Differential roles of interleukin‐1β and interleukin‐8 in neutrophil transendothelial migration in patients with Helicobacter pylori infection

Background: Little information is currently available on the contribution of locally generated inflammatory and chemotactic cytokines to endothelial cell activation and subsequent neutrophil transendothelial migration in patients with Helicobacter pylori (H. pylori)‐associated gastritis. Methods: The contents of interleukin (IL)‐1β and IL‐8 in the organ culture supernatants of antral mucosal tissues were measured with an enzyme‐linked immunosorbent assay. The effects of the endogenous IL‐1β and IL‐8 in mucosal tissues on neutrophil adherence and transendothelial migration were investigated using an experimental model of human umbilical vein endothelial cells (HUVEC). Results: The contents of IL‐1β and IL‐8 in organ cultures of antral mucosal tissues were significantly higher in patients with H. pylori infection than in those without infection. The organ culture supernatants from H. pylori‐positive patients induced the expression of intercellular adhesion molecule‐1 mRNA in HUVEC with increased binding of neutrophils, and these stimulatory effects were inhibited when HUVEC were pretreated with a nuclear factor‐κB inhibitor, MG‐132. Moreover, neutrophil adherence to HUVEC induced by the supernatants decreased after preincubation with neutralizing anti‐IL‐1β antibody. As compared with the supernatants from H. pylori‐negative patients, the samples from H. pylori‐positive patients exhibited a significantly higher chemotactic activity for neutrophils, which was inhibited almost completely by preincubation of the supernatants with anti‐IL‐8 antibody. Conclusions: Locally generated IL‐1β and IL‐8 could coordinate with each other during the process of neutrophil infiltration into the gastric mucosa in patients with H. pylori infection.     

Gastric metaplasia in the duodenal bulb shows increased mucosal interleukin-8 activity in Helicobacter pylori-positive duodenal ulcer patients

BACKGROUND: Although increased levels of interleukin (IL)-8 are known to be associated with infiltration of neutrophils in the gastric mucosa with Helicobacter pylori infection, no study has investigated the relationship between local IL-8 levels and neutrophil infiltration in the duodenal mucosa of patients with duodenal ulcer (DU). METHODS: Duodenal mucosal biopsy specimens with and without gastric metaplasia (GM) were obtained from patients with DU and controls with an endoscopic methylene blue (MB) staining method. Levels of IL-8 secreted in the organ cultures of biopsy specimens were measured with an enzyme-linked immunosorbent assay. The number of myeloperoxidase-positive neutrophils infiltrating the lamina propria was determined in immunohistochemically stained tissue sections. RESULTS: Histologic assessment showed that there was a strong correlation between the absence of endoscopic MB staining and the extent of GM. The levels of IL-8 in both duodenal and antral mucosal tissues were significantly higher in patients with H. pylori infection than in those without infection. In patients with DU the duodenal mucosal tissues with GM (MB-unstained mucosa) showed significantly higher levels of IL-8 than those without GM (MB-stained mucosa) or the antral mucosa. The number of neutrophils showed similar variations among DU and control patients with a positive correlation with IL-8 activity. The levels of IL-8 and the number of neutrophils decreased after H. pylori eradication in both duodenal and antral mucosal tissues, and these changes were more remarkable in the duodenal mucosal tissues with GM. CONCLUSIONS: Increased IL-8 activity in the duodenal mucosa with GM may be important for ulcerogenesis in H. pylori-positive DU patients.     

Gastric Metaplasia in the Duodenal Bulb Shows Increased Mucosal Interleukin-8 Activity in Helicobacter pylori-Positive Duodenal Ulcer Patients

