Regulation of murine intestinal inflammation by reactive metabolites of oxygen and nitrogen: divergent roles of superoxide and nitric oxide.

Several reports have implicated reactive oxygen and nitrogen metabolites (RONS) in the initiation and/or progression of inflammatory bowel diseases (IBDs). We have investigated the role of three key RONS-metabolizing enzymes (inducible nitric oxide synthase [iNOS], superoxide dismutase [SOD], nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) in a murine model of IBD. Mice genetically deficient ((-/-)) in either iNOS or the p47phox subunit of NADPH oxidase, transgenic (Tg) mice that overexpress SOD, and their respective wild-type (WT) littermates were fed dextran sulfate sodium (DSS) in drinking water for 7 days to induce colitis. In addition, the specific iNOS inhibitor 1400W was used in DSS-treated WT and p47phox(-/-) mice. WT mice responded to DSS feeding with progressive weight loss, bloody stools, elevated serum NO(X) and colonic mucosal injury with neutrophil infiltration. Both the onset and severity of colitis were significantly attenuated in iNOS(-/-) and 1400W-treated WT mice. While the responses to DSS did not differ between WT and p47phox(-/-) mice, enhanced protection was noted in 1400W-treated p47phox(-/-) mice. Interestingly, SOD(Tg) mice exhibited more severe colitis than their WT littermates. These findings reveal divergent roles for superoxide and iNOS-derived NO in intestinal inflammation.

Role of CD11/CD18 in shear rate-dependent leukocyte-endothelial cell interactions in cat mesenteric venules.

In vivo microscopy was used to assess the relationships among shear rate (and shear stress), leukocyte rolling velocity, and leukocyte adherence in a cat mesentery preparation. Shear rate in individual venules and arterioles of 25-35 microns diameter were varied over a wide range by graded occlusion of an arterial loop. There was a linear decline in leukocyte rolling velocity (Vwbc) as red cell velocity (Vrbc) was reduced. The ratio Vwbc/Vrbc remained constant despite variations in shear stress from 5-25 dyn/cm2. A reduction in shear stress was associated with an increased leukocyte adherence, particularly when Vwbc was reduced below 50 microns/s. Reduction in wall shear rate below 500 s-1 in arterioles allowed 1-3 leukocytes to adhere per 100 microns length of vessel, while venules exposed to the same shear rates had 5-16 adherent leukocytes. In arterioles, leukocyte rolling was only observed at low shear rates. At shear rates less than 250 s-1 leukocyte rolling velocity was faster in arterioles than venules, and the ratio Vwbc/Vrbc for arterioles was 0.08 +/- 0.02, which was fourfold higher than the ratio obtained in venules at similar shear rates. Pretreatment with the CD18-specific antibody (mAb) IB4 increased leukocyte rolling velocity in venules by approximately 20 microns/s at red cell velocities below 2,000 microns/s. mAb IB4 largely prevented the leukocyte adherence to arterioles and venules, and increased the ratio Vwbc/Vrbc observed in venules at low shear elicit a CD18-dependent adhesive interaction between leukocytes and microvascular endothelium, and that differences in shear rates cannot explain the greater propensity for leukocyte rolling and adhesion in venules than arterioles.

Histamine and histidine decarboxylase are correlated with mucosal repair in rat small intestine after ischemia-reperfusion.

The aim of this experiment was to demonstrate whether histamine and histidine decarboxylase (HDC) contribute to mucosal repair in small intestine subjected to ischemia-reperfusion (I/R). The superior mesenteric artery was occluded for 15 min followed by reperfusion. In jejunal mucosa, histamine content and HDC activity increased after I/R. Histamine output in mesenteric lymph was also elevated after I/R. These increases in HDC activity, and mucosal and lymph histamine levels were suppressed by pretreatment of alpha-fluoromethylhistidine (alpha-FMH), a suicide inhibitor of HDC. alpha-FMH also attenuated the increase of ornithine decarboxylase (ODC) activity normally observed after I/R. Transport of dietary lipid into lymph markedly decreased at 24 h after I/R, yet it was restored to normal at 48 h after I/R. alpha-FMH inhibitor led to a sustained deficit in lipid transport at 48 h after I/R. This sustained functional impairment in alpha-FMH treated animals was associated with blunted responses of HDC activity and histamine content to I/R. Our results suggest that histamine and HDC contribute to the restoration in mucosal function observed at 48 h after I/R. This response may be related, at least in part, to stimulation of ODC activity by histamine.

