The immune response against Helicobacter pylori--a direct linkage to the development of gastroduodenal disease.

Helicobacter pylori infects about half of the world's population. H. pylori elicits marked immune responses, but the infection is commonly life-long. Some infected individuals remain asymptomatic, while others develop significant gastroduodenal disease. We review the underlying host immune response to H. pylori which programs for persistence and evolution of gastroduodenal disease.

Preventive and therapeutic vaccines against Helicobacter pylori: current status and future challenges.

Approximately 50% of humanity is infected with Helicobacter pylori. Normally, this is a life-long infection indicating that the host response to natural infection fails to yield protective immunity. Moreover, the chronic inflammatory response associated with this infection can contribute to tissue damage and the pathogenesis of gastroduodenal disease including atrophic gastritis, peptic ulcer and gastric cancer. These damaging immune responses are attributed to a subset of helper T cells, so-called Th1 cells, that enhance cell-mediated immunity and induce damage to the gastric epithelium. Thus, it is desirable to have effective vaccines that could prevent and cure infection or at least, modify the host response in a manner that prevents immune-mediated disease. Using animal models as a tool to understand the immunobiology of Helicobacter infections, several investigators have shown that effective vaccines can be developed. Thus, prophylactic and even therapeutic vaccines have been described in various animal models. The basis for the effectiveness of these vaccines seems to be found in their ability to alter the gastric immune response, perhaps away from a homogeneous Th1 response towards a mixed Th1 and Th2 response. Using these encouraging approaches, vaccines are being developed for use in humans for the treatment and prevention of H. pylori infection and the gastroduodenal diseases associated with this infection.

Intrathymic changes in murine CD3, CD5 and CD8 expression after in vivo administration of anti-CD4.

Intrathymic development of murine cortical and medullary thymocyte subpopulations was examined after in vivo administration of anti-CD4 mAb. Four days after mice received 1 mg anti-CD4, expression of CD4 was reduced to about 25% the levels of controls. On cortical CD8+/PNAhigh thymocytes, CD5 and CD8 expression both decreased, but not in parallel, whereas CD3 expression increased almost 2-fold. Partial shifts in CD3 expression were seen 3 and 6 h after injection, but modulation of CD4 preceded the increase in CD3 expression. On medullary CD8-/PNAlow thymocytes, both CD3 and CD5 were down regulated. On nontargeted CD8+/PNAlow medullary thymocytes, expression of these molecules was decreased less than 20%, but the decrease in CD8 expression was primarily due to the appearance of a CD8low subpopulation. The results indicate that anti-CD4 mAb differentially affects the intrathymic development of T cell populations.

Lymphocyte compartments in antigen-sampling regions of rabbit mucosal lymphoid organs.

Uptake and delivery of antigens to immunocompetent cells in the gut are critical factors for the development of oral vaccines. Particulate antigens are transported within minutes by M cells to intraepithelial lymphocytes and into the follicle dome. The dome contains B cells, CD4+ T cells, and macrophages, indicating that the cells involved in antigen presentation are located below the dome's epithelium. The high number of M cells in rabbits and the development of monoclonal antibodies against rabbit lymphocytes have enabled the detailed study of lymphocytes associated with M cells. The follicle epithelium of rabbit Peyer's patches contains B cells and a population of CD4-/CD8-, major histocompatibility complex class II+ mononuclear cells of unknown function. These cells are phenotypically distinct from T cells in follicle domes, in T cell-dependent areas, in villus epithelium, or in villus lamina propria. In addition, lymphocytes in M-cell pockets express an activation antigen (3B6) not found on CD4+ or CD8+ cells in T cell-dependent areas. These results indicate that M-cell pocket lymphocytes in follicle epithelium form a phenotypically distinct compartment situated at the interface between M-cell-driven antigen uptake and the mucosal immune system.

Generation and characterization of monoclonal antibodies recognizing follicle epithelial M cells in rabbit gut-associated lymphoid tissues.

