T cell-associated α4ß7 but not α4ß1 integrin is required for the induction and perpetuation of chronic colitis.

Anti-adhesion therapies that target α(4) integrins (e.g., natalizumab) are thought to work by blocking T-cell recruitment to the intestinal tissues in patients with Crohn's disease (CD); however, little direct evidence is available to confirm this contention. We wished to evaluate the importance of T cell-associated α(4) integrins in a chronic colitis model in mice and to determine the effect of natalizumab treatment on intestinal tissue T-cell accumulation in human CD. Adoptive transfer of T cells lacking α(4) (α(4)(-/-)) but not ß(1) integrin into immunodeficient mice produced significantly attenuated disease. This was correlated with reduced numbers of colon CD4 T cells compared with the control mice; however, tissue distribution of T helper type 1 (Th1) and T helper type 17 (Th17) cells and regulatory T cells (Tregs) was not affected by the lack of α(4). Furthermore, α(4)(-/-) T cells demonstrated defective homing to the chronically inflamed small intestines and colons. Finally, patients treated with natalizumab showed significant reduction in mucosal CD4 T cells and no skewing in the foxp3(+) Treg or T-bet(+)Th1 fractions thereof. These results demonstrate a direct role for T cell-associated α(4)ß(7) but not α(4)ß(1) integrins during initiation and perpetuation of chronic colitis. Moreover, our data demonstrated that natalizumab treatment reduced mucosal CD4 T-cell accumulation in CD patients.

Evaluation of the immunoregulatory activity of intraepithelial lymphocytes in a mouse model of chronic intestinal inflammation.

Intraepithelial lymphocytes (IELs) represent the first line of lymphocyte defense against the intestinal bacteria. Although previous studies have demonstrated a protective role of IELs in the development of colitis, the data supporting a regulatory role for IELs are limited. The objective of this study was to examine the suppressive activity of IELs in vitro and in vivo using a mouse model of chronic small and large bowel inflammation. Adoptive transfer of CD8α(+) IELs isolated from small intestines of wild-type (WT) mice into TCR ßxδ-deficient (TCR ßxδ(-/-)) recipients did not prevent or delay the onset of the disease induced by WT CD4(+)CD45RB(high) T cells. On the contrary, we observed a more rapid onset of wasting and clinical signs of intestinal inflammation when compared with animals injected with CD4(+)CD45RB(high) T cells alone. Histopathological scores of small and large bowel did not differ significantly between the two groups. Transfer of IELs alone did not produce any pathological changes. Real-time PCR analysis of intestinal tissues showed up-regulation of message for T(h)1- and macrophage-derived cytokines in colon and small bowel. Using Foxp3-GFP reporter mice, we were unable to detect any Foxp3(+) cells within the CD8α(+) IELs but did find a small population of Foxp3(+)CD4(+) IELs in the small and large bowel. Using in vitro suppression assay, we found that neither TCRαß(+)CD8αα(+), TCRαß(+)CD8αß(+) nor TCRγδ(+)CD8αα(+) IELs were capable of suppressing CD4(+) T-cell proliferation. Taken together, our data do not support an immunoregulatory role for CD8α(+) IELs in a mouse model of small and large bowel inflammation.

Emerging roles of lymphatics in inflammatory bowel disease.

The mobilization and recruitment of blood and lymphatic vasculatures are widely described in inflammatory bowel diseases (IBDs). Although angiogenesis contributes to intense gut inflammation, it remains unclear whether and when lymphangiogenesis amplifies or protects in IBD. The prolonged maintenance of lymphatic (over blood vessels) in inflammation indicates that lymphatic-blood vessel interactions may regulate IBD pathogenesis and restitution. Although lymphatic expansion helps to restore fluid balance and clear cytokines and immune cells, lymphatic failure results in accumulation of these factors and exacerbates IBD. Lymphatic obstruction and remodeling may impair lymphatic pumping, leading to repeated rounds of lymphangiogenesis. Early descriptions of Crohn's disease and ulcerative colitis describe colon lymphatic congestion, remodeling, expansion, and many other features that are recapitulated in experimental IBD and also by intestinal lymphatic obstruction, supporting lymphangitis as a cause and consequence of IBD. Growth factors, cytokines, gut flora, Toll receptors, and leukocytes all regulate inflammation and gut lymphatic remodeling in IBD. This review summarizes the importance of lymphatics and lymphangiogenesis in IBD etiology that may be useful in diagnosis and therapy of gut inflammation.

