Toll-like receptor 4 regulates colitis-associated adenocarcinoma development in interleukin-10-deficient (IL-10(-/-)) mice.

Chronic inflammatory conditions such as ulcerative colitis and Crohn's disease are associated with an increased risk of developing adenocarcinoma. It has been hypothesized that this increased risk may be related to soluble mediators present in the inflammatory environment and that factors involved in exacerbating the inflammatory response could increase the risk of developing colitis-associated cancer. There is a growing body of evidence from both clinical studies and animal models which suggests that colitis occurs due to an aberrant immune response to enteric flora in genetically susceptible individuals. It is well documented that bacterial toxins such as endotoxin have potent pro-inflammatory effects through activation of TLR4 (Toll-like receptor 4) and therefore this molecule could potentially play a prominent role in the initiation/exacerbation of colitis and adenocarcinoma development. Using genetic mutant mice, we have examined the role of TLR4 in a spontaneously developing mouse model of colitis-associated adenocarcinoma: the IL-10(-/-) (interleukin-10-deficient) mouse. Surprisingly, our evidence suggests that the absence of TLR4 promotes colitis-associated adenocarcinoma in IL-10(-/-) mice. TLR4-dependent chemokine induction may play a part in modulating the development of colitis-associated neoplasia through altered leucocyte recruitment.

Hepatic venous dysregulation contributes to blood volume pooling in cirrhotic rats.

BACKGROUND AND AIMS: In cirrhosis, despite increased total blood volume, the circulation behaves as if it were volume depleted, a phenomenon termed "decreased effective circulating volume". As the gut/liver veins are the major blood reservoir, this suggests hepatosplanchnic venous pooling. We therefore aimed to elucidate the vasoactive responses of the hepatic veins in cirrhosis. METHODS: Cirrhosis was induced by chronic bile duct ligation in rats. The in vivo responses of postsinusoidal venules and sinusoids to vasoactive drugs, 20% haemorrhage, and 20% mannitol (volume expansion) were examined by intravital microscopy. In isolated perfused livers, change in liver weight was measured as an index of the hepatic vascular volume response. RESULTS: Blood volume was significantly increased in cirrhotic rats. In the cirrhotic hepatic vasculature, constrictive responses to norepinephrine and haemorrhage were blunted compared with controls. In contrast, the dilatory responses to the nitric oxide (NO) donor sodium nitroprusside and volume expansion were enhanced. Both constrictive and dilatory abnormalities were reversed by the NO synthase inhibitor N-nitro-L-arginine methyl ester. CONCLUSIONS: The hepatic sinusoidal and venous bed of cirrhotic rats showed an enhanced dilatory capacity to buffer volume increases but inadequately constricted in response to volume depletion or catecholamines. Both abnormalities may contribute to volume pooling and are mediated by NO.

Absence of Fer protein tyrosine kinase exacerbates endotoxin induced intestinal epithelial barrier dysfunction in vivo.

BACKGROUND AND AIMS: Fer kinase is activated by a number of growth factors and cytokines, and phosphorylates cortactin during cell shape change induced cortical actin reorganisation. In addition, Fer participates in cytoskeletal interactions mediated by cadherins, platelet endothelial cell adhesion molecule 1 (PECAM-1), and integrins, and has recently been implicated in limiting the innate immune response. Here we examined the role of Fer in modulating leucocyte recruitment and epithelial barrier function in the gut in response to lipopolysaccharide (LPS). METHODS: Mice targeted with a kinase inactivating mutation (FerDR) or strain matched wild-type (129Sv/J) mice were studied after intraperitoneal injection of LPS. Intravital microscopy was used to examine intestinal leucocyte kinetics, and leucocyte infiltration was assessed by fluorescence activated cell sorting. Systemic inflammation was assessed by measuring lung myeloperoxidase activity. Epithelial barrier function was assessed in vivo using blood to lumen 51Cr-EDTA clearance, with or without antibody based depletion of circulating neutrophils. RESULTS: LPS induced a significant increase in leucocyte adhesion and neutrophil infiltration into the intestinal tissue, and increased blood to lumen 51Cr-EDTA clearance. Pretreatment with neutrophil depleting antibody completely abrogated this response in wild-type mice. In FerDR mice, LPS induced leucocyte adhesion within the intestinal venules was exacerbated and associated with a trend towards increased neutrophil transmigration relative to wild-type mice. Surprisingly, LPS induced epithelial barrier permeability was increased 2.5-fold in FerDR mice relative to wild-type mice, and this barrier defect was only partly attenuated by depleting circulating neutrophils by >93 %. CONCLUSIONS: Fer plays a role in regulating LPS induced epithelial barrier dysfunction in vivo through both neutrophil dependent and neutrophil independent mechanisms.

