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- MATERIALS AND METHODS
H. pylori elicits specific humoral and cellular immune responses in the mucosal immune system. However, the type and extent of T lymphocyte response in the systemic immune system is not clear for H. pylori positive patients. In this study, peripheral blood T lymphocyte phenotypes and serum Th1/Th2 based cytokines of 32 H. pylori positive patients were analyzed and compared to those of healthy controls. While αβ TCR+ lymphocytes and their phenotype analysis were not significantly different to those of healthy controls, the percentage of pan γδ TCR+ lymphocytes was up to 2.4 times greater in the H. pylori positive group then in healthy controls. Furthermore, significant increases in IL-10 concentrations in serum samples of H. pylori patients indicated that their immune systems had switched toward a Th2 type immune response. The correlation between phenotype and type of T cell response in the peripheral blood during H. pylori infection is discussed.
H. pylori is a gram negative, microaerophilic and motile bacterium that causes persistent infection in the human gastrointestinal tract, especially the stomach, starting at an early age (1). It induces persistent infection in gastric mucosa, resulting not only in chronic gastritis which is asymptomatic in most individuals; but also in an association with pathologic development of gastric and duodenal ulcers. More seriously, it has also been associated with gastric adenocarcinoma, mucosa associated lymphoid tissue lymphoma and primary gastric non-Hodgkin's lymphoma (2, 3).
H. pylori infection induces vigorous humoral and cellular immune responses. Neutrophils, mast cells, eosinophils and B- and T- lymphocytes are all involved in that inflammatory process (4). Infection mediated chemokines and pro-inflammatory cytokines secreted by gastric epithelial cells stimulate the migration of granulocytes, monocytes and lymphocytes into the inflamed mucosa which results in more severe inflammatory pathology (5–7). In the case of the humoral immune response, infiltrating B lymphocytes and plasma cells give rise to H. pylori specific IgA and IgG antibodies. CD4+ T cells both help B lymphocytes to produce antibody, and induce inflammatory processes in the gastric mucosa by producing high amount of IFN-γ (8, 9). It has been shown that increased numbers of activated CD8+ T cells also accumulate in the gastric mucosa. The participation of mucosal CD8+ T cells in the inflammatory process may lead to development of more severe disease outcomes as described above (10, 11).
Although H. pylori specific IgG or IgA antibodies can be detected in the peripheral blood in the early stages of infection, it is a matter of debate as to whether circulating lymphocytes are increased and activated by H. pylori infection. On the one hand, some researchers have reported that peripheral blood lymphocytes of infected individuals show neither proliferation nor activation signals, and that peripheral blood lymphocytes isolated from patients with duodenal ulcer and chronic antral gastritis show no significant alteration in systemic immune system in response to H. pylori (12, 13). On the other hand, other groups have reported that stimulation of peripheral blood lymphocytes with distinct H. pylori antigenic fragments induces not only proliferation but also activation of CD4+ and CD8+ T lymphocytes (14–16).
Another debate concerns the type of T cell response both in the gastric mucosa and the periphery. Some groups have shown that the T cell response to H. pylori is dominated by a Th1 type T cell response in the gastric mucosa. The consequent release of IFN-γ by Th1 cells induces expression of pro-inflammatory cytokines TNF-α, IL-12 and IL-18 by T lymphocytes and granulocytes in the gastric mucosa (17, 18). However, a regulatory T cell response which dominates secretion of IL-4, IL-10 (19) and TGF-β (20) to down-regulate the inflammatory response and reduce bacterial load has also been reported. Despite the wide range of T cell responses in gastric mucosa, their impact on the systemic immune system is not known.
In view of the role of H. pylori in the pathogenesis of persistent bacterial infection, it is of interest to study the effect of H. pylori infection on the phenotypic distribution of lymphocytes, as well as on the pattern of cytokines involved both in inflammatory and anti-inflammatory T cell responses. Hence, it is reported here that H. pylori infection leads to expansion of pan gamma delta TCR positive T cells groups in the peripheral blood circulation, and that cytokine concentrations display a shift toward a Th2 type immune response as evidenced by high concentrations of IL-10 in the peripheral blood.
- Top of page
- MATERIALS AND METHODS
In the present study the phenotypic distribution of peripheral blood lymphocytes, and the balance between Th1-Th2 T cell responses in the systemic immune system of H. pylori infected individuals, was investigated. Our data demonstrates a significant expansion of granulocytes, gamma delta TCR+ T lymphocytes and high concentrations of IL-10 in the peripheral blood of Hp (+) subjects in comparison to healthy controls. No differences were detected between Hp (+) and Hp (−) subjects in regard to the frequencies of CD3+, CD19+, CD4+, CD8+ T cells and their subsets. Neither pro-inflammatory nor inflammatory cytokines were increased in Hp (+) patients. Accordingly, our data address three main points about the systemic immune response during H. pylori infection.
