Although Helicobacter pylori (Hp) plays an important role in the pathogenesis of chronic gastritis and gastric ulcer, little is known about the probable mechanisms of these types of gastrointestinal damage. To determine the precise mechanisms involved in ulcer formation, immune responses in patients with gastric ulcer (GUP) caused by Hp infection (Hp+) were compared with those of other gastritis patients (GP). The sensitivity and proliferation of peripheral blood mononuclear cells (PBMNCs) obtained from patients were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against exposure with complex Hp crude antigen (HPCA) and mitogen (phytohemagglutinin, PHA). Production of inflammatory cytokines, including interleukin (IL)-1β and IL-8, in serum and supernatants of PBMNCs were then measured by ELISA. It was found that, after stimulation with PHA, both IL-8 and IL-1β concentrations in sera and supernatants as well as proliferation and sensitivity were statistically greater in GUP Hp+ than GP Hp−. Furthermore, HPCA inhibited the proliferation of PBMNCs dose-dependently; however, it stimulated IL-8 and IL-1β production in supernatants of mononuclear cells. Therefore, the up-regulated concentrations of IL-8 and IL-1β may have been caused by increase in the size of mononuclear cell subpopulations or in their cytokine secretory activity, indicating the greatest cell responsiveness in GUP Hp+ patients. These results suggest that tissue damage and ulcers occur in patients who produce more IL-8 and IL-1β than patients who do not develop ulcers; the former consequently have more activated immune cells at the site of infection. Therefore, both host responses and Hp virulence factors may be involved in the development of gastric ulcers.
cytotoxin-associated gene A
cycline dependent kinase
- GP Hp+
gastritis patients with Helicobacter pylori infection
- GP Hp−
gastritis patients without Helicobacter pylori infection
gastric ulcer patients
- GUP Hp+
gastric ulcer patients with Helicobacter pylori infection
- GUP Hp−
gastritic ulcer patients without Helicobacter pylori infection; Helicobacter pylori
Helicobacter pylori crude antigen
inflammatory bowel disease
peripheral blood mononuclear cell
tumor necrosis factor
vacuolating cytotoxin A
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.
1 MATERIALS AND METHODS
RPMI 1640 media was purchased from Biosera (Gentaur, Austria), Lymphoprep from Axis-Shield (Oslo, Norway), MTT from Sigma (St Louis, MO, USA) and human IL-1β and IL-8 ELISA kits from U-CyTech Biosciences (Utrecht, the Netherlands). All other reagents were from Merck (Darmstadt, Germany).
1.2 Clinical samples
This uncontrolled study included 90 adults of Iranian origin (38 with gastritis caused by Hp infection, 14 with gastric ulcer caused by Hp infection, 28 with gastritis not caused by Hp infection and 10 with gastric ulcer not caused by Hp infection). All had been referred for endoscopy because of upper gastrointestinal symptoms (mostly recurrent abdominal pain) suggestive of organic disease and severe enough to require endoscopic evaluation. Endoscopic evaluation was carried out in the Motahari Clinic of Shiraz University of Medical Sciences. Patients with Hp infection were identified by gold standard methods such as the rapid urease test, bacterial culture and presence of Helicobacter-like organisms in biopsy samples obtained from them during endoscopy.
The average age of patients was 41 ± 14 years (range, 18–80 years). Exclusion criteria included treatment with antimicrobial, anti-inflammatory (nonsteroidal anti-inflammatory drugs and corticosteroids) or immunosuppressive medication during the 3 months preceding the endoscopy. Because endoscopy procedure is an invasive means of confirming Hp infection and identifying types of gastrointestinal diseases, healthy patients could not be used as controls.