A panel of mouse B cell hybridomas producing monoclonal antibodies (mAb) directed against rabbit M cell-containing epithelia was developed. By immunohistochemistry, the mAb 5D9, 5B11, 1D9, and 4G2 were found to label approximately 50% of the follicle-associated epithelial (FAE) cell populations overlying lymphoid follicles in Peyer's patches, cecal patch, sacculus rotundus, and appendix. The cell staining was localized to FAE cell basolateral surfaces outlining the M cell pockets which enclosed clusters of mononuclear leukocytes, and extended from the crypts of Peyer's patches and sacculus rotundus, and appendiceal crevices, to the apices of domes. In contrast, the stem cell and proliferative regions facing the lamina propria were devoid of immunologically reactive sites. The mAb 5D9, 1D9, and 4G2 did not recognize antigens associated with non-FAE cells in the intestinal lymphoid tissues examined. Only the mAb 5B11 labeled apical surfaces of Peyer's patch and cecal patch non-FAE. However, this mAb did not label interdomal colonic epithelial cells in sacculus rotundus and appendix. Besides recognizing FAE cells, the mAb 4G2 recognized a cross-reactive antigen displayed by dome and lymphoid follicle lymphocytes. By flow cytometry, the mAb 5D9, 5B11, and 1D9 were shown to stain from 14 to 29% of the cells in M cell-enriched populations prepared from Peyer's patches, sacculus rotundus, and appendix, whereas mAb 4G2 was found to recognize 44-54% of the cells. Two-color flow cytometric analysis showed that the mAb stained a functionally distinct subpopulation of Peyer's patch phagocytic cells and did not recognize spleen macrophages. These findings indicate that the panel of mAb recognized novel antigens expressed by FAE cells overlying intestinal lymphoid aggregates, and that the mAb allow identification of phagocytic M cells in suspensions of FAE cells.

Use of two-colour flow cytometry to assess killing of Giardia muris trophozoites by antibody and complement.

A two-colour flow cytometry technique was used to assess killing of Giardia muris trophozoites by rabbit anti-trophozoite antibodies and complement. Binding of rabbit antibody to trophozoites and killing of trophozoites were documented by flow cytometry, after incubating the organisms with fluorescein-conjugated anti-rabbit IgG and propidium iodide (PI). Percentages of PI+ (dead) trophozoites ranged from greater than 80%, after incubation with rabbit antiserum and complement, to less than 30%, after incubation with complement alone. The assay technique may be applicable to studies aimed at determining whether intestinal antibodies from Giardia-infected mammals can kill Giardia trophozoites.

Absence of secretory component expression by epithelial cells overlying rabbit gut-associated lymphoid tissue.

The expression of secretory component by epithelial cells overlying intestinal lymphoid aggregates was examined immunocytochemically in rabbits. Intensely labeled epithelial cells were distributed along surfaces of villi surrounding follicles in jejunal and ileal Peyer's patches and along interdomal epithelium in sacculus rotundus and appendix. Secretory component labeling extended from within crypts and appendiceal crevices to the tips of villi and interdomal regions. In contrast, no immunologically detectable secretory component sites were observed in follicle-associated epithelial cells. In crypts and crevices supplying follicles, epithelial cells facing the lamina propria of villi and interdomal epithelium expressed secretory component, but cells flanking the follicle domes lacked secretory component immunostaining, with a clear demarcation between positive and negative zones at the base of the stem cell regions. These findings demonstrate a unique difference in the expression of the receptor for immunoglobulin A antibody between follicle-associated and non-follicle-associated epithelium.

Recognition of a 30,000 MW antigen of Giardia muris trophozoites by intestinal IgA from Giardia-infected mice.