Role of p38 mitogen-activated protein kinase in chemokine-induced emigration and chemotaxis in vivo.

It has been proposed that L-selectin engagement with ligand activates p38 mitogen-activated protein kinase (MAPK) and can impact on downstream events of leukocyte rolling, including adhesion, and emigration. Using a novel chemotactic assay in vivo, we visualized slow release of chemokine from an agarose gel positioned 350 microm from a postcapillary venule, which induced directed migration (chemotaxis) of neutrophils. In this system, keratinocyte-derived cytokine induced phosphorylation of p38 MAPK, which phosphorylated a downstream protein (ATF-2). This latter event was blocked by the concentration of p38 inhibitors used in this study. Mice were treated with two different p38 inhibitors: SKF86002 and SB203580. Neither inhibitor affected rolling or adhesion in microvessels. Intravenous treatment with SFK86002 (5, 10, and 20 mg/kg) 30 min before the inflammatory stimulus inhibited the total number of emigrated cells at a dose of 20 mg/kg (62%, p < 0.05), despite the presence of many adherent cells within the vessels. A similar inhibition was observed with 20 mg/kg of a second p38 inhibitor SB203580 (67%, p < 0.05). In addition to emigration, both p38 inhibitors impaired the ability of emigrated cells to migrate through the tissue toward the chemotactic stimulus. In fact, the majority of emigrated leukocytes in p38 inhibitor-treated animals remained within 50 microm of the venule. Superfusion of the tissue with SKF86002 (0.7 mM) to impact only on emigrated and not vascular leukocytes resulted in no impairment in emigration, but in a significant reduction in chemotaxis away from the vessel wall. Again, the majority of emigrated leukocytes remained within 50 microm of the blood vessel. Our results suggest that p38 does not affect rolling or adhesion, but that it is involved in leukocyte emigration and chemotaxis through interstitium in response to keratinocyte-derived cytokine in vivo.

Exclusive neutrophil recruitment with oncostatin M in a human system.

Oncostatin M (OSM), a member of the IL-6 family has been postulated to be a potent recruiter of leukocytes, however information regarding the molecular mechanism(s) underlying this event is extremely limited. Therefore, the aim of this study was to investigate the role of OSM-mediated leukocyte recruitment in a human system in vitro under flow conditions. A parallel-plate flow chamber assay was used to examine leukocyte recruitment from whole blood by human umbilical vein endothelium treated for 24 hours with OSM. OSM in a dose-response manner revealed very significant leukocyte rolling and adhesion reaching optimal levels at a very low concentration of OSM (10 ng/ml). The OSM-induced leukocyte rolling and adhesion was comparable to levels seen with tumor necrosis factor. OSM was extremely selective for neutrophil recruitment (96%) with <3% lymphocyte recruitment. By contrast, tumor necrosis factor-alpha revealed no such selectivity, recruiting 70% neutrophils and at least 25% lymphocytes and detectable levels of eosinophils at 24 hours. The molecular mechanism underlying the leukocyte recruitment seemed to be entirely dependent on P-selectin as leukocyte recruitment could be completely blocked by the addition of a P-selectin-blocking antibody. An elevation in both P-selectin message and protein was observed with 24 hours of OSM stimulation of endothelium. By contrast, E-selectin and VCAM-1 were not detectable after OSM stimulation. Similar results were seen with passaged dermal microvascular endothelium that does not have a prestored pool of P-selectin. Based on these results, we conclude that OSM may be a very selective potent recruiter of neutrophils in more prolonged inflammatory conditions, an event exclusively dependent on P-selectin.

Neuronal nitric oxide synthase (NOS) regulates leukocyte-endothelial cell interactions in endothelial NOS deficient mice.

