S. Activated memory CD4 T cells repopulate the intestine early following antiretroviral therapy of simian immunodeficiency virus infected rhesus macaques but exhibit a decreased potential to produce IL-2. J Virol 1999;73: D Elios MM, Manghetti M, Almerigogna F, Amedei A, Costa F, Burroni D, Baldari CT, Romagnani S, Telford JL, Del Prete G. Different cytokine profile and antigen-specificity repertoire in Helicobacter pylori specific T cell clones from the antrum of chronic gastritis patients with or without peptic ulcer. Eur J Immunol 1997;27: Karttunen R, Karttunen T, Ekre HP, MacDonald TT. Interferon gamma and interleukin 4 secreting cells in the gastric antrum in Helicobacter pylori positive and negative gastritis. Gut 1995;36: D Elios MM, Manghetti M, De Carli M, Costa F, Baldari CT, Burroni D, Telford JL, Romagnani S, Del Prete G. T helper 1 effector cells specific for Helicobacter pylori in the gastric antrum of patients with peptic ulcer disease. J Immunol 1997;158: Hatz RA, Meimarakis G, Bayerdorffer E, Stolte M, Kirchner T, Enders G. Characterization of lymphocytic infiltrates in Helicobacter pylori associated gastritis. Scand J Gastroenterol 1996;31: Mohammadi M, Czinn S, Redline R, Nedrud J. Helicobacterspecific cell-mediated immune responses display a predominant Th1 phenotype and promote a delayed-type hypersensitivity response in the stomachs of mice. J Immunol 1996;156: Mohammadi M, Redline R, Nedrud J, Czinn S. Role of the host in pathogenesis of Helicobacter-associated gastritis: H. felis infection of inbred and congenic mouse strains. Infect Immun 1996;64: Madara JL, Stafford J. Interferon-gamma directly affects barrier function of cultured intestinal epithelial monolayers. J Clin Invest 1989;83: Mullin JM, Snock KV. Effect of tumor necrosis factor on epithelial tight junctions and transepithelial permeability. Cancer Res 1990; 50: Received May 13, Accepted October 25, Address requests for reprints to: Jay V. Solnick, M.D., Ph.D., Department of Medical Microbiology and Immunology, Room 3140, Tupper Hall, School of Medicine, University of California Davis, Davis, California jvsolnick@ucdavis.edu; fax: (530) Supported by grant R01 AI42081 from the National Institutes of Health. The authors thank Lori Hansen, Anne Ketchum, and Elizabeth Reay for their valuable assistance.