Combination of donor-specific blood transfusion with anti-CD28 antibody synergizes to prolong graft survival in rat liver transplantation.

Donor-specific blood transfusion (DST) has been shown to effectively induce tolerance to certain allografts. In addition, it is well known that blockade of costimulatory signals reduces the ability of T cells to respond to alloantigens, prolonging allograft survival in some transplant models. We assessed the effects of single or multiple DSTs in the absence or presence of anti-CD28 monoclonal antibodies (mAbs) on graft function and host survival in rat liver transplantation (LTx). Fully MHC-mismatched adult male Dark Agouti (DA) and Lewis (LEW) rats were used as donors and recipients, respectively. Heparinized DA blood was administered to naïve LEW rats 7 days before LTx [DST(-7d)], 14 and 7 days before LTx [DST(1 x 2)], twice a week for 2 weeks prior to LTx [DST(2 x 2)] and once a week for 4 weeks prior to LTx [DST(1 x 4)]. For some experiments, two different monoclonal antibodies (mAb) to rat CD28 (JJ316 and JJ319) were administered in combination with some DST treatments. We found that DST administration induced a time- and dose-dependent increase in host survival. Treatment of LEW rats with JJ316 or JJ319 mAb alone failed to prolong graft survival over untreated rats; however, the combination of DST(1 x 2) with JJ316 or JJ319 mAb induced indefinite survival at 100 days following surgery. We found that this protective effect was associated with increased numbers of splenic CD4+ CD45RC- but not CD4+ CD25+ foxp3+ T-cells in long-term survivors. Our data suggest that the combination of suboptimal DST with CD28 mAb induces donor-specific tolerance that correlates with enhanced numbers of regulatory T-cells.

Increased disease activity in eNOS-deficient mice in experimental colitis.

Oral dextran sodium sulfate (DSS, 3%) produces experimental colitis with many features of human inflammatory bowel disease (IBD), (leukocyte extravasation, cachexia, and histopathology). Previous studies suggest that the inducible nitric oxide synthase (iNOS) in blood cells or in the endothelium contribute to this injury. However, until now no study has been performed to directly evaluate the role of endothelial nitric oxide synthase (eNOS) in IBD. We compared disease activity in wild-type (eNOS+/+) and eNOS-deficient (eNOS-/-) mice in the DSS model of colitis. Administration of DSS induced weight loss, stool blood, and overt histopathology in both mouse strains. Disease activity was dramatically increased in eNOS-/- mice compared to wild types. Histologically, eNOS-deficient mice had greater leukocyte infiltration, gut injury, and expressed higher levels of the mucosal addressin, MAdCAM-1. These results demonstrate that eNOS plays an important role in limiting injury to the intestine during experimental colitis and altered eNOS content and/or activity may contribute to human IBD.

Polynitroxylated starch/TPL attenuates cachexia and increased epithelial permeability associated with TNBS colitis.

Free radicals play an important role in the initiation and progression of inflammatory bowel disease (IBD). Therefore, the reduction or elimination of adverse oxidant effects can provide novel therapy for IBD. Here, the antioxidant capacity and protective effects of a new class of chemically modified hetastarch (polynitroxyl starch, or PNS) plus 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol or TPL) (PNS/TPL) were assessed in a model of colitis. The superoxide scavenging capacity of PNS/TPL-that is, the inhibition of the reduction of cytochrome c in the presence of xanthine/xanthine oxidase (X/XO)-was evaluated in vitro. The effects of PNS/TPL on X/XO-induced neutrophil endothelial adhesion in vitro were investigated. Also, this study tested the protection produced by PNS/TPL in a mouse model of trinitrobenzene sulfonic acid (TNBS)-induced colitis. PNS/TPL was given intravenously immediately before (< 30 min) and intraperitoneally at 24 and 72 hr after TNBS induction. The body weight and survival rate of the mice were checked daily. Colonic mucosal damage was assessed on the 7th day by measuring intestinal permeability to Evans blue (EB) in vivo. The ability of PNS to reoxidize bioreduced TPL was documented by whole-body electron paramagnetic resonance (EPR) detection. We found that PNS or TPL exhibits superoxide dismutase (SOD)-like activity, with approximately 2% of SOD activity occurring on a molar basis. The endothelial-neutrophil adherence induced by X/XO was significantly inhibited by PNS/TPL but not by TPL alone. PNS/TPL protected against cachexia and mortality, both usually induced by TNBS. Epithelial permeability was increased significantly in TNBS mice but was ameliorated by the administration of PNS/TPL. In conclusion, PNS/TPL may be beneficial in the treatment or prevention of IBD through its antioxidant effects, which inhibit oxidant-mediated leukocyte adhesion and injury to endothelial cells.