Hepatic leukostasis and hypoxic stress in adhesion molecule-deficient mice after gut ischemia/reperfusion.

The concept that leukocyte-endothelial cell adhesion (LECA) is a major determinant of the tissue injury elicited by ischemia/reperfusion (I/R) is largely based on studies employing adhesion molecule-specific monoclonal antibodies. The objective of this study was to assess the contribution of LECA to I/R injury using mutant mice (all on a C57B1 background) that are deficient in either intracellular adhesion molecule-1, P-selectin, or CD11/CD18. The accumulation of fluorescently labeled leukocytes and the number of nonperfused sinusoids in livers of control and adhesion molecule-deficient mice were monitored by intravital microscopy for 1 h after release of the occluded (for 15 min) superior mesenteric artery. Autofluorescence of pyridine nucleotide (NADH) was measured as an indicator of mitochondrial O2 consumption and redox status. The number of stationary leukocytes in the liver after gut I/R was significantly elevated compared with baseline values in C57B1 (control) mice. Autofluorescence of NADH was also significantly increased (indicating hypoxia) after I/R in these mice, especially in the pericentral region. Intercellular adhesion molecule-1-, CD11/CD18-, and P-selectin-deficient mice all exhibited a blunted leukosequestration response to I/R and smaller increments in nonperfused sinusoids, relative to C57B1 mice. All adhesion molecule-deficient mice also exhibited an attenuated increment in NADH autofluorescence in the pericentral region, relative to control mice. These results from adhesion molecule-deficient mice provide additional support for the view that LECA is an important determinant of the liver dysfunction induced by gut I/R.

Clostridium difficile toxin A-induced microvascular dysfunction. Role of histamine.

Clostridium difficile toxin A (Tx-A) mediates secretion and inflammation in experimental enterocolitis. Intravital video microscopy was used to define the mechanisms that underlie the inflammatory reactions elicited by direct exposure of the microvasculature to Tx-A. Leukocyte adherence and emigration, leukocyte-platelet aggregation, and extravasation of FITC-albumin were monitored in rat mesenteric venules exposed to Tx-A. Significant increases in leukocyte adherence and emigration (LAE) and albumin leakage were noted within 15-30 min of Tx-A exposure. These responses were accompanied by mast cell degranulation and the formation of platelet-leukocyte aggregates. The Tx-A-induced increases in LAE and albumin leakage were significantly attenuated by pretreatment with either monoclonal antibodies (mAbs) directed against the leukocyte adhesion glycoproteins, CD11/CD18, intercellular adhesion molecule-1, and P-selectin (but not E-selectin) or with sialyl Lewis x, a counter-receptor for P-selectin. The mast cell stabilizer, lodoxamide, an H1- (but not an H2-) receptor antagonist, and diamine oxidase (histaminase) were also effective in reducing the LAE and albumin leakage elicited by Tx-A. The platelet-leukocyte aggregation response was blunted by an mAb against P-selectin, sialyl Lewis x, and the H1-receptor antagonist. These observations indicate that Tx-A induces a leukocyte-dependent leakage of albumin from postcapillary venules. Mast cell-derived histamine appears to mediate at least part of the leukocyte-endothelial cell adhesion and platelet-leukocyte aggregation by engaging H1-receptors on endothelial cells and platelets to increase the expression of P-selectin. The adhesion glycoproteins CD11/CD18 and intercellular adhesion molecule-1 also contribute to the inflammatory responses elicited by toxin A.

Inhibition of endotoxin-induced bacterial translocation in mice.