1.3 Preparation of complex Helicobacter pylori crude antigen
Several strains of Hp carrying different virulence factors (CagA, VacA and UreAB) obtained during previous studies of Iranian patients [15, 16] were used to prepare crude antigen. In brief, the bacteria were grown on Skirrow medium containing blood agar with the addition of vancomycin (10 µg/mL), polymixin B (0.25 µg/mL) and trimethoprim (5 µg/mL), after which the plates were incubated at 37°C under microaerophilic conditions (7% O2, 7.1% CO2, 7.1% H2, 79.8% N2) provided by Anoxomate (Mark II, Mart Microbiology BV, Drachten, the Netherlands) for up to 5 days. The organisms were identified as Hp using gold standard methods. Pure Hp colonies were then separated and re-cultured on enriched Columbia agar (48–72 hrs, 37°C, micro anaerobic conditions). The newly grown colonies were suspended in distilled water and disrupted by sonication for 5 mins on ice. The cell debris and unbroken cells were precipitated by centrifugation at 8000 g for 10 min at 4°C. The supernatant was aliquoted and stored at −70°C after determining the protein concentration by measuring OD at 280 nm.
1.4 Peripheral blood mononuclear cell isolation and cell culture
Five milliliters heparinized peripheral venous blood from each patient was added to a sterile tube containing 5 mL Ficoll (Lymphoprep) and centrifuged at 2500 g for 15 mins at 4°C. PBMNCs were then separated with a Pasteur pipette (dropper), washed twice with RPMI 1640 containing heat-inactivated FBS (10% V/V), L-glutamine (200 mM), penicillin (100 µg/mL), and streptomycin (100 µg/mL) and then re-suspended in CM10 medium (RPMI + 10% FBS). PBMNCs were separated and adjusted to 1.5 × 106 to 2 × 106 cells/mL CM10 medium. HPCA and PHA were added to the mononuclear cell culture medium to determine their ability to stimulate cytokine production.
Peripheral blood mononuclear cells (100 µL cell suspension/well; total number, 3 × 105 to 4 × 105 cells/well) were cultured in 96-well plates, 10 µL PHA (5 µg/well) or HPCA (10 µg/well) being added to three of the wells and 90 µL CM10 to each well for a final volume of 200 µL. One hundred µL CM10 was added to the wells containing only PBMNCs. Two types of wells were used: flat-shaped wells for determining cell proliferation and U-shaped wells for determining cytokine concentrations in the supernatant of the PBMNCs.
1.5 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for evaluation of cell proliferation
Cells were seeded in culture medium at 37°C in a 95% air and 5% CO2 humidified incubator. After 24 hrs culture, 20 µL MTT (final concentration 5 µg/well) was added to each well and the plates incubated for 2 hrs. After formazan crystals had formed, 150 µL of culture medium supernatant was removed from the wells without disrupting the formazan precipitate and 150 µL detergent reagent (DMSO) added, the wells were then left at room temperature in darkness for 2 hrs on a shaker. Absorbance was measured at 570 nm using a micro plate spectrophotometer (in flat-shaped wells).
1.6 Cytokine assay
Concentrations of IL-1β and IL-8 were assayed by the U-Cytech diagnostic technique as follows. For assays of cytokines in serum, 5 mL venous blood without an anticoagulant agent was obtained from each patient, separated and then frozen at −70°C until determination of cytokine content. For assays of cytokines in supernatants of PBMNCs, 24 hrs after the initiation of mononuclear cells culture with HPCA or PHA, 150 µL of supernatants from six plate samples were pooled and frozen at −70°C until required. Cytokine content was determined using commercial ELISA kits for IL-1β and IL-8 according to the manufacturer's instructions. Samples were thawed only once for analysis of each cytokine. Standard curves were constructed according to the manufacturer's instructions. Minimum detectable cytokine concentrations were estimated to be 2 pg/mL for both IL-1β and IL-8.
1.7 Statistical analysis
Data are presented as mean ± SEM. The significance among the groups was calculated by the ANOVA method. P < 0.05 was considered significant. For statistical analysis, the SPSS 13 (SPSS, Chicago, IL, USA) program was used.