Background: Although increased levels of interleukin (IL)-8 are known to be associated with infiltration of neutrophils in the gastric mucosa with Helicobacter pylori infection, no study has investigated the relationship between local IL-8 levels and neutrophil infiltration in the duodenal mucosa of patients with duodenal ulcer (DU). Methods: Duodenal mucosal biopsy specimens with and without gastric metaplasia (GM) were obtained from patients with DU and controls with an endoscopic methylene blue (MB) staining method. Levels of IL-8 secreted in the organ cultures of biopsy specimens were measured with an enzyme-linked immunosorbent assay. The number of myeloperoxidase-positive neutrophils infiltrating the lamina propria was determined in immunohistochemically stained tissue sections. Results: Histologic assessment showed that there was a strong correlation between the absence of endoscopic MB staining and the extent of GM. The levels of IL-8 in both duodenal and antral mucosal tissues were significantly higher in patients with H. pylori infection than in those without infection. In patients with DU the duodenal mucosal tissues with GM (MB-unstained mucosa) showed significantly higher levels of IL-8 than those without GM (MB-stained mucosa) or the antral mucosa. The number of neutrophils showed similar variations among DU and control patients with a positive correlation with IL-8 activity. The levels of IL-8 and the number of neutrophils decreased after H. pylori eradication in both duodenal and antral mucosal tissues, and these changes were more remarkable in the duodenal mucosal tissues with GM. Conclusions: Increased IL-8 activity in the duodenal mucosa with GM may be important for ulcerogenesis in H. pylori-positive DU patients.     

Helicobacter pylori-associated gastric ulcer exhibits enhanced mucosal chemokine activity at the ulcer site

BACKGROUND AND AIM: Although mucosal alpha- and beta-chemokines are considered to be involved in the pathogenesis of Helicobacter pylori-associated gastritis, little is known how these chemokines are related to the ulcerogenesis in peptic ulcer patients. We examined the levels of interleukin (IL)-8 and macrophage inflammatory protein-1alpha (MIP-1alpha) in organ cultures and the numbers of inflammatory cells infiltrating the lamina propria by using the mucosal tissues obtained from gastric ulcer (GU) patients with and without H. pylori infection. METHODS: Levels of IL-8 and MIP-1alpha secreted in organ cultures were measured by an enzyme-linked immunosorbent assay. Numbers of myeloperoxidase-positive neutrophils, CD68-positive macrophages, and mononuclear cells were determined in tissue sections. RESULTS: The mucosal tissues of both the gastric antrum and the ulcer site obtained from patients with H. pylori-positive GU showed significantly higher levels of IL-8 and MIP-1alpha and increased numbers of inflammatory cells compared with the corresponding mucosal tissues from those with H. pylori-negative GU or the antral mucosal tissues from H. pylori-negative controls. When the values were compared between the mucosal tissues from the gastric antrum and those from the ulcer site, the latter group of tissues showed significantly higher levels of IL-8 and MIP-1alpha and increased numbers of neutrophils and macrophages than the former group regardless of its healing process in patients with H. pylori-positive GU. CONCLUSION: Mucosal alpha- and beta-chemokines may be important to the ulcerogenesis in H. pylori-associated GU disease.     

Gastric mucosal cytokine levels in relation to host interleukin-1 polymorphisms and Helicobacter pyloricagA genotype

OBJECTIVE: The outcome of a Helicobacter pylori infection is related in part to interrelationships among H. pylori virulence factors and the H. pylori-induced mucosal response. The host inflammatory response is partly governed by polymorphisms in pro-inflammatory genes. MATERIAL AND METHODS: Cytokine levels (interleukin (IL)-1beta, IL-6 and IL-8) were examined in H. pylori-infected and uninfected normal-appearing mucosa from patients with non-ulcer dyspepsia (NUD), margins of gastric ulcers and cancer tissues. Cytokine levels were compared with cagA genotypes and host interleukin (IL)-1 polymorphisms. RESULTS: The study comprised 168 Thai patients. All infected patients possessed anti-CagA antibody. Gastric mucosal IL-8 levels were significantly higher in H.pylori-positive cases than in -negative cases in all three tissue types (e.g. 1115 versus 217 pg/mg protein for NUD) (p<0.001). Normal-appearing but H. pylori-infected antral mucosa of patients with cagA type 1a strains had higher IL-8 levels than those with type 2a strains (2632 versus 1036 pg/mg protein) (p<0.005). IL-1B-511T/T carriers had higher antral mucosal IL-1ss levels versus non-carriers (pg/mg protein) (T/T=221, T/C=178, C/C=70) (p=0.005). IL-1B-511T/T carriers also had higher IL-1beta levels versus non-carriers in H. pylori-negative patients. CONCLUSIONS: It was found that both the host factors (IL-1 polymorphisms) and bacterial factors (cagA type 1a versus type 2a) influenced gastric mucosal cytokine levels. Future studies should concentrate on interactions among host factors (e.g. genetics and tissue responses) and bacterial and environmental factors.     