Firstly, many studies have reported that H. pylori infection results in massive infiltration of mononuclear cells into the gastric mucosa. However, the pathogen is rarely eliminated and, in the absence of treatment, infection persists for life. That is because, during persistent infection, the pathogen may induce either inflammatory or regulatory cellular immune responses in the gastric mucosa (2, 3). In order to determine the impact of that local immune response on mononuclear cells in the systemic circulation, increase in peripheral blood mononuclear cells, T-cells and their sub-groups and the expression status of activation markers have been investigated by several groups. Kondo et al. have analyzed peripheral blood mononuclear cell counts; specifically neutrophil and monocyte counts, in the peripheral blood of Hp (+) patients. They observed an increase in neutrophil and monocyte counts and therefore concluded that H. pylori infection plays a role in systemic disorders (23). Besides that, Karttunen et al. observed a significant increase in peripheral blood basophiles in H. pylori patients (24). According to our data H. pylori infection induces a significant expansion in the granulocyte population, however we do not know which type of granulocytes are involved nor at which phase of the disease their proportion increases.
With respect to the analysis of peripheral blood T cells; Yuceyar et al have investigated the expansion of peripheral blood lymphocytes and the expression of activation markers in H. pylori associated duodenal ulcer and chronic antral gastritis. They observed neither expansion nor activation of T- and B- lymphocytes and NK cells in peripheral blood samples from Hp(+) subjects (12). Conversely, Soares et al. have investigated co-stimulatory marker CD28 expression on CD4+ and CD8+ T cells together with activation marker HLA-DR. They observed an increase in the number of CD3+ and CD4+ cells, and significant expression of co-stimulatory marker CD28 over CD4+ and CD8+ T cells in the Hp (+) group. Moreover, they did not detect an increase in the expression of HLA-DR on T cells, in agreement with other studies and the present data (25). With respect to the effector function of T cells in the systemic circulation, while some studies have reported a proliferative and effective T cell response after in vitro stimulation with or without H. pylori antigen/s; some have observed no significant activation or co-stimulation response of peripheral blood lymphocyte subgroups (26, 27). Karttunen et al. detected a proliferative response of mononuclear cells after stimulation with intact H. pylori. However those responses do not correlate with infection status, since in both antibody positive (H. pylori infected) and antibody negative (non-infected subjects) samples showed a similar degree of T cell proliferative response. In another study Chmiela et al. also demonstrated that T lymphocytes from healthy volunteers show a significant proliferative response after stimulation with H. pylori and distinct antigenic fragments. According to our data, H. pylori does not induce a significant expansion of B-, T- lymphocytes (carrying αβ TCR) and their subgroups.
Another conflict concerns the expansion status of γδ TCR+ T cell populations in the gastric mucosa during H. pylori infection. Trejdosiewicz et al. observed no significant change from normal in percentages of γδ TCR+ in H. pylori associated gastritis (28). On the other hand, Futagami et al. detected accumulation of γδ TCR+ cells in gastric mucosa and that accumulation corresponded to an increase in IL-7 and IL-1beta in gastric mucosa (29). The present study is the first report of expansion of γδ TCR+ T-cells in the peripheral blood of Hp (+) patients. We think that expansion of γδ T cells may be sourced by extraordinary expansion in the gastric mucosa. Nevertheless, we do not know if our expanded clone originates from gastric mucosa or peripheral blood, since our γδ TCR+ specific antibody's specificity covers pan-γδ TCR+ T cells clones.
It has been suggested that γδ TCR+ cells perform an autologous surveillance role, removing damaged cells by recognition of stress or heat shock proteins. Thus it is predictable that there should be an increase in γδ TCR+ cells as part of an inflammatory response. Additionally, both mucosal and peripheral blood γδ TCR+ T cells differentiate into either Th1 or Th2 cells depending on the microenvironment (30, 31), which means γδ T cells can function as inflammatory or anti-inflammatory T cells. Given that H. pylori can induce either inflammatory or regulatory T cell responses, the expansion of that T cell population appears to be meaningful since an increased γδ T cell frequency could explain the significant elevation of IL-10 in our study. On the other hand, no significant correlation between IL-10 levels and γδ TCR+ T cell frequencies (r = 0.027, p = 0.888) was found in the present study.
Thirdly, during H. pylori infection an anti-inflammatory T cell response in the systemic circulation was observed by us. That kind of T cell response could be initiated by one of the regulatory phases of persistent infection. Lundin et al. have demonstrated that high production of the suppressive cytokine IL-10 in H. pylori infected gastric adenocarcinoma patients leads to a diminished cytotoxic anti-tumor T cell response in the stomach (32). That phenomenon may contribute to tumor progression in gastric adenocarcinoma patients. The role of peripheral blood originated γδ T cells on that progression should be investigated in future studies. Another view is that an anti-inflammatory T cell response can be associated with a regulatory function of T-lymphocytes during persistent infection (33). H. pylori makes use of some features of the host immune response to its benefit. However, Lundgren et al. have shown that it also induces expansion of CD4+ CD25highFoxp3+ regulatory T cells in H. pylori infected gastric mucosa, which weakens host lymphocyte proliferation and anti-inflammatory responses (34). Concerning that, a significant increase in IL-10 levels of Hp (+) patients may be the result of regulatory T-lymphocytes.
In conclusion, it has been shown that a regulatory T cell response is dominant in the systemic immune system during H. pylori infection, and that response is linked to the existence of γδ TCR+ T cells and high level of IL-10 in Hp (+) patients. These results could have important consequences both for understanding the role of systemic immune response during H. pylori infection and for protection against infection.