1. The present study was designed to examine the possible role of neuronal nitric oxide synthase (nNOS) in regulation of leukocyte - endothelial cell interactions in the absence of endothelial nitric oxide synthase (eNOS), using intravital microscopy of the cremasteric microcirculation of eNOS(-/-) mice. 2. Baseline leukocyte rolling and adhesion revealed no differences between wild-type and eNOS(-/-) mice in either the cremasteric or intestinal microcirculations. 3. Superfusion with L-NAME (100 microM) caused a progressive and significant increase in leukocyte adhesion in both wild-type and eNOS(-/-) mice, without detecting differences between the two strains of mice. 4. Superfusion with 7-nitroindazole (100 microM), a selective inhibitor of nNOS, had no effect on leukocyte adhesion in wild-type animals. However, it increased leukocyte adhesion significantly in eNOS(-/-) mice, which was reversed by systemic L-arginine pre-administration. 5. Stimulation of the microvasculature with H(2)O(2) (100 microM) induced a transient elevation in leukocyte rolling in wild-type mice. Conversely, the effect persisted during the entire 60 min of experimental protocol in eNOS(-/-) mice either with or without 7-nitroindazole. 6. Semi-quantitative analysis by RT - PCR of the mRNA for nNOS levels in eNOS(-/-) and wild-type animals, showed increased expression of nNOS in both brain and skeletal muscle of eNOS(-/-) mice. 7. In conclusion, we have demonstrated that leukocyte-endothelial cell interactions are predominantly modulated by eNOS isoform in postcapillary venules of normal mice, whereas nNOS appears to assume the same role in eNOS(-/-) mice. Interestingly, unlike eNOS there was insufficient NO produced by nNOS to overcome leukocyte recruitment elicited by oxidative stress, suggesting that nNOS cannot completely compensate for eNOS.

Nitric oxide and intestinal inflammation.

Inflammation of the intestinal tract remains a very serious concern in the clinical setting. Unfortunately, to date, the mechanisms underlying many inflammatory conditions such as sepsis or inflammatory bowel diseases are poorly understood and our therapeutic interventions are less than ideal. Over the past decade, an abundance of research has been directed toward the role of nitric oxide (NO) in intestinal inflammation. It has become apparent that NO might have a dichotomous role as both a beneficial and detrimental molecule. Nitric oxide is a weak radical produced from L-arginine via the enzyme nitric oxide synthase (NOS). NOS exists in three distinct isoforms; constitutively (cNOS) expressed neuronal NOS (NOS1 or nNOS) and endothelial NOS (NOS3 or eNOS) or an inducible isoform (NOS2 or iNOS) capable of high production output of NO during inflammation. Constitutively expressed NOS has been shown to be critical to normal physiology and inhibition of these enzymes (nNOS or eNOS) caused damage. It has been proposed that the high output production of NO from iNOS causes injury, perhaps through the generation of potent radicals such as peroxynitrite and hence may explain the apparent dichotomous role of NO. However, recent studies have challenged this simple paradigm providing evidence that iNOS may have some protective role in some inflammatory models. Moreover, the importance of peroxynitrite has been questioned. In this review we discuss the role of cNOS and iNOS in intestinal inflammation and provide an overview of peroxynitrite in intestinal inflammation, highlighting some of the controversy that exists.

E/P-selectin-deficient mice: an optimal mutation for abrogating antigen but not tumor necrosis factor-alpha-induced immune responses.

Leukocyte recruitment in cremaster microcirculation was visualized by intravital microscopy, either in ovalbumin sensitized and challenged animals, or in response to TNF-alpha. In antigen-challenged mice a significant increase in leukocyte rolling (approximately 50 to 200-300 cells/min) and adhesion (2 to 15-20 cells/100 microm), and a very dramatic increase in emigration ( approximately 1 to >40 cells/field) was observed over 24 h. Although rolling and adhesion was dramatically blunted in P-selectin- or P selectin/ICAM-1-deficient mice, emigrated cell number was similar to that observed in wild-type mice. Leukocyte rolling, adhesion and emigration was almost entirely abrogated over 24 h in E/P-selectin-deficient mice, demonstrating that antigen-induced leukocyte recruitment can be entirely disrupted in the absence of both endothelial selectins. However, E/P-selectin-deficient mice were able to recruit leukocytes at 24 h after TNF-alpha challenge. Rolling 24 h post-TNF-alpha in E/P-selectin-deficient mice was not inhibitable with anti-L-selectin antibody, suggesting an entirely selectin-independent pathway of rolling. We identified this pathway to be alpha (4)-integrin dependent and demonstrated that VCAM-1 expression was increased only in mice challenged with TNF-alpha. These data demonstrate that, in vivo, sufficient amounts of TNF-alpha can recruit leukocytes independently of selectins, whereas inhibition of endothelial selectins is the optimal intervention in reducing the immune response to antigen.

