Yasunari Nakamura

Rebamipide protects against activation of neutrophils by Helicobacter pylori

Our objectives were to determine whether rebamipide, a unique antiulcer agent, would inhibit adhesive reactions between neutrophils and endothelial cells as well as the production of active oxygen species from neutrophils elicited by an extract ofH. pylori. A water extract ofH. pylori that was prepared from biopsy materials obtained from a patient with gastric ulcer increased the surface expression of CD18 on human neutrophils isolated from peripheral blood, the adhesion of neutrophil-endothelial cells, and the production of active oxygen species by neutrophils. Rebamipide, at concentrations of 10−5 and 10−6 M, reduced the adherence of neutrophils to endothelial cells as well as the CD18 expression on neutrophils induced by this bacterial extract. Rebamipide also inhibited the production of active oxygen species from neutrophils stimulated byH. pylori extract. These results suggest that rebamipide protects against the gastric mucosal inflammation associated withH. pylori by inhibiting neutrophil function.

Neutrophils, Lipid Peroxidation, and Nitric Oxide in Gastric Reperfusion Injury in Rats

Nitric oxide (NO) modulation of ischemia-reperfusion injury was investigated by measuring lipid peroxide and neutrophil accumulation in rat stomachs treated with NG-nitro-L-arginine (L-NNA), a specific NO synthase inhibitor. Ischemia-reperfusion injury was induced in the rat stomach. Treatment with L-NNA for 3 days at a dose of 3 mg/kg/day significantly enhanced this injury. This enhancement was reversed by the simultaneous administration of L-arginine at a dose of 30 mg/kg/day. Both thiobarbituric acid (TBA)-reactive substances, an index of lipid peroxidation, and myeloperoxidase (MPO) activity, an index of tissue-associated neutrophil accumulation, were increased in the gastric mucosa after ischemia-reperfusion. L-NNA treatment enhanced these increases in TBA-reactive substances and MPO activity. The increase in the area of gastric erosions correlated closely with accumulation of TBA-reactive substances as well as the increase in MPO activity. Enhancement of ischemia-reperfusion injury by L-NNA treatment was inhibited by injection with anti-neutrophil antibody, anti-platelet activating factor (PAF) antagonist, and anti-leukotriene B4 (LTB4) receptor antagonist. In addition, the increase in TBA-reactive substances and MPO activity was decreased by these antibodies or antagonists. Enhancement of reperfusion-induced gastric mucosal injury associated with inhibition of NO synthesis may involve neutrophil infiltration and lipid peroxide accumulation in the gastric mucosa, mediated by PAF and LTB4.

Role of neutrophil-mediated inflammation in aspirin-induced gastric mucosal injury.

The objectives of this study were to determine the roles of neutrophil-endothelial cell interactions and oxygen-derived free radicals in the pathogenesis of aspirin-induced gastric mucosal injury in rats. Oral administration of acidified aspirin (200 mg/kg) resulted in linear hemorrhagic erosions and an increase in myeloperoxidase activity, an index of neutrophil infiltration, in the gastric mucosa. Aspirin-induced gastric damage and the increase in myeloperoxidase activity were significantly inhibited by the injection of anti-CD11a, anti-CD11b, anti-intercellular adhesion molecule-1 monoclonal antibodies, and the combination of superoxide dismutase and catalase, which are scavengers of active oxygen species. These results suggest that neutrophil-endothelial adhesive interactions, which occur via CD11a/CD18-and CD11b/CD18-dependent interactions with intercellular adhesion molecule-1, and oxygen-derived free radicals produced by neutrophils are implicated in the production of aspirin-induced gastric mucosal injury.