The primary functions of the gut are to absorb nutrients and exclude bacteria and their products. However, under certain circumstances the gut may lose its barrier function and serve as a reservoir for systemic microbial infections. These experiments were performed to determine the mechanisms whereby endotoxin causes bacteria to escape (translocate) from the gut. Bacteria translocated from the gut to the mesenteric lymph nodes of mice challenged with nonlethal doses of Escherichia coli 026:B6 or E. coli 0111:B4 endotoxin. Physical disruption of the gut mucosal barrier appears to be the primary mechanism whereby endotoxin promotes bacterial translocation. Mucosal injury and endotoxin-induced bacterial translocation were reduced by inhibition (allopurinol) or inactivation (tung-sten diet) of xanthine oxidase activity (P less than 0.01), but were not affected by the platelet-activation factor antagonists, SRI 63-441 or BN 52021. Because the inhibition or inactivation of xanthine oxidase activity reduced both the extent of mucosal injury and endotoxin-induced bacterial translocation, the effect of endotoxin on the gut appears to be mediated, at least to some degree, by xanthine oxidase-generated, oxygen-free radicals.

Role of H1 receptors and P-selectin in histamine-induced leukocyte rolling and adhesion in postcapillary venules.

The objective of this study was to define the nature, magnitude, and mechanisms of histamine-induced leukocyte-endothelial cell interactions in postcapillary venules of the rat mesentery using intravital microscopic techniques. Superfusion of the mesentery with histamine (10(-7)-10(-5) M) resulted in a dose-related increase in the number of rolling leukocytes, a reduction in rolling velocity, and an increased clearance of FITC-labeled rat albumin from blood to superfusate. The histamine-induced recruitment of rolling leukocytes and increased albumin clearance were prevented by histamine H1 (hydroxyzine, diphenhydramine) but not H2 (cimetidine) receptor antagonists. Because histamine induces expression of the adhesion molecule P-selectin in cultured endothelial cells, a monoclonal antibody directed against rat P-selectin and soluble sialyl-LewisX oligosaccharide (the carbohydrate ligand to P-selectin) were also tested as inhibitors. Both were effective in preventing the histamine-induced recruitment of rolling leukocytes, but neither agent attenuated the increased albumin clearance. These observations suggest that (a) histamine recruits rolling leukocytes and increases albumin leakage in postcapillary venules via H1 receptor activation, (b) histamine-induced recruitment of rolling leukocytes is mediated in part by P-selectin expressed on the endothelial cell surface, and (c) the histamine-induced vascular albumin leakage is unrelated to leukocyte-endothelial cell adhesion. Our results are consistent with the view that histamine may act as a mediator of acute inflammatory reactions.

Platelet activation In mice and human Helicobacter pylori infection.

Extracts of Helicobacter pylori (HP) have been shown to induce leukocyte adhesion in mesenteric venules, but the effects of HP infection on gastric microvessels are unknown. Inflammatory cell interactions in the gastric microcirculation were studied by intravital videomicroscopy in mice inoculated with either saline or fresh isolates of HP. Platelet aggregates were detected and quantified in murine portal blood, while endothelial P-selectin expression was determined using the dual radiolabeled mAb technique. Platelet activation and aggregation were studied in HP-infected patients and controls by measuring the platelet-aggregate ratio and platelet P-selectin expression. HP infection induced a marked increase in the flux of rolling leukocytes and the appearance of platelet and leukocyte- platelet aggregates in murine gastric venules. The HP-induced rolling and platelet aggregate formation was abrogated by mAbs against L- or P-, but not E- selectin. Endothelial cell expression of P-selectin was not altered, but platelet P-selectin expression was enhanced in HP-infected mice. Circulating platelet aggregates and activated platelets were also detected in HP-infected patients. These findings indicate that platelet activation and aggregation contribute to the microvascular dysfunction and inflammatory cell recruitment associated with HP infections.

Increased chymotrypsin-like protease (chymase) expression and activity in placentas from women with preeclampsia.