Role of appendix and spleen in experimental colitis.

There is growing clinical evidence suggesting that certain secondary lymphoid tissues (e.g., appendix and spleen) contribute to the initiation and/or perpetuation of ulcerative colitis. In this study, the importance of secondary lymphoid tissues in inducing colitis was assessed experimentally by removing the spleen and/or appendix (or sham operation) prior to inducing colitis in mice. Feeding 2.5% dextran sulphate sodium (DSS) in drinking water over 7 days induced colitis. Clinical disease activity was assessed based on weight loss, stool consistency, and presence of blood in stools. Additional measurements included white blood cell count and hematocrit, and myeloperoxidase activity (MPO) in colon samples. Colonic injury was assessed by histology and computerized image analysis. DSS treatment in sham-operated mice produced colitis associated with weight loss, bloody diarrhea, and mucosal ulceration. Clinical assessment of DSS-treated mice subjected to appendectomy or combined appendectomy/splenectomy exhibited a delayed onset and course of disease activity. Histomorphologic examination revealed significantly lower damage scores and a reduction in ulcerated mucosal surface area. Colonic MPO activity, which correlated with tissue injury and disease activity, was lowest in appendectomized mice. No beneficial effects of splenectomy were observed after 7 days of colitis. These findings support the hypothesis that appendicular lymphoid tissue, but not the spleen, contributes to the development of colitis.

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.

Immunological basis of inflammatory bowel disease: role of the microcirculation.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the intestine and/or colon of unknown etiology in which patients suffer from severe diarrhea, rectal bleeding, abdominal pain, fever, and weight loss. Active episodes of IBD are characterized by vasodilation, venocongestion, edema, infiltration of large numbers of inflammatory cells, and erosions and ulcerations of the bowel. It is becoming increasingly apparent that chronic gut inflammation may result from a dysregulated immune response toward components of the normal intestinal flora, resulting in a sustained overproduction of proinflammatory cytokines and mediators. Many of these Th1 and macrophage-derived cytokines and lipid metabolites are known to activate microvascular endothelial cells, thereby promoting leukocyte recruitment into the intestinal interstitium. This review discusses the basic immune mechanisms involved in the regulation of inflammatory responses in the gut and describes how a breakdown in this protective response initiates chronic gut inflammation.

Selected contribution: Effects of gender on reduced-size liver ischemia and reperfusion injury.

Hepatic resection with concomitant periods of ischemia and reperfusion (I/R) is a common occurrence in resectional surgery as well as reduced-size liver transplantation (e.g., split liver or living donor transplantation). However, the I/R induced by these types of surgical manipulations may impair liver regeneration, ultimately leading to liver failure. The objectives of the study were to develop a murine model of reduced-size liver I/R and assess the role of gender in this model of hepatocellular injury. We found that 100% of female mice survived the surgery indefinitely, whereas all male mice had greater initial liver injury and died within 5 days after surgery. The protective effect observed in females appeared to be due to ovarian 17beta-estradiol, as ovariectomy of females or administration of a selective estrogen antagonist to female mice resulted in enhanced liver injury and greater mortality following reduced-size liver I/R. Conversely, 17beta-estradiol-treated male mice exhibited less hepatocellular damage and survived indefinitely. Taken together, these data demonstrate an estrogen-mediated protective pathway(s) that limits or attenuates hepatocellular injury induced by reduced-size liver I/R.