Helicobacter pylori infection potentiates aspirin induced gastric mucosal injury in Mongolian gerbils

Background: Helicobacter pylori infection and non-steroidal anti-inflammatory drugs are two major causes of gastric ulceration but interactions between H pylori and these drugs in gastric mucosal injury are unclear. Aims: We studied the influence of experimental H pylori infection on gastric mucosal injury induced by aspirin. Subjects: Male Mongolian gerbils free of specific pathogens were used. Methods: H pylori ATCC43504 culture broth was administered by oral gavage at seven weeks of age. After three weeks, acidified aspirin (400 mg/kg) was administered orally, and three hours later the total area of gastric erosions, myeloperoxidase (MPO) activity (an index of neutrophil accumulation), thiobarbituric acid reactive substances (TBARS, an index of lipid peroxidation), and KC/GRO (a chemoattractive cytokine in rodents) were measured in gastric mucosa. To determine the role of neutrophils in these circumstances, antigerbil neutrophil rabbit serum (ANS) was administered to some animals 18 hours before aspirin. Results: Aspirin caused more extensive haemorrhagic erosions (33.1 (12.3) mm2) associated with greater MPO activity (1887.7 (598.5) μU/mg protein) and TBARS (0.33 (0.14) nmol/mg protein) and KC/GRO concentrations (28.3 (9.5) pg/mg protein) in infected than in uninfected gerbils (13.7 (2.3); 204.0 (68.9); 0.12 (0.06); 3.1 (0.8), respectively) Pretreatment with ANS inhibited the increases in gastric erosions, MPO activity, and TBARS but not KC/GRO concentration. The reduction in aspirin induced mucosal injury by administration of ANS was much greater in H pylori infected animals (65%) than in uninfected animals (31%). Conclusions: H pylori infection potentiates aspirin induced gastric mucosal injury by mechanisms that include accumulation of activated neutrophils.

INTERACTIONS OF NEUTROPHILS AND ENDOTHELIAL CELLS UNDER LOW FLOW CONDITIONS IN VITRO

The interactions of polymorphonuclear leukocytes (PMN) and endothelial cells are modulated by adhesion molecules, inflammatory cytokines, and shear stress. We investigated the changes in PMN-endothelial cell interactions induced by interleukin (IL)-1β under low flow conditions. PMN were isolated from the venous blood of healthy adults, and endothelial cells were obtained from human umbilical veins. The number of PMN that adhered to the endothelial cell monolayer that was treated with IL-1 increased significantly at shear stresses from .5 to 4.0 dyn/cm2 as compared with untreated endothelial cells. Anti-intercellular adhesion molecule (ICAM)-1 monoclonal antibody (mAb), anti-E-selectin mAb, and anti-CD18 mAb each significantly inhibited the increase in PMN adherence induced by IL-1 at a low shear stress (1.0 dyn/cm2). Anti-CD18 mAb significantly reduced the number of PMN that migrated through the endothelial monolayer by blocking the adherence of PMN to the luminal surface of the endothelial cells, as well as their transendothelial migration. In contrast, anti-ICAM-1 and anti-E-selectin mAb each reduced the number of PMN that migrated by reducing the number of PMN that adhered to the luminal surface without significantly influencing the percent of the adherent PMN that had migrated. Although anti-L-selectin mAb reduced the adherence and migration of PMN, these effects were not statistically significant. These results indicated that under low flow conditions, as well as in the nonflow state, PMN-endothelial cell interactions were elicited via CD11/CD18 and ICAM-1 without the involvement of selectins.

Methylprednisolone inhibits neutrophil-endothelial cell interactions induced by interleukin-1 β under flow conditions

The effects of methylprednisolone (m-PSL) on IL-1beta-induced neutrophil-endothelial cell interactions, which are normally mediated by increased expression of both intercellular adhesion molecule-1 (ICAM-1) and E-selectin on endothelial cells, were examined using an in vitro flow system. Human neutrophilic polymorphonuclear leukocytes (PMN) were perfused at a shear stress of 1 dyne/cm2 on human umbilical vein endothelial cells (HUVEC) pretreated with IL-1beta (20 U/mL) for 4 hours. Many PMN adhered to IL-1-stimulated HUVEC and then migrated beneath endothelial cell monolayers. Treatment of HUVEC with m-PSL inhibited adherence and migration of PMN in a dose dependent manner. M-PSL also inhibited IL-1beta-induced upregulation of E-selectin and ICAM-1 on HUVEC in a dose dependent manner. These results suggest that m-PSL works as an anti-inflammatory agent through inhibiting PMN-endothelial cell interactions.