Placenta-derived chymotrypsin-like protease (CLP/chymase) promotes endothelial P-selectin and E-selectin expression, which may be responsible for the increased neutrophil/endothelial interactions in preeclampsia (PE). However, little is known about this protease expression and production in human placenta. This study was undertaken to determine the distribution and gene expression of CLP in human placenta. Human placental tissues were obtained immediately after delivery from normal and PE pregnancies. We examined (1) CLP/chymase immunoactivity by immunohistochemical staining of villous tissue sections; (2) trophoblast mRNA and protein expression for chymase by RT-PCR and Western blot analysis; (3) chymase cDNA sequencing in isolated trophoblast cells (TCs); and (4) release of CLP by placental villous tissue cultured under 2% and 20% O(2). We found (1) CLP expression is mainly localized in the epithelial layer of syncytiotrophoblasts; (2) both mRNA and protein expression are significantly (p<0.05) upregulated in TCs isolated from PE vs. normal placentas; (3) TC chymase cDNA sequence and the deduced amino acid sequence are 100% identical to that reported for the human heart; and (4) villous tissue releases more chymotrypsin when cultured with 2% O(2). We conclude that (1) the DNA and protein sequence for chymase in placental trophoblast cells are the same as those reported in the human heart; (2) CLP/chymase expression is upregulated in TCs during PE; and (3) lowered oxygen condition promotes CLP release by placental TCs. Since chymase is a potent non-ACE angiotensin II producing enzyme, our data suggest that if placenta-derived CLP/chymase is released into the maternal circulation, it may contribute to the cardiovascular complications associated with PE.

Postanoxic T lymphocyte-endothelial cell interactions induce tumor necrosis factor-alpha production and neutrophil adhesion: role of very late antigen-4/vascular cell adhesion molecule-1.

The objective of this study was to define the influence of postanoxic T-lymphocyte-endothelial cell interactions on anoxia-reoxygenation (A/R)-induced neutrophil-endothelial cell adhesion and cell adhesion molecule (CAM) expression on human umbilical vein endothelial cells (HUVECs). HUVEC monolayers were exposed to 60 minutes of anoxia, followed by 24 hours of reoxygenation, wherein freshly isolated human T lymphocytes were added at 6 hours during reoxygenation. After an additional 18 hours of incubation (ie, total of 24 hours of reoxygenation), the T-cell/endothelial cell (TC/EC) coculture media were collected and added to naive HUVEC monolayers incubated with neutrophils. Although the A/R-conditioned media per se had no effect on neutrophil adhesion, the media from TC/EC cocultures significantly increased the adhesion response. This enhanced adhesive interaction was associated with significant increases in tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) levels in the TC/EC coculture media and was accompanied by a pronounced increase in endothelial E-selectin expression. Treatment of the TC/EC coculture media with anti-TNF-alpha or anti-IL-8 antibodies reduced the media-induced neutrophil adhesion response. The enhanced neutrophil adhesion and the elevated medium levels of TNF-alpha, but not IL-8, were markedly reduced by inserts that prevented direct TC/EC contact and by monoclonal antibodies directed against vascular cell adhesion molecule-1 (VCAM-1) or very late antigen-4 (VLA-4). Collectively, these findings show that VLA-4-/VCAM-1-mediated interactions between T lymphocytes and postanoxic endothelial cells stimulates TNF-alpha production, which in turn elicits endothelial cell adhesion molecule expression and a corresponding increase in neutrophil adhesion.

T-lymphocyte-derived tumor necrosis factor exacerbates anoxia-reoxygenation-induced neutrophil-endothelial cell adhesion.

