G. Webb McKnight

A Monoclonal Antibody' Against the CD18 Leukocyte Adhesion Molecule Prevents Indomethacin-Induced Gastric Damage in the Rabbit

The role of leukocyte adherence in the mechanism of gastropathy induced by nonsteroidal antiinflammatory drugs was investigated using a rabbit model. Gastric damage was induced by intragastric instillation of indomethacin [5 mg/mL] for a period of 30 minutes. Histologically, this treatment resulted in extensive vascular congestion and leukocyte margination within the mucosa. Pretreatment with a monoclonal antibody [IB-4] directed against the common beta subunit of the CD11/CD18 adhesion glycoprotein complex significantly (P less than 0.05) reduced both the vasocongestion and the prevalence of leukocyte margination. Macroscopically, indomethacin treatment resulted in the formation of numerous hemorrhagic lesions in the corpus region of the stomach. Pretreatment with IB-4 reduced the extent of gastric hemorrhagic damage by approximately 85% (P less than 0.001). Damage in the group pretreated with IB-4 did not differ significantly from that in rabbits that did not receive indomethacin. In separate experiments, the dose of IB-4 used was shown to completely suppress the recruitment of granulocytes in response to two different agonists. These results support the hypothesis that leukocyte adherence to the vascular endothelium is an important event in the pathogenesis of ulceration induced by nonsteroidal antiinflammatory drugs. Leukocytes might contribute to ulceration by occluding microvessels, thereby reducing mucosal blood flow, and by releasing various mediators, proteases, and free radicals that can produce tissue necrosis.

Hydrogen sulphide synthesis in the rat and mouse gastrointestinal tract

AIMS Hydrogen sulphide (H2S) exerts several anti-inflammatory effects, accelerates the healing of experimental gastric ulcers, and can stimulate intestinal secretion. Little is known about H2S synthesis in the gastrointestinal tract. The aim of this study was to characterize H2S synthesis throughout the gastrointestinal tract. METHODS H2S synthesis in various gastrointestinal tissues of rats and mice was determined. The effects and selectivity of inhibitors of two key enzymes for H2S synthesis, cystathionine-gamma-lyase and cystathionine-beta-synthase, were examined. Cystathionine-gamma-lyase and cystathionine-beta-synthase expression was evaluated by Western blotting and immunohistochemistry. Cystathionine-gamma-lyase and cystathionine-beta-synthase expression in biopsies of human colon was also examined. RESULTS H2S synthesis was variable throughout the gastrointestinal tract in parallel with variations in cystathionine-gamma-lyase and cystathionine-beta-synthase expression. The efficacy of cystathionine-beta-synthase and cystathionine-gamma-lyase inhibitors to reduce H2S synthesis in these tissues was also variable. Cystathionine-beta-synthase is the predominant source of H2S synthesis in the colon of rodents. Cystathionine-gamma-lyase and cystathionine-beta-synthase were also expressed in healthy human colon biopsies. CONCLUSIONS The capacity for H2S synthesis varies throughout the rodent gastrointestinal tract, as does the distribution and contribution of the two key enzymes. Investigation of additional enzymatic sources of H2S and the development of more selective inhibitors are suggested.

Prevention and reversal of experimental colitis by a monoclonal antibody which inhibits leukocyte adherence.

The role of neutrophils in the pathogenesis of acute colitis was investigated using a rabbit model. Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid in 30% ethanol. Myeloperoxidase activity was measured at various times after induction of colitis as an index of neutrophil infiltration, and this was confirmed by histology. The permeability of the colonie epithelium to [51Cr]EDTA was also measured at various times after induction of colitis. The most marked increase in neutrophil infiltration of the colon occurred during the period 3–6 h after induction of colitis. This was also the period in which the greatest increase in colonie permeability was observed. Pretreatment with a monoclonal antibody (IB-4) directed against the leukocyte adhesion molecule, CD18, markedly suppressed neutrophil infiltration into the colonie tissue after induction of colitis. This pretreatment also significantly reduced the extent of epithelial injury. Administration of IB-4 to rabbits 12 h after induction of colitis resulted in a rapid decline in tissue myeloperoxidase activity. When measured 12 h after IB-4 administration (3 mg/kg), colonie myeloperoxidase activity was reduced by about 80% compared to the control group treated with the vehicle. These results are consistent with the hypothesis that neutrophils contribute significantly to the epithelial dysfunction that characterizes colitis and suggest that antibodies directed against adhesion molecules may represent a novel approach to the treatment of intestinal inflammatory disorders.