Role of nitric oxide in inflammation.

A number of laboratories have sought to elucidate the role of nitric oxide (NO) in both acute and chronic inflammatory diseases. It is now well appreciated that NO can influence many aspects of the inflammatory cascade ranging from its own expression to recruitment of leucocytes to the effected tissue. With the advent of mice selectively deficient in the various isoforms of nitric oxide synthase (NOS), the role that NO may play in various disease states can now be examined in vivo. One such syndrome that has gained much attention in recent years is ischaemia and reperfusion-induced tissue injury. Ischaemia-reperfusion (I/R) injury is an important clinical consideration in situations such as transplantation, trauma, liver or bowel resection and haemorrhagic shock. A hallmark of I/R is the production of reactive oxygen species (ROS) during the reperfusion phase and it is thought that the production of ROS mediate much of the post-ischaemic tissue injury. This review will examine the current state of knowledge regarding the regulatory mechanisms by which NO can influence various aspects of the inflammatory cascade as well as its role in a model of I/R injury in vivo.

MAdCAM mediates lymphocyte-endothelial cell adhesion in a murine model of chronic colitis.

Previous studies have revealed that the expression of several endothelial cell adhesion molecules [e.g., intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and mucosal addressin cell adhesion molecule 1 (MAdCAM-1)] is dramatically elevated in the chronically inflamed colonic vasculature of severe combined immunodeficient (SCID) mice reconstituted with congenic CD4+, CD45RB(high) T lymphocytes. The objective of this study was to define the contribution of different endothelial cell adhesion molecules to the lymphocyte-endothelial cell (L/E) adhesion observed in the colonic microvasculature in this experimental model of inflammatory bowel disease. Fluorescently labeled T lymphocytes, isolated from spleens of normal BALB/C mice, were injected intravenously into SCID mice that had been reconstituted with CD4+, CD45RB(high) T lymphocytes either before (3 wk after reconstitution) or after (7 wk postreconstitution) the onset of clinical signs of colitis (i.e., diarrhea, loss of body wt). Intravital fluorescence microscopy was used to quantify L/E adhesion in different-sized venules of the colonic submucosa during the development of colitis. L/E adhesion was noted in some segments of the vasculature in precolitic SCID mice (3 wk after reconstitution) but not in similar-sized vessels of control (wild type and SCID) mice. L/E adhesion was observed in a greater proportion of venules and occurred with greater intensity in the mucosa of colitic mice (7 wk postreconstitution). Pretreatment with a blocking monoclonal antibody against MAdCAM-1, but not ICAM-1 or VCAM-1, significantly and profoundly reduced L/E adhesion in colitic mice. Immunohistochemical staining also revealed the localization of T cells on colonic endothelial cells expressing MAdCAM-1. These findings indicate that MAdCAM-1 is largely responsible for recruiting T lymphocytes into inflamed colonic tissue.

Regulation and distribution of MAdCAM-1 in endothelial cells in vitro.

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a 60-kDa endothelial cell adhesion glycoprotein that regulates lymphocyte trafficking to Peyer's patches and lymph nodes. Although it is widely agreed that MAdCAM-1 induction is involved in chronic gut inflammation, few studies have investigated regulation of MAdCAM-1 expression. We used two endothelial lines [bEND.3 (brain) and SVEC (high endothelium)] to study the signal paths that regulate MAdCAM-1 expression in response to tumor necrosis factor (TNF)-alpha using RT-PCR, blotting, adhesion, and immunofluorescence. TNF-alpha induced both MAdCAM-1 mRNA and protein in a dose- and time-dependent manner. This induction was tyrosine kinase (TK), p42/44, p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-kappa B/poly-ADP ribose polymerase (PARP) dependent. Because MAdCAM-1 is regulated via MAPKs, we examined mitogen/extracellular signal-regulated kinase (MEK)-1/2 activation in SVEC. We found that MEK-1/2 is activated by TNF-alpha within minutes and is dependent on TK and p42/44 MAPKs. Similarly, TNF-alpha activated NF-kappa B through TK, p42/44, p38 MAPKs, and PARP pathways in SVEC cells. MAdCAM-1 was also shown to be frequently distributed to endothelial junctions both in vitro and in vivo. Cytokines like TNF-alpha stimulate MAdCAM-1 in high endothelium via TK, p38, p42/22 MAPKs, and NF-kappa B/PARP. MAdCAM-1 expression requires NF-kappa B translocation through both direct p42/44 and indirect p38 MAPK pathways in high endothelial cells.