Spontaneously developing chronic colitis in IL-10/iNOS double-deficient mice.

Mice deficient in both inducible nitric oxide synthase (iNOS) and interleukin (IL)-10 (iNOS(-/-)/IL-10(-/-)) were created to examine the role of iNOS in spontaneously developing intestinal inflammation. IL-10(-/-)/iNOS(-/-) mice were compared with IL-10(-/-) (iNOS(+/+)) littermates over 6 mo. RT-PCR, Western blot analysis, and immunohistochemistry were performed to measure iNOS message and protein levels. Plasma nitrate/nitrite (NO(x)) levels were assessed by HPLC. Damage scores (macroscopic and microscopic) and granulocyte infiltration were assessed. At 3-4 wk, IL-10(-/-) and IL-10(-/-)/iNOS(-/-) mice had no signs of colonic inflammation or granulocyte infiltration. Plasma NO(x) levels were not different from controls. By 3-4 mo, IL-10(-/-) mice had increased damage scores and granulocyte infiltration concurrent with increased mRNA and protein synthesis (restricted to the epithelium) for iNOS in intestinal tissues but not other tissues. Plasma NO(x) levels increased fivefold. Interestingly, in the absence of iNOS induction or increased plasma NO(x), iNOS(-/-)/IL-10(-/-) mice had damage and granulocyte infiltration equivalent to those observed in IL-10(-/-) littermates. These data suggest that iNOS does not impact on the development or severity of spontaneous chronic inflammation in IL-10-deficient mice.

Role of inducible nitric oxide synthase in trinitrobenzene sulphonic acid induced colitis in mice.

BACKGROUND: Studies using inhibitors of nitric oxide synthase (NOS) to date are inconclusive regarding the role of inducible NOS (iNOS) in intestinal inflammation. AIMS: (1) To examine the role of iNOS in the development of chronic intestinal inflammation; (2) to identify the cellular source(s) of iNOS. METHODS: Colitis was induced by an intrarectal instillation of trinitrobenzene sulphonic acid (TNBS, 60 mg/ml, 30% ethanol), in wild type (control) or iNOS deficient mice. Mice were studied over 14 days; the colons were scored for injury and granulocyte infiltration was quantified. Blood to lumen leakage of (51)Cr-EDTA was measured as a quantitative index of mucosal damage. RESULTS: At 24 and 72 hours, iNOS deficient mice had significantly increased macroscopic inflammation compared with wild type mice. Granulocyte infiltration increased significantly at 24 hours and remained elevated in iNOS deficient mice at 72 hours, but significantly decreased in controls. However, by seven days post-TNBS macroscopic damage, microscopic histology, granulocyte infiltration, and mucosal permeability did not differ between wild type and iNOS deficient mice. A four- to fivefold increase in iNOS mRNA was observed in wild type mice at 72 hours and seven days post-TNBS and was absent in iNOS deficient mice. Immunohistochemistry techniques showed that iNOS expression was predominantly localised in neutrophils, with some staining also in macrophages. CONCLUSIONS: These results suggest that leucocyte derived iNOS ameliorates the early phase, but does not impact on the chronic phase of TNBS induced colitis despite the presence of iNOS.

Intestinal inflammation in adhesion molecule-deficient mice: an assessment of P-selectin alone and in combination with ICAM-1 or E-selectin.