Effects of Leukotrienes on Susceptibility of the Rat Stomach to Damage and Investigation of the Mechanism of Action

The ability of various leukotrienes to alter the susceptibility of the rat gastric mucosa to injury by 20% ethanol and the possible mechanism of action were examined using an ex vivo gastric chamber preparation. Intraarterial infusions of leukotriene B4 or N-acetyl leukotriene E4 (0.01-1.0 microgram/kg per min for 10 min) had no significant effect on the extent of damage induced by topically applied 20% ethanol. However, infusion of leukotriene C4, D4, or E4 (1.0 micrograms/kg per min) significantly increased ethanol-induced damage, as measured macroscopically, histologically, and functionally. Lower doses of leukotriene C4, D4, or E4 were without significant effect in this model. The increase in damage induced by these three leukotrienes could be blocked by pretreatment with either of two structurally unrelated leukotriene D4 antagonists (L-649,923 or L-660,711). The augmentation of damage by leukotriene C4 was not affected by pretreatment with indomethacin or with a specific thromboxane A2-receptor antagonist (L-670,596). At the dose that increased ethanol-induced damage, none of the leukotrienes tested significantly altered gastric vascular permeability, as measured by Evans blue leakage. However, using laser-Doppler flowmetry, leukotrienes C4 and D4 were found, when administered intraarterially at doses in the 0.05-1.0 micrograms/kg per min range, to produce dose-dependent reductions of gastric blood flow while N-acetyl leukotriene E4 was without effect and leukotriene B4 induced slight increases. The effects of leukotrienes C4 and D4 on gastric blood flow could be inhibited by the two leukotriene D4 antagonists but not by the thromboxane antagonist. These results demonstrate that although they do not produce damage by themselves, leukotrienes C4, D4, and E4 are capable of augmenting ethanol-induced injury to the gastric mucosa. Changes in vascular permeability do not appear to play a role in the mechanism of action of the leukotrienes, while their effects on gastric blood flow are likely to be important. Under certain condition it is therefore possible that local release of leukotrienes could, at least in part through reducing vascular perfusion, predispose the surrounding tissue necrosis.

Reduction of gastrointestinal injury in acute endotoxic shock by flurbiprofen nitroxybutylester

Nitric oxide has been reported to have paradoxical effects in experimental endotoxic shock, contributing to the hemodynamic consequences of endotoxin administration, but apparently protecting the gastrointestinal mucosa. A novel class of nitric oxide-releasing nonsteroidal anti-inflammatory drug (NSAID) derivatives has recently been described which exert anti-inflammatory activities but produce significantly less gastrointestinal injury than the parent nonsteroidal anti-inflammatory drugs from which they are derived. Thus, the present study was performed to determine the effects of one of these derivatives, flurbiprofen 4-nitroxybutylester, compared to the native nonsteroidal anti-inflammatory drug, flurbiprofen, in an experimental model of endotoxic shock. Intravenous administration of endotoxin from Salmonella typhosa to rats pretreated with flurbiprofen produced a profound decrease in systemic arterial blood pressure, an increase in hematocrit and extensive gastric and small intestinal damage. In rats pretreated with flurbiprofen 4-nitroxybutylester, endotoxin produced comparable changes in blood pressure and hematocrit to those seen in rats treated with flurbiprofen; however, the severity of gastrointestinal damage was significantly reduced. Gastric blood flow was profoundly decreased following endotoxin administration, but was significantly higher in rats pretreated with flurbiprofen 4-nitroxybutylester than in rats pretreated with flurbiprofen. These results demonstrate that despite not affecting the acute systemic effects of endotoxin administration, flurbiprofen 4-nitroxybutylester is capable of protecting the gastrointestinal mucosa from injury, possibly through preservation of mucosal blood flow.

Attenuation of epithelial injury in acute experimental colitis by immunomodulators.