Transendothelial migration of neutrophils involves integrin-associated protein (CD47).

Inflammation is a primary pathological process. The development of an inflammatory reaction involves the movement of white blood cells through the endothelial lining of blood vessels into tissues. This process of transendothelial cell migration of neutrophils has been shown to involve neutrophil beta 2 integrins (CD18) and endothelial cell platelet-endothelium cell adhesion molecules (PECAM-1; CD31). We now show that F(ab')2 fragments of the monoclonal antibody B6H12 against integrin-associated protein (IAP) blocks the transendothelial migration of neutrophils stimulated by an exogenous gradient of the chemokine interleukin 8 (IL-8; 60% inhibition), by the chemotactic peptide N-formyl-methionylleucylphenylalanine (FMLP; 76% inhibition), or by the activation of the endothelium by the cytokine tumor necrosis factor alpha (98% inhibition). The antibody has two mechanisms of action: on neutrophils it prevents the chemotactic response to IL-8 and FMLP, and on endothelium it prevents an unknown but IL-8-independent process. Blocking antibodies to IAP do not alter the expression of adhesion proteins or production of IL-8 by endothelial cells, and thus the inhibition of neutrophil transendothelial migration is selective. These data implicate IAP as the third molecule essential for neutrophil migration through endothelium into sites of inflammation.     

Relationship of Helicobacter pylori-associated Gastritis and Polymorphonuclear Leukocyte Elastase

Abstract: Background: Much remains to be clarified about the mechanisms of Helicobacter pylori (H. pylori)-associated gastritis. We considered the possibility of neutrophil involvement and investigated the role of polymorphonuclear leukocyte elastase (PMN-E) in the etiology of H. pylori-associated gastritis. Methods: In 60 patients with gastritis, infection by H. pylori was diagnosed by a combination of the ELISA method and conventional culture method. The hexosamine contents of the mucosal tissues and plasma polymorphonucler leukocyte elastase complexes (PMN-EC) were determined. Results: PMN-EC was found to be elevated in H. pylori-positive gastritis patients, and the degree of elevation was proportionately related to the grade of gastritis inflammation. The hexosamine concentration of the gastric antral mucosa was significantly lower in H. pylori-positive patients than in the H. pylori-negative patients. The ratio of PMN-EC/gastric mucosal hexosamine concentration was significantly higher in the H. pylori-positive group than in the negative group. Conclusion: These data suggest that one of the mechanisms of the H. pylori-associated gastritis may be the infiltration of neutrophils into the gastric mucosa followed by the release of PMN-E which is responsible for the degradation of gastric mucosal proteins and resultant tissue damages.     

Mucosal concentrations of proinflammatory cytokines and chemokines at gastric cardia: implication of Helicobacter pylori infection and gastroesophageal reflux

OBJECTIVES: The pathogenesis of carditis remains unclear, although gastroesophageal reflux disease (GERD) and Helicobacter pylori infection have been proposed. Little is known about the profile of proinflammatory cytokines and chemokines in the pathogenesis of carditis. METHODS: We studied 28 patients with GERD and 40 controls. Two biopsy specimens were taken endoscopically from the cardiac mucosa within 5 mm from the squamocolumnar junction; one was snap frozen for measurement of mucosal levels of interleukin 1beta (IL-1beta), tumor necrosis factor-alpha, IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), regulated on activation normal T-cell expressed and presumably secreted (RANTES) by enzyme-linked immunosorbent assays, while another was processed for histopathology. H. pylori infection was assessed by serology, rapid urease test, and histology with Giemsa staining. Samples were taken from the cardia of 18 H. pylori-positive patients, before and after eradication treatment. RESULTS: Carditis was significantly associated with H. pylori infection, but not GERD. IL-8, MCP-1, and RANTES levels were significantly higher in cardiac mucosa of patients with carditis than in those without it and in patients with than without H. pylori infection. IL-8 concentrations were significantly associated with the degree of neutrophil infiltration within the cardiac mucosa and decreased after cure of the infection. Mucosal MCP-1 and RANTES levels correlated positively with the grades of mononuclear cell infiltration and IL-1beta concentrations. CONCLUSION: Our results indicate that chemokines produced locally in the cardiac mucosa may be involved in the development of H. pylori-associated carditis.     

