Helicobacter pylori, a flagellated, spiral shaped, microaerophilic gram-negative bacillus, is one of the most prevalent human pathogens worldwide. It causes chronic gastritis and may predispose infected individuals to developing gastric ulcers. Although it stimulates specific cellular and humoral immune responses, these responses do not eliminate Hp infection, which can persist for a long time . The exact mechanism by which Hp resists the immune response and causes damage to gastrointestinal tissue remains unclear. One immune response that occurs in gastritis associated with Hp infection and GUP is increased production of pro-inflammatory cytokines, including IL-8  and IL-1 .
Interleukin-8 is the best characterized member of the CXC subfamily (α-chemokine). It acts primarily on polymorphonuclear cells, having potent stimulatory and chemotactic effects on basophils, eosinophils and T cells . On exposure to inflammatory stimuli such as LPS, IL-1 or TNF, a wide variety of cell types, including T lymphocytes, monocytes, macrophages, neutrophils, fibroblasts, endothelial cells and epithelial cells, can produce IL-8 [5, 6]. IL-8 may be involved in various biological processes such as hematopoiesis  and angiogenesis . Furthermore, it reportedly plays an important role in the pathogenesis of various inflammatory diseases including psoriasis, rheumatoid arthritis, pancreatitis, asthma, acute respiratory distress syndrome and sepsis . The amount of IL-8 produced can correlate with severity of pathology and disease outcome . Its production is also increased in Hp-infected patients .
Various cells such as fibroblasts, keratinocytes, synoviocytes, neuronal cells, endothelial cells, immune cells like macrophages and mast cells, and glial cells like Schwann cells, microglia and astrocytes can produce another multifunctional pro-inflammatory cytokine, IL-1β, also known as catabolin . IL-1β plays an important role in inducing tissue damage such as corneal damage  and in regulating inflammation in various diseases such as breast cancer , inflammatory bowel disease , rheumatoid arthritis, osteoarthritis, neuropathic pain, multiple sclerosis, vascular disease and Alzheimer disease .
We therefore aimed to (i) determine and compare serum concentrations of IL-1β and IL-8 in GUP and GP; (ii) determine whether HPCA can activate release of IL-1β and IL-8 from PBMNCs; (iii) determine and compare the concentrations of IL-1β and IL-8 in mononuclear cells supernatants after stimulation with HPCA and PHA; and (iv) examine the cell responsiveness and compare the proliferation of mononuclear cells after stimulation with HPCA or PHA in vitro.
- Top of page
- 1 MATERIALS AND METHODS
- 2 RESULTS
- 3 DISCUSSION
- 4 DISCLOSURE
In this study, serum concentrations of IL-8 and IL-1β of GUP Hp+, GUP Hp−, GP Hp+ and GP Hp− groups were compared. Furthermore, IL-8 and IL-1β produced by PBMNCs of GUP and GP groups following in vitro stimulation with PHA or HPCA were determined. The ELISA method was then used to determine the concentrations of cytokines, and MTT assay to evaluate the proliferation response of PBMNCs following PHA or HPCA exposure.
In this study, it was found that concentrations of IL-8 and IL-1β in serum and PBMNC culture supernatants of GUP were higher than those of GP. In addition, patients infected with Hp produced more of these cytokines than did non-infected patients. Thus, the strongest host inflammatory response was found in the GUP Hp+ group. Hp virulence factors that may be present in complex HPCA, including LPS , CagA and VacA may be responsible for this inflammatory effect in GUP Hp+ . However, the precise role of each virulence factor in this situation is not yet clear and whether gastric ulcers occur only because of the presence of such virulence factors is yet to be determined.