Role of P-Selectin and Intercellular Adhesion Molecule-1 in TNB-Induced Colitis in Rats

It has been proposed that neutrophil-endothelial cell interactions mediated by adhesion molecules are involved in the pathogenesis of inflammatory bowel disease. The objective of the present study was to determine the effects of monoclonal antibodies (MAbs) directed against endothelial adhesion molecules, P-selectin and intercellular adhesion molecule-1 (ICAM-1), in rats with colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNB). Colonic inflammation was induced by administering an enema of TNB dissolved in 50% ethanol (120 mg/ml) to male Wistar rats (at a total volume of 0.25 ml per rat) after a 48-hour fast. Anti-P-selectin MAb or anti-ICAM-1 MAb was injected via the tail vein at a dose of 1 mg/kg after the induction of colitis. Rats in the control group received nonbinding mouse immunoglobulin G1. The plasma level of soluble P-selectin showed an increase within 48 h after the TNB enema. Colonic inflammation was assessed at 1 week after TNB administration. The colonic damage score and the wet weight of the colon were significantly decreased by treatment with either MAb. The increase of myeloperoxidase (MPO) activity, an index of neutrophil accumulation, and the increase of thiobarbituric acid-reactive substances (TBA-RS), an index of lipid peroxidation, in the colonic mucosa were inhibited by both MAbs. These results suggest that neutrophil-endothelial cell interactions via P-selectin and ICAM-1 play an important role in the development of TNB-induced colitis in rats.

Role of elastase and active oxygen species in gastric mucosal injury induced by aspirin administration in Helicobacter pylori-infected Mongolian gerbils

H. pylori infection potentiates aspirin‐induced gastric mucosal injury by mechanisms that include accumulation of activated neutrophils.

Effect of shear stress and a stable prostaglandin I2 analogue on adhesive interactions of colon cancer cells and endothelial cells

In the process of cancer metastasis, adhesion between cancer cells and endothelial cells is an important early step. In the present study, the effects of shear stress and the adhesion molecules responsible for cancer cell interactions with endothelial cells were investigated in a system similar to in vivo microcirculation. The effect of prostaglandin I2 (PGI2) also was determined. Human colon cancer cell line Colo 201 and human umbilical vein endothelial cells (HUVEC) were used. After HUVEC on a glass slide were incubated with IL‐1β for 4 h, cancer cells in suspension were perfused on HUVEC at wall shear stresses of 5–40 μN/cm2. Experiments were videotaped, and the number of adherent cells were counted. Additionally, the effects of anti‐sialyl Lewis a (SLea) MoAb, anti‐E‐selectin MoAb, and a PGI2 analogue were investigated. Expression of adhesion molecules on cancer cells and HUVEC was assessed using flow cytometry and enzyme immunoassay, respectively. Few cancer cells adhered to HUVEC without IL‐1β; however, many cancer cells adhered to IL‐1β‐stimulated HUVEC at low shear stress (5–20 μN/cm2). Cancer cells did not migrate beneath HUVEC. The increased adhesion was inhibited by anti‐E‐selectin MoAb, anti‐SLea MoAb, and a PGI2 analogue. In addition, the PGI2 analogue decreased the surface expression of SLea on Colo 201 cells. These results suggest that Colo 201 cells adhere to IL‐1β‐stimulated endothelial cells via SLea and E‐selectin under low flow conditions; PGI2 analogues may protect against metastasis by inhibiting cancer cell–endothelial cell interactions.

Effect of rebamipide, a novel anti-ulcer agent, on acute gastric mucosal injury induced by ischemia-reperfusion in rats

The protective effect of a novel anti-ulcer agent rebamipide on the acute gastric mucosal injury induced by ischemia-reperfusion was studied in rats. Ischemia/reperfusion injury was produced on the rat stomach by applying a small clamp to the celiac artery for 30 min and by removal of the clamp for 60 min. The decrease in the gastric mucosal blood flow was not influenced by the treatment with rebamipide. The increase in the total area of erosions on the stomach after ischemia-reperfusion and the increase in lipid peroxides in the gastric mucosa were significantly inhibited by the oral administration of rebamipide at doses of 30 and 100 mg/kg. These results suggest that the protective effect of rebamipide against the ischemia/reperfusion-induced aggravation of gastric mucosal injury may be the result of its inhibitory effect on lipid peroxidation.