Lafutidine inhibits Helicobacter pylori-induced interleukin-8 production in human gastric epithelial cells

BACKGROUND AND AIM: Attachment of Helicobacter pylori to gastric epithelial cells leads to the production of chemokines, such as interleukin-8 (IL-8), which in turn activate and recruit neutrophils to the site of infection. Lafutidine [(+/-)-2-(furfurylsulfinyl)-N-(4-(4-(piperidinomethyl)-2-pyridyl)oxy-(Z)-2-butenyl)acetamide] is a new type of antiulcer drug that possesses an antisecretory action as well as gastroprotective activity, independent of its antisecretory action. In the present study, we examined the effects of lafutidine on H. pylori-induced IL-8 release and H. pylori adhesion to MKN45 cells. METHODS: MKN45 cells were stimulated with H. pylori, tumor necrosis factor (TNF)-alpha, or IL-1beta, then IL-6 and IL-8 levels in the culture supernatants were determined with a specific enzyme-linked immunosorbent assay kit. RESULTS: Lafutidine significantly inhibited both the release of IL-8 induced by H. pylori and the adhesion of H. pylori to cells in a dose-dependent manner. These properties of lafutidine are unrelated to the blockade of histamine H(2)-receptors, because the same effects have not been observed with other H(2)-receptor antagonists, such as cimetidine and famotidine. Lafutidine also significantly inhibited H. pylori-induced IL-6 release. Both TNF-alpha and IL-1beta-induced IL-8 releases, conversely, were little affected by lafutidine up to a concentration of 10(-5) M. CONCLUSIONS: These results suggest that lafutidine inhibits IL-8 release by inhibiting H. pylori adherence to gastric epithelial cells, indicating a novel mechanism by which lafutidine protects against the mucosal inflammation associated with H. pylori infection.     

Gastric mucosal secretion of interleukin-10: relations to histopathology, Helicobacter pylori status, and tumour necrosis factor-alpha secretion.

BACKGROUND: Interleukin-10 (IL-10) is an 18 kDa peptide with a range of anti-inflammatory and immunosuppressive properties. AIM: To determine whether this cytokine is involved in gastric mucosal inflammation in Helicobacter pylori infection. METHODS: The production of IL-10 by antral mucosal biopsy specimens during short term in vitro culture was determined by measuring IL-10 content of supernatants by enzyme linked immunosorbent assay (ELISA). H pylori status was determined by serology and histology, with gastritis scored using the Sydney system. Tumour necrosis factor-alpha (TNF-alpha) content of supernatants was also determined in a subgroup of patients. RESULTS: IL-10 secretion was significantly greater in patients with H pylori associated chronic gastritis than in patients who were H pylori negative with normal mucosa/reactive changes, and those with H pylori negative chronic gastritis (p < 0.01 and < 0.05 respectively). There was a significant correlation overall between IL-10 secretion and chronic inflammation score (r = 0.40). Secretion of TNF-alpha, which was significantly higher in H pylori infected patients than uninfected patients with a normal mucosa (p < 0.04), correlated with scores for chronic inflammation and activity (r = 0.39 and 0.38 respectively), but was only weakly correlated with IL-10 secretion (r = 0.22, NS). CONCLUSIONS: Gastric mucosal production of IL-10 and TNF-alpha are increased in chronic gastritis associated with H pylori infection, and mucosal cytokine secretion varies with important histopathological aspects of gastric inflammation. Whereas the secretion of IL-10 in H pylori infection may be protective, limiting tissue damage caused by inflammation, it may also contribute towards failure of the immune response to eliminate the organism.     

Mucosal tumour necrosis factor alpha and interleukin-6 in patients with Helicobacter pylori associated gastritis.