Biopsy specimens from patients with inflammatory bowel disease have demonstrated an up-regulation of P-selectin, suggesting a role for P-selectin in intestinal inflammation. We examined the role of P-selectin in experimental intestinal inflammation using mice deficient in P-selectin alone or in combination with either ICAM-1 or E-selectin. Colitis was induced using acetic acid or trinitrobenzene sulfonic acid (TNBS). Damage scores and neutrophil infiltration 24 h post acetic acid were not different between wild-type and P-selectin- or P-selectin/ICAM-1-deficient mice, whereas P/E-selectin-deficient mice had enhanced leukocyte recruitment and damage. At 72 h an attenuation in damage scores and a slight decrease in neutrophil infiltration was observed in the P- and P/ICAM-deficient animals. The P/E-selectin-deficient mice maintained enhanced leukocyte recruitment and damage. In wild-type mice P-selectin expression was elevated 48 and 72 h post acetic acid-induced inflammation. Surprisingly, P-selectin or P-selectin/ICAM-1 deficiency did not improve the inflammation induced by TNBS over 7 days. In fact, increased mortality was observed. Anti-adhesion therapy may play only a limited, beneficial role and often a detrimental role in intestinal inflammation.

Varying roles of E-selectin and P-selectin in different microvascular beds in response to antigen.

Expression of E-selectin and P-selectin is critical in the effector phase of leukocyte recruitment in response to Ag. Whether their relative roles differ between tissues in response to the same Ag is unknown. In this study, a type I hypersensitivity response was elicited in C57BL/6 mice by systemic sensitization with OVA. Following local Ag challenge, endothelial selectin expression was examined in the skin and cremaster muscle microvasculature using a dual-radiolabeled mAb technique. Next, the dermal and muscle microcirculations were visualized using intravital microscopy to establish roles for P-selectin and/or E-selectin. In untreated mice, leukocyte recruitment in both skin and skeletal muscle was mediated entirely by P-selectin. Following Ag challenge, leukocyte rolling flux and adhesion were dramatically increased and leukocyte rolling velocity was unchanged in muscle. Only P-selectin expression increased in muscle, and leukocyte recruitment was entirely dependent upon this selectin. In contrast, in Ag-challenged skin, leukocyte rolling flux did not increase, but rolling velocity dropped profoundly. In skin, only E-selectin expression increased, and blockade of either E-selectin or P-selectin had minimal effect on either rolling flux or rolling velocity. Blockade of both selectins reduced rolling flux by 80% and increased rolling velocity sevenfold. These data highlight striking differences in expression of the endothelial selectins in separate microvascular beds in response to the same stimulus and demonstrate that these differences underlie very different patterns of leukocyte recruitment. The data underscore the importance of studying individual microvascular beds to understand tissue-specific leukocyte recruitment in vivo.

Effect of intracolonic benzalkonium chloride on trinitrobenzene sulphonic acid-induced colitis in the rat.

BACKGROUND: We investigated the effects of benzalkonium chloride (BAC) on trinitrobenzene sulphonic acid (TNBS)-induced colitis in rats. METHODS: TNBS was administered intrarectally before and/or after BAC treatment. In the first study, the effects of treatment with BAC 6, 12 or 24 h after TNBS were examined. In the second study, animals were treated with BAC before, after or before and after TNBS, and were examined 7 days later. The severity of colitis was assessed by macroscopic and histological scoring of the colonic damage and by determination of colonic myeloperoxidase (MPO) activity. Macrophages and CD4+ and CD8+ T cells were examined by immunohistochemistry. RESULTS: When BAC was instilled into the colon 6, 12 or 24 h after TNBS, weight loss and macroscopic and histological features of the colon were similar to that of controls (TNBS alone). In contrast, MPO activity was significantly reduced in all three groups post-treated with BAC. In the groups examined 7 days after TNBS treatment, rats post-treated with BAC exhibited increased weight gain and significantly reduced macroscopic damage and MPO activity compared to the TNBS control group. Rats pre-treated with BAC exhibited less macroscopic damage of the colon than rats receiving only TNBS, but histological damage, MPO and weight gain were unchanged from TNBS controls. Immunohistochemistry revealed that BAC pre-treatment increased the numbers of macrophages and T cells in the colon. After TNBS treatment, macrophage accumulation was evident in the colon, but T cells were scarce. However, these cells were preserved or enhanced in the colonic mucosa in TNBS-treated rats that had been pre-treated with BAC. CONCLUSIONS: Treatment with BAC, particularly after induction of colitis, produces a significant reduction in the severity of tissue injury and inflammation through mechanisms that are not fully understood.

Lack of beneficial effect of a tachykinin receptor antagonist in experimental colitis.