The overall objective of this study was to determine whether T lymphocytes can modulate the increased neutrophil adherence and upregulation of endothelial cell adhesion molecules in human umbilical vein endothelial cells (HUVECs) exposed to anoxia/reoxygenation (A/R). HUVEC monolayers were exposed to 60 minutes of anoxia, followed by 4 hours of reoxygenation in the absence or presence of human T lymphocytes. The A/R-induced neutrophil adhesion was significantly enhanced when T lymphocytes and HUVECs were cocultured for the first 45 minutes of reoxygenation. This was accompanied by a more pronounced increase in E-selectin expression. When T lymphocytes were cocultured with HUVECs by use of inserts that prevented direct cell-cell contact, a comparable A/R-induced enhancement of neutrophil adhesion and of E-selectin expression was observed, indicating that soluble factors produced by T lymphocytes mediate the exaggerated A/R-induced inflammatory responses. Treatment with either an anti-tumor necrosis factor-alpha antibody or catalase attenuated the T-cell-mediated responses in postanoxic HUVECs. Moreover, the T-cell-mediated neutrophil adhesion response was mimicked by exposure of naive HUVECs to H(2)O(2). These findings indicate that H(2)O(2) produced by postanoxic endothelial cells stimulates T cells to produce tumor necrosis factor-alpha, which in turn elicits endothelial cell adhesion molecule expression and a corresponding increase in neutrophil adhesion.

NAD(P)H oxidase-derived superoxide mediates hypercholesterolemia-induced leukocyte-endothelial cell adhesion.

Experimental animals placed on a high-cholesterol diet for 2 or more weeks exhibit an inflammatory response in postcapillary venules. The aims of this study were to determine (1) whether superoxide mediates the hypercholesterolemia-induced inflammatory response and (2) whether leukocyte and/or vessel wall NAD(P)H oxidase contributes to this response. Intravital videomicroscopy was used to quantify leukocyte-endothelial cell adhesion in cremasteric postcapillary venules of wild-type (WT) mice, CuZn-superoxide dismutase transgenic (SOD TgN) mice, and mice heterozygous (p47(phox)+/-) or homozygous (p47(phox)-/-) for NAD(P)H oxidase placed on either a normal diet or high-cholesterol diet (HCD) for 2 weeks. The number of adherent and emigrated leukocytes in postcapillary venules of WT HCD mice was significantly higher than that detected in venules of their normal-diet counterparts. However, the HCD-induced recruitment of adherent and emigrated leukocytes was not observed in SOD TgN mice. Whereas hypercholesterolemic p47(phox)+/- and WT mice exhibited similar inflammatory responses, p47(phox)-/- mice did not. Bone marrow chimeras were developed to selectively delete p47(phox) from either the vessel wall or circulating leukocytes. Whereas WT marrow transplanted into WT mice produced a normal inflammatory response of venules to HCD, chimeric mice with p47(phox) deficiency in either the vessel wall or leukocytes exhibited an attenuated inflammatory response to HCD that was comparable with that observed in p47(phox)-/- HCD mice. Our findings indicate that enhanced superoxide production is a critical event that initiates the leukocyte-endothelial cell adhesion in postcapillary venules of HCD mice. NAD(P)H oxidase appears to be an important source of this superoxide.

The microcirculation and inflammation: modulation of leukocyte-endothelial cell adhesion.

The accumulation of leukocytes in inflamed tissue results from adhesive interactions between leukocytes and endothelial cells within the microcirculation. These adhesive interactions and the excessive filtration of fluid and protein that accompanies an inflammatory response are largely confined to one region of the microvasculature: postcapillary venules. The nature and magnitude of the leukocyte-endothelial cell adhesive interactions that take place within postcapillary venules are determined by a variety of factors, including expression of adhesion molecules on leukocytes and/or endothelial cells, products of leukocyte (superoxide) and endothelial cell (nitric oxide) activation, and the physical forces generated by the movement of blood along the vessel wall. The contribution of different adhesion molecules to leukocyte rolling, adherence, and emigration in venules is discussed. Emerging views on potential endogenous antiadhesion molecules produced by endothelial cells as well as the influence of alterations in shear rate on leukocyte adhesion are addressed. Finally, the pathophysiological significance of the microvascular responses to inflammation are discussed in terms of adhesion-directed strategies for the treatment of different cardiovascular diseases and circulatory disorders.

Leukocyte-endothelial cell adhesive interactions: role of xanthine oxidase-derived oxidants.