The production of tumour necrosis factor alpha (TNF alpha) and interleukin-6 by human antral mucosa during short term culture in vitro has been measured by enzyme linked immunosorbent assay. TNF alpha and interleukin-6 concentrations in culture supernatants were significantly greater (p less than 0.001) in patients infected with Helicobacter pylori, all of whom had chronic gastritis, than in patients who were H pylori negative with histologically normal gastric mucosa. Among H pylori colonised patients, TNF alpha concentrations were significantly higher in those with active gastritis and neutrophil infiltration into the epithelium than in those with inactive gastritis. In contrast, interleukin-6 concentrations were raised in both active and inactive gastritis. This study shows that H pylori gastritis is associated with increased gastric mucosal production of TNF alpha and interleukin-6 and that the nature of the mucosal cytokine response varies with the immunohistology of the disease. Inflammatory cytokines generated locally within the gastric mucosa could be relevant to the gastric physiology of H pylori infection.     

Relation between clinical presentation, Helicobacter pylori density, interleukin 1beta and 8 production, and cagA status

BACKGROUND: It is not known whether cagA+ Helicobacter pylori in duodenal ulcer (DU) have enhanced virulence compared with non-DU cagA+ H pylori. AIMS: To investigate the relation between presentation, H pylori density, interleukin 1beta (IL-1beta) and IL-8 production, and cagA status. METHODS: Fifty DU and 50 gastritis patients with cagA+ H pylori and 11 with cagA- infections were studied. Bacterial density and cytokine production were assessed using the same biopsies. Cytokine production was also measured from supernatants of medium following coculture of H pylori with MKN-45 cells. RESULTS: There was no relation between H pylori density and cagA status. There was a dose dependent relation between mucosal cytokine levels and density of cagA+ H pylori. H pylori density increased to a threshold, followed by a rapid increase in cytokines and then a plateau. IL-1beta and IL-8 levels in the antrum were greater in DU than in gastritis; in the corpus the cytokine level/H pylori differed irrespective of similar H pylori densities. However, cytokine production was similar in vitro, independent of presentation or biopsy site, suggesting that host factors are critical determinants of the inflammatory response. Mucosal IL-8 and IL-1beta levels were low with cagA- and cagA+, cagE- H pylori infections. CONCLUSIONS: The increase in antral IL-1beta and IL-8 production and inflammation in DU is related to increased numbers of bacteria and not to an increase in cytokine production per cagA+ isolate. There was no evidence of enhanced virulence of H pylori from DU compared with cagA+ non-DU H pylori.     

Neutrophil chemotaxis in gastric mucosa

This prospective study compares levels of neutrophil chemotactic activity (NCA) in gastric juice to the neutrophil count in gastric biopsies. Sixty-three male patients enrolled in the study and had antral biopsies following collection of gastric juice during esophagogastroduodenoscopy. Biopsies were examined for the magnitude of gastritis, tissue PMN count, and presence of Helicobacter pylori. Secretions were assayed for neutrophil chemotactic activity. Results show an increase in NCA and in tissue PMN counts with increasing severity of gastritis. H. pylori-positive patients had higher levels of NCA and PMN than H. pylori-negative patients. Wide variability in NCA levels preclude a direct correlation between NCA and PMN counts.     

Coordinate upregulation of interleukin-8 and growth-related gene product-alpha is present in the colonic mucosa of inflammatory bowel