The principal aims of this work were (i) to identify the molecular weight (MW) of Giardia muris trophozoite antigens that are recognized by IgA in small intestinal secretions from G. muris-infected mice, and (ii) to determine whether mouse intestinal Giardia-specific IgA is directed against trophozoite surfaces. BALB/c mice were infected with G. muris cysts, and intestinal secretions were harvested from these mice at various times after the start of Giardia infection, and from uninfected mice. Flow cytometry showed that intestinal IgA from G. muris-infected mice, but not from uninfected mice, became bound to trophozoite surfaces in vitro. Western blotting of trophozoite proteins with mouse intestinal secretions showed that IgA from Giardia-infected mice reacted specifically with a broad protein band of approximately 30,000 MW. This finding suggests that one or more trophozoite proteins of approximately 30,000 MW are targets for intestinal antibody in mice infected with G. muris.

Uptake and translocation of fluorescent latex particles by rabbit Peyer's patch follicle epithelium: a quantitative model for M cell uptake.

A quantitative, light microscopic morphometric model for uptake of particulates by Peyer's patch M cells was developed. Rabbit intestinal loops containing Peyer's patches were inoculated with fluorescent, non-degradable polystyrene microparticles (600-750 nm), and their localization in Peyer's patches was traced after varying time periods. The particles were localized sequentially at the FAE cell surface, spanning the entire width of FAE cells, and within the subepithelial dome as a function of time. The particles were associated with 5D9+ or 1D9+ M cells, but were not taken up or transported by villus epithelia. The kinetics suggested a synchronous wave of uptake and transepithelial transport. Quantitative analysis revealed a considerably greater uptake efficiency of polystyrene microspheres in comparison to other biological particles.

T-cell-mediated mucosal immunity in the absence of antibody: lessons from Helicobacter pylori infection.

Approximately 50% of humanity is infected with Helicobacter pylori. This lifelong infection elicits a marked host response, including a robust gastric IgA response. However, natural infection fails to yield protective immunity. Rather than providing protection, the chronic inflammatory response associated with natural infection can contribute to tissue damage and the pathogenesis of gastroduodenal disease, including atrophic gastritis, peptic ulcer, and gastric cancer. These immune responses are attributed to a subset of helper T cells, so-called Th1 cells, that enhance cell-mediated immunity and induce damage to the gastric epithelium. Thus, it is desirable to have effective vaccines that could prevent and cure infection and that may modify the host response in a manner that prevents immune-mediated disease. Using animal models as a tool to understand the immunobiology of Helicobacter infections, several investigators have shown that effective vaccines can be developed. Thus, prophylactic and even therapeutic vaccines have been described in various animal models. The basis for the effectiveness of these vaccines appears related to their ability to alter the gastric immune response, from a homogeneous Th1 response to a mixed Th1 and Th2 response. Interestingly, immunity can occur in the absence of B cells, suggesting that novel IgA-independent mechanisms exist that confer protection against a luminal infection. Thus, H. pylori infection provides a model with which new mechanisms of immunological protection can be identified and applied to other mucosal infections.

Follicle epithelial M cells are a source of interleukin-1 in Peyer's patches.

The production of interleukin-1 (IL-1) by rabbit Peyer's patch M cells populating the follicle-associated epithelium (FAE) was studied. Sorted 5D9+ phagocytic epithelial M cells synthesized IL-1 after stimulation with lipopolysaccharide (LPS) in vitro, as evidenced by the ability of culture supernatants to induce the proliferation of the T-cell line D10.G4.1. Fixed LPS-stimulated M cells were less effective at mediating T-cell proliferation than supernatants from LPS-activated M cells. The magnitude of T-cell proliferation was M-cell concentration dependent, and proportional to the dose of LPS. The M-cell-mediated D10.G4.1 cell proliferation was inhibited > 75% with anti-IL-1 alpha, but < 50% with similar concentrations of anti-IL-1 beta. The results show that M cells secrete IL-1, and suggest the participation of M cells in the delivery of a localized co-stimulatory signal for T-cell and B-cell proliferation in the microenvironment of gut-associated lymphoid tissue (GALT).