2.1 Comparison of patients' interleukin-8 and -1β serum concentrations
Serum concentrations of IL-8 and IL-1β were determined by the ELISA method. Figure 1 shows these according to study group. Serum IL-8 concentrations were significantly higher in GUP Hp+ than in GUP Hp− (P < 0.01), GP Hp+ (P < 0.01) and GP Hp− (P < 0.01). Serum IL-1β concentrations in GUP Hp+ were also significantly higher than in GUP Hp− (P = 0.023), GP Hp+ (P < 0.01) or GP Hp− (P < 0.01). Furthermore, IL-8 serum concentrations in GP Hp+ were significantly higher than in GP Hp− (P = 0.042) and IL-8 serum concentrations in GUP Hp+ were significantly higher than in GUP Hp− (P < 0.01). Furthermore, overall IL-8 and IL-1β serum concentrations in GUP were higher than in GP; and highest in Hp+ patients; thus, GUP Hp+ had the highest IL-8 and IL-1β serum concentrations. The data also show that overall IL-8 concentrations were higher than those of IL-1β in all groups: for example, in GP Hp,− IL-8 serum concentrations were significantly higher than those of IL-1β (P = 0.015) (Fig. 1).
2.2 Helicobacter pylori crude antigen stimulates production of interleukin-8 and -1β by mononuclear cells
Helicobacter pylori crude antigen, which contains complex water-soluble antigenic factors of Hp, activated production of IL-8 and IL-1β in vitro. Compared with the control group, HPCA significantly stimulated production of IL-8 and IL-1β by PBMNCs (P < 0.01). PBMNCs that had been exposed to HPCA (10 µg/mL) for 72 hrs showed significantly greater increases in IL-8 (P < 0.01) and IL-1β (P = 0.02) concentrations in GUP than in GP (Figs 2 and 3). Exposure of PHA to PBMNCs increased supernatant IL-8 and IL-1β concentrations significantly more in GUP than in GP (P < 0.05). Furthermore, IL-8 concentrations in HPCA-stimulated PBMNC supernatant were increased significantly more in both GUP (P = 0.035) and GP (P = 0.025) than in the groups that had been stimulated with PHA. IL-1β concentrations in supernatant of PBMNCs from both GUP and GP that had been stimulated with HPCA were significantly greater (P < 0.01) than in those that had been stimulated with PHA. Furthermore, as shown in Figure 4, the highest concentrations of IL-8 and IL-1β, which were produced after stimulation of mononuclear cells with HPCA, were found in the GUP Hp+ group. The aforementioned findings suggest that PBMNCs from the GUP Hp+ group in particular are more responsive to HPCA than to PHA regarding production of IL-8 and IL-1β.
2.3 Effect of Helicobacter pylori crude antigen on proliferation of mononuclear cells
As shown in Figure 5, in vitro exposure of PBMNCs to HPCA inhibits proliferation of these cells following PHA stimulation. HPCA inhibited proliferation of PBMNCs that had been obtained from GUP significantly more than those from GP (P = 0.042). To determine the optimal HPCA concentration, different concentrations (0.5, 1, 1.5, 3, 5, and 10 µg/well) of HPCA were added to the mononuclear cells culture. Concentrations above 5 µg/mL consistently caused inhibited PBMNC proliferation by more than 90% and this inhibitory activity was dose dependent (Fig. 6). In this study, 10 µg/well HPCA was found to be the optimal concentration for stimulating PMNCs (Fig. 6). To ensure the inhibitory activity of HPCA after exposure of PBMNCs to PHA for 2 hrs, 10 µg HPCA was added to the wells and proliferation evaluated after 72 hrs. As shown in Figure 5, ODs of PBMNCs with PHA and HPCA were not increased compared with those of PBMNCs plus PHA, indicating that HPCA blunts the stimulatory effect of PHA on PBMNC proliferation. Furthermore, following stimulation with PHA, the PBMNC proliferation rate was higher in the GUP than in the GP group (P = 0.024). Also, PBMNCs from GUP Hp+ demonstrated the greatest proliferation response, indicating that GUP Hp+ were more sensitive to this stimulation than other groups.
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.
Our thanks are due to Miss Esmat Kazemi for linguistic copy-editing. Funding of this work was supported by a grant from the Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. This is the thesis of Hamid Reza Rahimi towards a Doctor of Pharmacy degree.
The authors have no financial conflict of interest.