The objective of this study was to determine whether agents that either scavenge or inhibit the production of oxygen radicals can alter the adhesive interactions between leukocytes and venular endothelium elicited by ischemia-reperfusion. Cat mesenteric and intestinal blood flows were reduced to 20% of baseline for 1 hr, followed by 1 hr of reperfusion. Sixty minutes after reperfusion, red blood cell velocity (Vr), leukocyte rolling velocity (Vw), and the number of adherent leukocytes were measured in mesenteric venules. Then, either manganese-superoxide dismutase (Mn-SOD), catalase, desferrioxamine, or oxypurinol was administered intravascularly. Ten minutes later, repeat measurements were obtained and compared with pretreatment values. Catalase, Mn-SOD, and oxypurinol significantly attenuated neutrophil adherence while neither inactivated-catalase nor desferrioxamine altered the reperfusion-induced leukocyte adhesion. The ratio of Vw to erythrocyte velocity, an index of the fracture stress between rolling leukocytes and venular endothelium, was not altered by any of the agents studied. These results and data in the literature indicate that many of the agents that are commonly used to either scavenge or inhibit the production of oxygen radicals in postischemic tissues exert a significant inhibitory influence on leukocyte adhesion to microvascular endothelium in vivo. Our results are also consistent with the view that xanthine oxidase-derived oxidants contribute to the leukocyte-endothelial cell adhesive interactions associated with reperfusion of ischemic tissues.

P-selectin-dependent leukocyte recruitment and intestinal mucosal injury induced by lactoferrin.

Plasma concentrations of lactoferrin relevant to an inflammatory response are known to elicit leukocyte-endothelial cell adhesion in mesenteric venules. The objectives of this study were (1) to determine whether exogenously administered lactoferrin causes microvascular and mucosal injury in rat intestine and (2) to assess the contribution of adherent leukocytes to a lactoferrin-mediated injury process. Mucosal myeloperoxidase (MPO) activity and vascular protein clearance were monitored in the distal intestine of male Sprague-Dawley rats. Macroscopic erosive lesions of the mucosa and increases in mucosal MPO and intestinal vascular protein were observed 2 h following the lactoferrin infusion, results consistent with granulocyte accumulation and microvascular protein leakage. These lactoferrin-induced alterations were significantly attenuated in animals pretreated with a monoclonal antibody (mAb) directed against P-selectin but not by an E-selectin-specific mAb. In another series of experiments, leukocyte adherence/emigration and leakage of fluorescein isothiocyanate (FITC)-labeled albumin were measured in rat mesenteric venules using intravital video microscopy. Lactoferrin elicited increases in both leukocyte adhesion/emigration and albumin extravasation, which were attenuated by mAbs directed against P-selectin but not E-selectin. These observations indicate that (1) the lactoferrin released by activated neutrophils may lead to significant microvascular and mucosal injury or dysfunction and (2) the lactoferrin-induced injury is related to P-selectin-mediated adhesion of leukocytes to microvascular endothelium. Our results raise the possibility that neutrophil-derived lactoferrin contributes to the inflammatory response by promoting further granulocyte accumulation and activation and that mAbs to P-selectin may be therapeutically beneficial in inflammatory disorders.

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.

Reperfusion-induced oxidative stress in diabetes: cellular and enzymatic sources.

Reactive oxygen metabolites (ROMs) have been implicated in the pathogenesis of the inflammatory response to ischemia/reperfusion (I/R), which is exacerbated in diabetes. This study revealed an increased (P < 0.01) ROMs production in mesenteric tissue (measured using the oxidant-sensitive fluorochrome dihydrorhodamine 123) after I/R in control and diabetic rats, with larger increments (P <0.0001) observed in the latter group, that was associated with an increased inflammatory response measured by intravital microscopy. Either xanthine oxidase inhibition, superoxide scavenging, ICAM-1 immunoneutralization, or blockade of platelet-activating factor or leukotrienes effectively reduced leukocyte recruitment and ROMs production in control and diabetic rats. Moreover, neutrophils from diabetic rats showed an enhanced production of ROMs in vitro in basal and stimulated conditions. We conclude that the oxidative stress during reperfusion is markedly enhanced in diabetes and this appears to result from increased leukocyte recruitment and a higher capacity of diabetic leukocytes to generate ROMs in response to stimulation.

Expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in acute and chronic inflammation.

The objective of this study was to quantify, in vivo, constitutive and tumor necrosis factor alpha (TNF-alpha)-enhanced expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in different tissues from healthy wild-type mice (C57BL/6) as well as interleukin-10 (IL-10)-deficient mice with and without active colitis. Using the dual radiolabel monoclonal antibody technique, we found substantial constitutive expression of MAdCAM-1 in the intestine, colon, and mesenteric lymph nodes. MAdCAM-1 expression in these tissues was significantly enhanced, in a time-dependent manner, by systemic administration of TNF-alpha. Maximum surface expression was observed at 18 h after TNF-alpha administration and remained significantly elevated at 48 h post-TNF-alpha injection. No significant constitutive nor TNF-alpha-induced expression of MAdCAM-1 was detected in skeletal muscle, brain, or heart. In IL-10-deficient (IL-10 k/o) mice with no clinical or histological evidence of colitis, constitutive and TNF-alpha-induced expression of MAdCAM-1 in the intestine, cecum, and colon was not different from those values obtained with healthy wild-type controls. IL-10-deficient mice with active colitis exhibited a four- to fivefold greater expression of MAdCAM-1 in the cecum and colon compared with their healthy controls or to IL-10 k/o mice with no evidence of colitis. Taken together, these data demonstrate that TNF-alpha enhances surface expression of MAdCAM-1 in intestinal and colonic tissues to the same extent in both wild-type and IL-10 k/o mice with no colonic inflammation, whereas IL-10 k/o mice with active colitis exhibited a profound up-regulation of MAdCAM-1 in the colon.

Intercellular cell adhesion molecule-1 regulates lymphocyte movement into intestinal microlymphatics of rat Peyer's patches.

The objective of this study was to determine whether specific adhesion molecules modulate lymphocyte movement from Peyer's patches into intestinal microlymphatics. The fluorochrome acridine orange was injected via a micropipette into Peyer's patches to fill lymphatics. The flux of labeled lymphocytes into intestinal microlymphatics was monitored with intravital fluorescence microscopy. The lymphatic microvessels in the perifollicular area of Peyer's patches were filled with lymphocytes, most of which remained within the lymphatics. Some lymphocytes became detached and were drained into intestinal lymph. Administration of antibodies directed against ICAM-1 significantly increased lymphocyte flux into interfollicular lymphatics. The immunohistochemical study showed intense ICAM-1 expression on the lymphocytes densely packed in the lymphatics surrounding follicles in Peyer's patches. A large number of lymphocytes are normally sequestered in the lymphatic network of Peyer's patches. This sequestration of lymphocytes is largely mediated by ICAM-1-dependent cell-cell interactions.

Leukocyte-endothelial cell interactions evoked by mast cells.

In this review we have summarized some of the evidence to support the view that mast cells play a critical role in leukocyte recruitment to sites of inflammation. Initially, data using a pharmacological tool, compound 48/80, which directly activates mast cells, is reviewed, demonstrating that this reagent can induce the multi-step recruitment of leukocytes (rolling, adhesion and emigration) to sites of inflammation. The adhesive mechanisms and pro-inflammatory mediators implicated in mast cell-induced leukocyte recruitment are discussed. Additionally, data are presented to implicate mast cells in delayed-type hypersensitivity reactions as they pertain to leukocyte recruitment. There is a growing body of evidence to suggest that mast cells also recruit leukocytes in IgE-independent leukocyte recruitment. Ischemia/reperfusion- and bacterial toxin- (Helicobacter pylori and Clostridium difficile) induced leukocyte recruitment is at least in part mast cell dependent. Future directions including preliminary work highlighting the role of nitric oxide as a modulator of mast cell function and subsequent leukocyte recruitment is also discussed.