Generation and characterization of monoclonal antibodies against Giardia muris trophozoites.

Mouse monoclonal antibodies (mAb) were produced against Giardia muris trophozoite surface antigens. To generate B-cell hybridomas, P3/NS1/1-Ag4-1 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized parenterally with G. muris trophozoites. Hybridoma culture supernatants were screened for mAb by flow cytometry of G. muris trophozoites incubated with culture supernatant followed by fluorescein-conjugated anti-mouse IgG and IgM. Flow cytometry showed three types of trophozoite staining by mAb: (i) bright staining of greater than 90% of trophozoites, with aggregation of the organisms; (ii) bright staining of approximately 90% of trophozoites, with little or no aggregation; (iii) dull staining of approximately 20% of trophozoites, without aggregation. Western blotting of mAb on G. muris trophozoite antigens separated by polyacrylamide gel electrophoresis showed that a mAb exhibiting the third of these flow cytometry staining patterns recognized trophozoite antigens of MW approximately 31,000 and 35,000. Immunoprecipitation studies indicated that the same mAb specifically precipitated two 125I-labelled trophozoite surface antigens of MW approximately 30,000. Monoclonal antibodies generated in this study may facilitate the purification and biochemical characterization of trophozoite antigens that are targets for protective intestinal antibody in G. muris-infected mice.

Secretory immune responses in ageing rats. II. Phenotype distribution of lymphocytes in secretory and lymphoid tissues.

The distribution of lymphocyte phenotypes was examined in various tissues from weanling (21-35 days), adult (3-4 months), mid-life (10-12 months) and senescent (18-20 months) rats. Lymphoid tissues included peripheral blood, spleen, cervical, mesenteric and inguinal lymph nodes. Tissues associated with secretory immune responses were also examined, including submandibular and parotid salivary glands, extraorbital lacrimal glands and Peyer's patches. IgA, IgG and IgM B cells were determined by surface Ig staining. Total T cells (W3/13), T helper/inducer (Th) (W3/25), T suppressor/cytotoxic (Ts) (OX8) and immature T cells (Thy 1.1; OX7) were also evaluated. IgG B cells were significantly decreased in lymphoid tissues from the senescent rats, while the weanling group exhibited decreased levels of all three B-cell isotypes compared to adult animals. IgA B cells were significantly decreased in the secretory tissues of the senescent rats, while IgM B cells were increased in both the weanling and senescent groups. Total T-cell percentages were unaffected by ageing in any of the tissues. The only consistent alteration in the lymphoid tissues was a decrease in Thy 1.1-positive cells in the older groups compared to the weanling group. A decreased Th cell percentage was demonstrated in the salivary and lacrimal glands of the weanling and senescent groups. Decreases in Th/Ts ratios, as well as decreased numbers of plasma cell precursors in the secretory tissues of the aged rats, suggests that alterations in normal secretory immune responses may be expected to accompany the ageing process.

Phenotype of mononuclear leucocytes resident in rat major salivary and lacrimal glands.

The phenotypic distribution of lymphocytes and mononuclear phagocytes resident in rat secretory glands was examined. Isolated exocrine gland mononuclear leucocyte populations contained 50-61% W3/13+ T cells and greater proportions of W3/25+ T helper cells relative to OX8+ T suppressor cells. Surface Ig+ cells (sIg) constituted from 32% to 34% of the cells and their distribution was sIgM greater than sIgA greater than sIgG. The macrophage populations comprised from 0.02% to 0.1% of the unfractionated gland cells. Fractionated secretory gland-adherent cells consisted primarily of non-specific esterase+, phagocytic and Fc receptor-bearing cells. From 35% to 79% of the macrophages in exocrine glands expressed I-A molecules. The results suggest that exocrine glands have the ability to respond locally to an antigenic challenge independently of a central mucosal immune response.