TNF-alpha induced endothelial MAdCAM-1 expression is regulated by exogenous, not endogenous nitric oxide.

BACKGROUND: MAdCAM-1 is an adhesion molecule expressed in Peyer's patches and lymphoid tissues which is mobilized by cytokines like TNF-alpha and is a major determinant of lymphocyte trafficking to the gut in human inflammatory bowel disease (IBD). It has been suggested that both reactive oxygen and nitrogen metabolites participate in regulating adhesion molecule expression in response to TNF-alpha. METHODS: To examine how exogenous and endogenous sources of NO modulate MAdCAM-1 induction by TNF-alpha, we pre-treated mouse lymphatic endothelial cells with either long or short acting NO donors prior to TNF-alpha-stimulation, and measured MAdCAM-1 induction at 24 h. RESULTS AND DISCUSSION: DETA-NO, a long-acting NO donor, and SperNO, a rapid releasing NO donor both inhibited TNF-alpha-stimulated MAdCAM-1 expression in a concentration dependent manner. Both NO donors also reduced a4b7-dependent lymphocyte endothelial adhesion. Inhibition of endogenous NO production by either L-NAME, a non-selective NOS inhibitor, or by 1400 w, a selective iNOS inhibitor failed to induce, or potentiate TNF-alpha regulated MAdCAM-1 expression. CONCLUSIONS: Exogenous NO donors may be beneficial in the treatment of IBD, while endogenous nitric oxide synthases may be less effective in controlling adhesion molecule expression in response to cytokines.

Myoglobin-catalyzed tyrosine nitration: no need for peroxynitrite.

The nitration of tyrosine residues in protein to yield 3-nitrotyrosine derivatives has been suggested to represent a specific footprint for peroxynitrite formation in vivo. However, recent studies suggest that certain hemoproteins such as peroxidases catalyze the H(2)O(2)-dependent nitration of tyrosine to yield 3-nitrotyrosine in a peroxynitrite-independent reaction. Because 3-nitrotyrosine has been shown to be present in the postischemic myocardium, we wished to assess the ability of myoglobin to catalyze the nitration of tyrosine in vitro. We found that myoglobin catalyzed the oxidation of nitrite and promoted the nitration of tyrosine. Both nitrite oxidation and tyrosine nitration were H(2)O(2)-dependent and required the formation of ferryl (Fe(+4)) myoglobin. In addition, nitrite oxidation and tyrosine nitration were pH-dependent with a pH optimum of approximately 6.0. Taken together, these data suggest that the acidic pH and low oxygen tension produced during myocardial ischemia will facilitate myoglobin-catalyzed, peroxyntrite-independent formation of 3-nitrotyrosine.

Glucocorticoids and IL-10, but not 6-MP, 5-ASA or sulfasalazine block endothelial expression of MAdCAM-1: implications for inflammatory bowel disease therapy.

BACKGROUND: Enhanced MAdCAM-1 (mucosal addressin cell adhesion molecule-1) expression is associated with the aetiology of inflammatory bowel disease, but little is known about MAdCAM-1: regulation, or how inflammatory bowel disease therapies modulate MAdCAM-1. AIM: To examine how agents currently used to treat inflammatory bowel disease affect MAdCAM-1: induced by tnf-alpha in an in vitro model of inflammatory bowel disease. METHODS: Endothelial monolayers were pretreated with dexamethasone (DEX): 5-aminosalicylic acid (5-ASA), 6-mercaptopurine (6-MP), sulfasalazine or interleukin-10: (IL-10: prior to TNF-alpha (20 ng/mL), and MAdCAM-1: measured by Western blotting, RT-PCR, EMSA and lymphocyte adhesion assays. RESULTS: MAdCAM-1: was induced dose- and time-dependently by TNF-alpha on endothelial cells. Either dexamethasone or IL-10: reduced TNF-alpha-induced MAdCAM-1: protein, mRNA and lymphocyte adhesion. However, neither 5-ASA, sulfasalazine nor 6-MP blocked MAdCAM-1 induction. CONCLUSIONS: Our data indicate that dexamethasone or IL-10 can exert therapeutic activity in inflammatory bowel disease through MAdCAM-1 inhibition. 5-ASA, sulfasalazine and 6-MP, while beneficial in inflammatory bowel disease, do not directly control MAdCAM-1, and are beneficial through inhibition of other inflammatory processes.