Nerves within the wall of the intestine may contribute to inflammatory responses, such as those occurring in inflammatory bowel disease. Studies in an experimental model of colitis have demonstrated that neuromodulation, through chemical sympathectomy or administration of lidocaine, can markedly attenuate granulocyte infiltration and tissue injury. Given the many pro-inflammatory effects of substance P, we have evaluated the effects of a tachykinin receptor (NK-1) antagonist, RP 67580, in models of acute colitis in the rat and guinea pig. While administration of RP 67580 and a second NK-1 antagonist (CP-96,345-1) significantly reduced the infiltration of granulocytes into colonic tissue during the first 12 h after induction of colitis in the rat, repeated administration of RP 67580 over a three day period failed to significantly affect granulocyte recruitment or the severity of tissue injury. In contrast, lidocaine enemas were effective in reducing both indices of inflammation/injury. In the guinea pig, similar observations were made. These observations demonstrate that blockade of NK-1 receptors over a three day period failed to significantly modify the course of experimental colitis. It remains possible that the beneficial effects of lidocaine may be due, in part, to inhibition of substance P release, and that the contribution of substance P to inflammation in experimental colitis occurs through NK-1 receptor-independent mechanisms.

Inducible nitric oxide synthase plays a critical role in resolving intestinal inflammation.

BACKGROUND & AIMS: Overproduction of nitric oxide by inducible nitric oxide synthase (iNOS) has been proposed as a pathogenic factor in colitis. The objective of this study was to examine the role of iNOS using iNOS-deficient mice in experimental colitis. METHODS: Colitis was induced by intrarectal instillation of 3% acetic acid and assessed for neutrophilic infiltration and intestinal injury over 7 days. iNOS messenger RNA expression was also measured. RESULTS: At 24 hours, acetic acid induced a mild colitis in wild-type mice. An increase in neutrophil infiltration and tissue edema was also observed. In the iNOS-deficient mice, a twofold increase in macroscopic damage was observed. Neutrophil infiltration and tissue edema were similar to those in wild-type animals at this time point. Although inflammation in wild-type mice had resolved by 7 days, a sevenfold increase in damage score and elevated myeloperoxidase level were still evident in iNOS-deficient mice. A striking increase in the message for iNOS was observed in inflamed wild-type mice at 24 hours and was still present at 72 hours. No message was found in iNOS-deficient mice. CONCLUSIONS: Induction of iNOS seems to be a critical protective response to injury in intestinal inflammation possibly by reducing leukocytic infiltration.

Effects of chemical sympathectomy and sensory nerve ablation on experimental colitis in the rat.

We assessed the effects of primary afferent nerve ablation (systemic treatment with capsaicin during adult or neonatal periods), primary afferent nerve activation (intracolonic capsaicin), and sympathectomy [6-hydroxydopamine (6-OHDA)] on the development of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) in rats. We also examined whether lidocaine was effective after ablation of primary afferent nerves or sympathectomy. Colitis was assessed by macroscopic scoring, measurement of myeloperoxidase (MPO) activity, and histology. Systemic capsaicin treatment in adults increased the macroscopic damage score. Capsaicin treatment of neonates did not significantly increase damage score or MPO activity compared with vehicle-treated controls. However, all capsaicin-treated groups had a higher mortality. Intracolonic capsaicin treatment did not alter the severity of colitis. Chemical sympathectomy resulted in a decreased damage score and improved histology compared with controls. In 6-OHDA pretreated rats, lidocaine administration reduced the macroscopic and histological scores and MPO activity almost to control levels. However, lidocaine administration in capsaicin-treated rats attenuated the macroscopic damage but did not improve MPO activity or histology. These data suggest that capsaicin-sensitive nerves play a protective role in experimental colitis and sympathetic nerves contribute to the development of colitis. The beneficial effects of lidocaine appear to be due primarily to its action on enteric nerves.

Flavonoid-induced gastroprotection in rats: role of blood flow and leukocyte adherence.