BACKGROUND: Although ?lpha-chemokines, such as interleukin (IL)-8 and epithelial neutrophil-activating peptide 78, are implicated in the pathogenesis of inflammatory bowel disease (IBD), little information is currently available on the expression and cellular source of growth-related gene product-alpha (GROalpha) and its functional relationship to other ?lpha-chemokines in the intestinal mucosa of patients with IBD. METHODS: The contents of IL-8 and GROalpha in organ cultures, the expression of IL-8 and GROalpha mRNA, and the modulatory effects of inflammatory mediators on IL-8 and GROalpha-producing cells were examined using colonic mucosal tissues. In vitro stimulatory effects of IL-8 and GROalpha on neutrophils were investigated in terms of chemotactic migration and superoxide anion generation. RESULTS: The contents of IL-8 and GROalpha in organ cultures were elevated in patients with IBD, especially in those with active ulcerative colitis (UC). Both IL-8 and GROalpha contents increased according to an increase in histological disease activity in patients with UC, but not in those with Crohn disease. In contrast, no significant correlation was found between the contents of these alpha-chemokines and clinical disease activity. In situ hybridization detected increased expression of IL-8 and GROalpha mRNA in macrophages, pericrypt myofibroblasts, and the epithelium of tissue specimens with active lesions of IBD. The secretion of IL-8 and GROalpha from macrophages and myofibroblasts obtained from control patients was upregulated by inflammatory cytokines and bacterial products. The concentrations of recombinant (r)-IL-8, which covered the levels of activity detected in individual organ cultures or cell cultures of fractionated mucosal cells, could induce chemotactic migration and superoxide anion generation in neutrophils in vitro, and r-GROalpha had synergistic effects on r-IL-8-induced neutrophil activation. CONCLUSIONS: A coordinate upregulation of IL-8 and GROalpha may be involved in the tissue injury in patients with IBD through their stimulatory effects on neutrophils.     

Suppression of Helicobacter pylori-induced interleukin-8 production in gastric cancer cell lines by an anti-ulcer drug,geranylgeranylacetone

BACKGROUND: Geranylgeranylacetone (GGA) is an antigastritis and anti-ulcer agent, with as yet an unknown mechanism of action. In this study, we investigated the effect of GGA on Helicobacter pylori-induced interleukin (IL)-8 production and IL-8 mRNA expression in KATOIII cells, an established gastric cell line. METHODS: Interleukin-8 production in H. pylori-infected KATOIII cells was measured by using enzyme-linked immunoassay. The cytotoxicity of H. pylori on KATOIII cells was measured by a 51Cr release assay. The effect of GGA on H. pylori-induced IL-8 mRNA expression was measured by using northern blotting. RESULTS: Interleukin-8 production increased with time and H. pylori dose; the most significant increase was seen within 6-24 h of coculture with H. pylori. A dose of 0.1 mmol GGA suppressed IL-8 production (P = 0.0077) and inhibited H. pylori-induced IL-8 mRNA expression (P = 0.0019). Furthermore, H. pylori-induced gastric mucosal cell injury associated with IL-8 and neutrophil activation was enhanced by NH3, and this enhancement was suppressed by GGA (P = 0.0043). CONCLUSIONS: Helicobacter pylori-infected gastric mucosal cells produce IL-8, which can promote neutrophil activation, thus contributing to mucosal tissue injury associated with H. pylori infection. Agents like GGA, which can suppress IL-8 production may have a protective role in the treatment of mucosal tissue damage seen in H. pylori infection.     

Effect of acid-suppressive therapy on Helicobacter pylori production of interleukin-8 in the gastric mucosa.

BACKGROUND: Recent studies have shown that acid-suppressive therapy increases the severity of Helicobacter pylori- associated gastritis in the corpus. PURPOSE: To evaluate interleukin (IL)-8 production in the gastric corpus mucosa before and during acid-suppressive therapy in H pylori-infected patients. PATIENTS AND METHODS: Ten patients with reflux esophagitis (five H pylori-positive and five H pylori-negative) were treated with omeprazole 20 mg. Serum gastrin concentrations, H pylori colonization density and mucosal IL-8 levels in the corpus were investigated at entry and two weeks after starting therapy. IL-8 levels were measured by ELISA. The organism density was determined, and scored according to area occupied by the bacterial colonies. The presence of H pylori was assessed by rapid urease testing and histological finding of gastric biopsy specimens. RESULTS: In H pylori-positive patients, concentrations of IL-8 during therapy significantly exceeded those before therapy (36.2+/-6. 8 versus 18.3+/-3.8 pg/mg protein; P<0.05) without altering H pylori density. In H pylori-negative patients, IL-8 levels were similar before and during therapy (6.1+/-2.7 versus 6.3+/-3.0 pg/mg protein). Concentrations of gastrin during therapy were significantly higher than those before therapy in all patients. CONCLUSIONS: The results suggest that acid suppression increases mucosal IL-8 levels in H pylori-infected patients with reflux esophagitis.