Enhanced post-ischemic liver injury in iNOS-deficient mice: a cautionary note.

The objective of this study was to assess the role of inducible nitric oxide synthase (iNOS) in ischemia- and reperfusion (I/R)-induced liver injury. We found that partial hepatic ischemia involving 70% of the liver resulted in a time-dependent increase in serum alanine aminotransferase (ALT) levels at 1-6 h following reperfusion. Liver injury at 1, 3, and 6 h post-ischemia was not due to the infiltration of neutrophils as assessed by tissue myeloperoxidase (MPO) activity and histopathology. iNOS-deficient mice subjected to the same duration of ischemia and reperfusion showed dramatic and significant increases in liver injury at 3 but not 6 h following reperfusion compared to their wild type controls. Paradoxically, iNOS mRNA expression was not detected in the livers of wild type mice at any point during the reperfusion period and pharmacological inhibition of iNOS using L-N(6)(iminoethyl)-lysine (L-NIL) did not exacerbate post-ischemic liver injury at any time post-reperfusion. These data suggest that iNOS deficiency produces unanticipated genetic alterations that renders these mice more sensitive to liver I/R-induced injury.

Mechanisms of the antioxidant effects of nitric oxide.

The Janus face of nitric oxide (NO) has prompted a debate as to whether NO plays a deleterious or protective role in tissue injury. There are a number of reactive nitrogen oxide species, such as N2O3 and ONOO-, that can alter critical cellular components under high local concentrations of NO. However, NO can also abate the oxidation chemistry mediated by reactive oxygen species such as H2O2 and O2- that occurs at physiological levels of NO. In addition to the antioxidant chemistry, NO protects against cell death mediated by H2O2, alkylhydroperoxides, and xanthine oxidase. The attenuation of metal/peroxide oxidative chemistry, as well as lipid peroxidation, appears to be the major chemical mechanisms by which NO may limit oxidative injury to mammalian cells. In addition to these chemical and biochemical properties, NO can modulate cellular and physiological processes to limit oxidative injury, limiting processes such as leukocyte adhesion. This review will address these aspects of the chemical biology of this multifaceted free radical and explore the beneficial effect of NO against oxidative stress.

Role of intercellular adhesion molecule 1 in indomethacin-induced ileitis.

Adhesion molecules have been implicated in the pathogenesis of inflammatory bowel diseases. We investigated their expression and contribution to leukocyte recruitment in experimental intestinal inflammation. Ileitis was induced in Sprague-Dawley rats by two injections of indomethacin (7.5 mg/kg), given 24 h apart. Endothelial intercellular adhesion molecule-1 (ICAM-1) expression was quantified using the dual radiolabeled monoclonal antibody technique and Mac-1 (CD11b/CD18) expression on leukocytes by flow cytometry. Leukocyte infiltration was monitored by tissue myeloperoxidase (MPO) activity. The first indomethacin injection induced a time- and site-dependent increase of ICAM-1 expression in ileal mucosa and muscularis. The second injection resulted in a reduction of ICAM-1 expression below constitutive levels whereas Mac-1 was upregulated. MPO changes paralleled lesion development over 48 h. ICAM-1 and MPO values were correlated for the first 24 h. Immunoneutralization of either ICAM-1 or Mac-1 attenuated mucosal injury. We conclude that (i) indomethacin-induced ileitis is associated with a temporally disassociated upregulation of ICAM-1 and (ii) despite a reduction in ICAM-1 after 24 h, ICAM-1, in concert with Mac-1, contributes to mucosal injury and leukocyte infiltration elicited by indomethacin.