To elucidate the mechanisms of flavonoid-induced protection against nonsteroidal anti-inflammatory drug (indomethacin)-induced acute gastric damage, the effects of 5-methoxyflavone and 5-methoxyflavanone on the gastric vasculature were compared both in vivo (using laser Doppler flowmetry in anesthetized rats) and in vitro on rat superior mesenteric arteries. The effects of the compounds on indomethacin-induced leukocyte adherence to mesenteric venules were investigated by intravital videomicroscopy. Oral 5-methoxyflavone reduced indomethacin-induced macroscopic damage by 38 to 99% (ED50 = 5.5 mg/kg). Damage was not significantly reduced by 5-methoxyflavanone. Light microscopy studies also demonstrated a reduction in damage severity. 5-Methoxyflavone, but not 5-methoxyflavanone, increased the gastric conductance significantly. The effects on isolated mesenteric arteries correlated with the effects on in vivo conductance. Finally, indomethacin-induced leukocyte adherence was inhibited to a greater extent by 5-methoxyflavone than by 5-methoxyflavanone. In conclusion, the flavonoid 5-methoxyflavone provides gastroprotection against nonsteroidal anti-inflammatory drug-induced gastric damage. A structurally similar compound, 5-methoxyflavanone, demonstrated minimal gastroprotective activity, suggesting that the double bond of 5-methoxyflavone is required for biological activity. The finding that 5-methoxyflavone (but not 5-methoxyflavanone) significantly increased gastric vascular perfusion and reduced leukocyte adherence to mesenteric venules suggests that these mechanisms may contribute to the flavonoid's gastroprotective activity.

Tumor necrosis factor mediation of NSAID-induced gastric damage: role of leukocyte adherence.

Neutrophil adherence to the vascular endothelium has been suggested to be a critical event in the pathogenesis of nonsteroidal anti-inflammatory drug (NSAID)-induced gastric damage. Recently, increased plasma levels of tumor necrosis factor-alpha (TNF-alpha), which can increase leukocyte adherence, have been reported after administration of indomethacin. This study was performed to determine the relationship between plasma TNF-alpha levels, leukocyte adherence, and NSAID-induced gastric injury. Administration of indomethacin to rats resulted in a significant elevation of plasma TNF-alpha levels within 30 min and the development of gastric erosions. Pretreatment with dexamethasone and prostaglandin E2 almost completely prevented gastric injury and abolished the rise in plasma TNF-alpha. Pentoxifylline dose dependently reduced both gastric damage and plasma TNF-alpha. Similar effects were observed with three other TNF-alpha synthesis inhibitors and with an anti-TNF-alpha antisera. Pentoxifylline also significantly reduced the extent of antral ulceration induced by naproxen. However, pentoxifylline did not significantly affect indomethacin-induced leukocyte adherence. These results suggest that TNF-alpha plays a critical role in the pathogenesis of NSAID-induced gastric injury, but this cytokine may not be responsible for NSAID-induced leukocyte adherence.

Indomethacin-induced gastric injury and leukocyte adherence in arthritic versus healthy rats.

BACKGROUND & AIMS: Arthritic patients are at greater risk of developing nonsteroidal anti-inflammatory drug (NSAID)-induced ulcers than other NSAID users. Leukocyte adherence to the vascular endothelium has been suggested to play an important role in the pathogenesis of experimental NSAID-associated gastropathy. The aim of this study was to examine the role of leukocyte adherence in NSAID-induced gastric injury in healthy and adjuvant-induced arthritic rats. METHODS: Leukocyte adherence was examined using intravital microscopy before and after indomethacin administration. The role of CD18 and intercellular adhesion molecule 1 (ICAM-1) in indomethacin-induced gastric injury and leukocyte adherence was examined using specific monoclonal antibodies against these molecules. RESULTS: Indomethacin (5-10 mg/kg) caused significantly more gastric damage in arthritic than normal rats, and the former group had significantly greater levels of leukocyte adherence to mesenteric postcapillary venules under basal conditions. Dexamethasone markedly reduced basal and indomethacin-induced leukocyte adherence and the extent of gastric damage. In arthritic rats, pretreatment with a monoclonal antibody directed against ICAM-1 significantly reduced gastric damage and leukocyte adherence to the levels observed in healthy rats, whereas an antibody directed against CD18 had no effect. CONCLUSIONS: Indomethacin-induced damage in healthy and arthritic rats is largely dependent on ICAM-1 expression. The increased susceptibility of arthritic rats to indomethacin-induced gastric injury may be partly related to elevated levels of ICAM-1-dependent leukocyte adherence.