The correlation between Hp virulence factors and host responses such as the production of IL-8 and IL-1β by immune cells is very important. In patients with Hp infection, various bacterial products reportedly directly damage the surfaces of gastric epithelial cells . Furthermore, the concentrations of IL-8 in sera and supernatants of PBMNCs are higher than those of IL-1β, which demonstrates the major role of IL-8 in the inflammatory responses and pathogenesis of Hp infection. In one study, amounts of cytokines, including IL-2, IL-8, IL-4, and TNF-α, were determined by ELISA in homogenates of gastric mucosa from 51 children with abdominal pain . The amounts of all of these cytokines were significantly greater in the patients than in a control group. These findings suggest that cytokines, especially IL-8, play important roles in determining the severity of Hp infection . The same study used immunohistochemistry techniques to examine expression of different cytokines in antral biopsy specimens that had been obtained from 10 Hp infected and 10 uninfected children. In this study, correlations between expression of cytokines and histopathology scores were evaluated . The degree of expression of IL-8 and other cytokines, including IFN-γ, IL-4, transforming growth factor beta and TNF-α was greater in the Hp infected group than in controls . Other studies have also suggested that IL-8 plays an important role in the pathogenesis of Hp infection and that its concentration correlates with the severity and outcome of the disease [10, 22, 23].
In the present study, HPCA induced cell responsiveness, as evidenced by IL-1β and IL-8 production and PBMNC proliferation in vitro. HPCA stimulates production of IL-8 and IL-1β more strongly than does PHA, showing that mononuclear cells are more responsive to HPCA than to PHA. Although HPCA powerfully stimulated production of pro-inflammatory cytokines, it prevented proliferation of PBMNCs following PHA stimulation. This suggests that, in these cells, HPCA uses different signaling pathways for cell proliferation than for cytokine production. However, because PHA mainly induces propagation of lymphocytes, HPCA probably inhibits PHA-driven proliferation of the lymphocyte subpopulation. If so, cells of monocyte/macrophage lineage may be the cellular source of IL-1 and IL-8. Also, PBMNCs from GUP Hp+ showed more proliferation following stimulation with PHA than did PBMNCs from other groups. Because there were higher concentrations of IL-8 and IL-1β in the GUP Hp+ group, this could be attributable to pre-exposure of these cells to inflammatory cytokines in vivo.
T helper cells are a group of lymphocytes that assist activation of other immune cells by releasing cytokines. The two main types of Th cells, Th1 and Th2, are classified based on their cytokine profiles. Th1 cells induce production of IL-2, IFN-γ and TNF-β whereas Th2 cells induce production of IL-4, IL-5, the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokines IL-8, TNF-α, IL-6, IL-1β and IL-12 [24, 25]. It has been shown that some Hp antigens that are known to be involved in the pathogenesis of this infection, such as UreaAB, CagA, VacA and neutrophil-activating protein, can inhibit activation of Th1 cells by inducing production of cytokines like IL-10 and changing the number of Th cells in favor of Th2-type responses [26, 27]. Thus, it seems that Hp infection can suppress Th1 cells and stimulate an inflammatory immune response. This inflammatory immune response can induce damage to gastric tissue.
As described, HPCA inhibited PBMNC proliferation in a dose-dependent manner (Fig. 6), this inhibitory effect being greatest in the GUP Hp+. This finding is compatible with another report showing an inhibitory effect of Hp on lymphocyte proliferation [28, 29]. Studies have shown that this anti-proliferative activity is related to some of the components of HPCA, including CagA , VacA [31-33], arginase  and Hp LPS . In this regard, Chen et al.  showed that Hp produces heat-labile proteins or peptides of approximately 100 kDa weight that suppress T cell mitogen-induced proliferation of lymphoid cells. However, another study has shown that inhibition of PBMNC proliferation may be mediated by the inhibitory effect of Hp on Cdk factor, a cell cycle regulator: Hp can inhibit immune cell proliferation via Cdk without affecting cytokine production . Gerhard et al. have also shown that proliferation of lymphocytes is abolished when they are co-incubated with different Hp strains or with protein extracts of culture supernatants. It seems that this inhibition is due to a protein or protein complex (30–60 kDa) that is independent of virulence factors like VacA . These authors suggested that an arrest in the G1 phase of the cell cycle is the major cause of inhibition of proliferation; thus cytokine production is not affected nor significant apoptosis induced . Therefore, IL-8 and IL-1β up-regulation may be the result of increased numbers of mononuclear cells or enhancement of cellular cytokine response.
In conclusion, our study demonstrated that tissue damages and ulcers occur in the patients who produce more IL-8 and IL-1β than others and consequently have more numerous activated immune cells at the site of infection. Furthermore, components of HPCA can inhibit PBMNCs proliferation in a dose-dependent and reversible manner. Although HPCA can inhibit proliferation of PBMNCs, it does not affect production of IL-1β and IL-8.