Intestinal epithelial permeability can be modulated by the immune system and can be greatly increased by transepithelial migration of neutrophils. Since immunosuppressants have been reported to inhibit the ability of neutrophils to migrate, we assessed the effects of two immunosuppressants on epithelial permeability and granulocyte infiltration in a model of acute colitis. Epithelial permeability was measured at 3 and 6 h after induction of colitis in the rabbit by intracolonic administration of trinitrobenzene sulfonic acid. At these times, blood-to-lumen leakage of 51Cr-EDTA was elevated by approximately 8- and 18-fold, respectively, above levels observed in healthy controls. Pretreatment with either of the immunosuppressants (cyclosporin A and L-683,590) significantly reduced the changes in 51Cr-EDTA leakage observed at the latter time point. These drugs also significantly attenuated granulocyte infiltration of the colon after induction of colitis, as measured by tissue myeloperoxidase activity. Unlike the immunosuppressants, misoprostol, a prostaglandin analogue, attenuated the increases in colonic permeability but had no effect on granulocyte infiltration in this model. These results demonstrate that two structurally unrelated immunosuppressants are capable of markedly reducing neutrophil infiltration and the colonic permeability changes observed in an experimental model of acute colitis, although the mechanisms through which these effects are produced remain unclear.

Comparison of the effects of endothelin-1 and endothelin-3 on the rat stomach

The effects of systemic administration of endothelin-1 and endothelin-3 on the susceptibility of the stomach to injury were compared in the anesthetized rat, as were their effects on gastric vascular tone, systemic blood pressure and hematocrit. When infused at concentrations in the 10(-7)-10(-6) M range, endothelin-1 was a far more potent hypertensive agent than endothelin-3. Endothelin-1 caused significant hemoconcentration, while endothelin-3 did not Endothelin-1 was approximately 5- to 10-times more potent as a vasoconstrictor in the stomach and a similar difference in potencies was observed when the ability of these peptides to increase the susceptibility of the stomach to ethanol-induced damage was compared. The two peptides were equipotent in producing gastric mucosal hemorrhage in the absence of any exogenous irritant. These results demonstrate that like endothelin-1, endothelin-3 has ulcerogenic and vasoconstriction actions in the stomach. While there are very large differences in the potencies of the two peptides in terms of producing systemic hypertension and hemoconcentration, the differences in the potency of the gastric ulcerogenic and vasoconstrictor effects are much less marked.

The Modulation of Gastric Mucosal Integrity by Endothelin-1 and Prostacyclin

Summary The interaction of the vasoconstrictor peptide, endothelin-1 (ET-1), and the endothelium-derived vasodilator eicosanoid, prostacyclin, was examined as it pertains to the modulation of gastric mucosal integrity. Using an ex vivo chamber preparation of the rat stomach, the effects of intravenous ET-1 on the susceptibility of the mucosa to damage induced by topical application of an irritant, 20% ethanol, were examined. ET-1 significantly augmented gastric hemorrhagic damage induced by the irritant when administered at concentrations in the 10–7 to 10–6 M range. Pretreatment with indomethacin at a dose that inhibited gastric prostacyclin synthesis by over 85% resulted in significant augmentation of the ulcerogenic actions of ET-1. The damaging actions of ET-1 could be significantly reduced by topical pretreatment of the gastric mucosa with prostacyclin (5–50

The mucoid cap over superficial gastric damage in the rat: A high-pH microenvironment dissipated by nonsteroidal antiinflammatory drugs and endothelin

mUg/ml). This pretreatment also significantly reduced the hypertension and hemoconcentration observed following ET-1 administration. ET-1 also significantly augmented the susceptibility of the gastric mucosa to injury induced by hydrochloric acid. Oral administration of 150 mM HC1 produced little or no gastric damage in control rats. However, a 5-min intravenous infusion of ET-1 produced significant increases in the severity of acid-induced gastric damage in a concentration-dependent manner (10–7 to 10–6 M). These results demonstrate that ET-1 is a potent ulcerogenic agent in the rat stomach. The ulcerogenic actions of ET-1 can be significantly augmented by indomethacin and significantly reduced by prostacyclin, suggesting that the balance between endothelial cell release of ET-1 and prostacyclin may be an important factor in modulating gastric mucosal integrity.