Differential adherence of epithelium overlying gut-associated lymphoid tissue. An ultrastructural study.

The relative adherence of follicle associated and nonfollicle associated epithelial cells in intestinal lymphoid tissues was compared morphologically. Incubation of Peyer's patches and appendix with hyaluronidase resulted in detachment of M cells and other epithelial cells overlying lymphoid follicle surfaces but not of villus or colonic epithelial cells. Enzymatic treatment of intestinal lymphoid tissues produced superficial erosions of follicle epithelium which exposed a porous and fenestrated basal lamina. After enzymatic treatment, detached M cell-containing follicle epithelium was characterized by intracellular vacuoles, widening of intercellular spaces, cell rounding, disappearance of microvilli, and loss of M cell-lymphocyte associations. Enzymatic treatment was responsible for removal of follicle epithelium, since Peyer's patches and appendix tissues incubated with medium alone did not exhibit appreciable epithelial detachment. These results, showing that the adherence of epithelial cells overlying intestinal lymphoid follicles is more labile than that of villus and colonic epithelial cells, may be of pathophysiologic significance in follicle ulcerative processes.

Distribution of Langerhans cells in normal and carcinogen-treated mucosa of buccal pouches of hamsters.

The distribution of Langerhans cells in normal and carcinogen-treated mucosa of buccal pouches of hamsters was studied. Decrease in density and in focal aggregates of Langerhans cells and loss of their complex dendritic networks were found in carcinogen-treated mucosa.

Resident salivary gland macrophages function as accessory cells in antigen-dependent T-cell proliferation.

The function of salivary gland macrophages in the induction of local immunity in secretory organs was investigated in Fischer 344 rats. Macrophages obtained from dispersed submandibular gland (SMG) cells were characterized and examined for their ability to present antigen to T cells. Populations of SMG-adherent cells contained approximately 80% macrophages, of which 46-62% were I-A+ cells. These numbers were from five to 10-fold greater than the I-A+ cells in macrophage populations from peritoneal exudates (5-11%). SMG macrophages functioned effectively as antigen-presenting cells. Antigen presentation was antigen specific, macrophage dose dependent and inhibitable by monoclonal anti-I-A antibodies. These studies suggest that a functional salivary-gland immune-response pathway exists that can function independently of a gut-associated lymphocyte-homing mechanism.

Phenotypically distinct subpopulations of T cells in domes and M-cell pockets of rabbit gut-associated lymphoid tissues.

Follicle epithelium and domes of gut-associated lymphoid tissue (GALT) contain populations of lymphocytes which first contact antigen taken up from the intestine. In order to study the association of lymphocytes with M cells in follicle epithelium, monoclonal antibodies (mAb) were generated by immunizing BALB/c mice with lymphocytes populating GALT domes from NZW rabbits, and their specificity was assessed by immunohistochemistry and flow cytometry. mAb 3C10 (IgM) and 3B6 (IgG3) recognized subpopulations of intraepithelial lymphocytes associated with M cells. mAb 3C10 also identified macrophage-lymphocyte clusters in domes and tangible body macrophages in germinal centres of GALT but did not react with cells in T-dependent areas (TDA) or B cells in follicles. mAb 3B6 recognized lymphocytes in domes and B cells in follicles but not T cells in TDA of GALT. The distribution of 3B6+ cells overlapped with, but was more restricted than, that of Ia+ cells. Analysis of lymphocytes in follicle epithelium showed that greater than 95% of lymphocytes associated with M cells were Ia+. T cells represented approximately 95% of intraepithelial lymphocytes in the appendix and approximately 65% in Peyer's patches. A majority of intraepithelial lymphocytes was recognized by mAb 3B6, but mAb 3C10 identified only approximately 30%. Because neither 3C10 nor 3B6 recognized lymphocytes in TDA of GALT, these results indicate that most lymphocytes associated with M cells are a distinct phenotype of Ia+ T cells.