Human eosinophil adherence to vascular endothelium mediated by binding to vascular cell adhesion molecule 1 and endothelial leukocyte adhesion molecule 1.

Adherence of human eosinophils to cytokine-stimulated endothelial cells, which was only partially due to CD18-dependent pathways, was also mediated by binding to endothelial leukocyte adhesion molecule 1 (ELAM-1) and vascular cell adhesion molecule 1 (VCAM-1). Eosinophils bound specifically to both recombinant soluble ELAM-1 and recombinant soluble VCAM-1. Eosinophil binding to recombinant soluble VCAM-1 and to transfected CHO cells expressing VCAM-1 was inhibited with anti-VCAM-1 (4B9) and anti-very late activation antigen 4 (anti-VLA-4; HP1/2 or HP2/1) monoclonal antibodies. Eosinophils, but not neutrophils, expressed VLA-4 detected by cytofluorography. Eosinophil adherence to tumor necrosis factor alpha-stimulated human umbilical vein endothelial cells was partially blocked by monoclonal antibodies against ELAM-1 (BB11) and VCAM-1 (4B9) and against VLA-4 (HP2/1). Thus, while both eosinophils and neutrophils can bind to activated endothelial cells by adherence to ICAM-1 and ELAM-1, only eosinophils expressed VLA-4 and adhered to VCAM-1 on activated endothelial cells. Eosinophil adherence to VCAM-1 might provide a mechanism contributing to the selective recruitment of eosinophils into tissue sites of inflammation.     

siRNA silencing reveals role of vascular cell adhesion molecule-1 in vascular smooth muscle cell migration

Vascular cell adhesion molecule-1 (VCAM-1) is an adhesion molecule expressed by endothelial cells for recruitment of leukocytes during inflammation. It is also abundantly expressed by smooth muscle cells in atherosclerotic lesions and in injured arteries. In this study, we examined the role of VCAM-1 in smooth muscle cell migration. Smooth muscle cells were isolated from the aorta of C57BL/6 mice and transfected with short interfering RNAs (siRNAs) targeting VCAM-1. Inhibition on VCAM-1 expression by siRNAs was assessed by Western blot analysis, RT-PCR and by measuring soluble VCAM-1 concentrations in the incubation medium. One siRNA that showed greater suppression on VCAM-1 expression was used for migration assay. A single scratch wound was made on 70% confluent cells and cells migrated from wounded monolayer were counted 24 and 48h after injury. Treatment with VCAM-1 siRNA resulted in a significant reduction in the number of migrated cells. This siRNA also exhibited a minor effect on smooth muscle cell proliferation. Thus, our findings indicate that VCAM-1 is necessary for the migration of smooth muscle cells and interfering VCAM-1 expression could be an effective approach to prevention and treatment of atherosclerosis and restenosis.     

The role of intercellular adhesion molecule 1 and neutrophils in acute pancreatitis and pancreatitis-associated lung injury

BACKGROUND & AIMS: Intercellular adhesion molecule 1 (ICAM-1) and neutrophils play important roles in many inflammatory processes, but their importance in both acute pancreatitis and pancreatitis-associated lung injury has not been defined. METHODS: To address this issue, mice that do not express ICAM-1 were used and depleted of neutrophils by administration of antineutrophil serum. Pancreatitis was induced by administering either supramaximal doses of the secretagogue cerulein or feeding a choline-deficient, ethionine-supplemented diet. The severity of pancreatitis was evaluated by quantitating serum amylase, pancreatic edema, acinar cell necrosis, and pancreas myeloperoxidase activity (i.e., neutrophil content). Lung injury was evaluated by quantitating lung myeloperoxidase activity and pulmonary microvascular permeability. ICAM-1 was quantitated by enzyme-linked immunosorbent assay and was localized by light-microscopic immunohistochemistry. RESULTS: It was found that serum, pancreas, and lung ICAM-1 levels increase during pancreatitis. Both pancreatitis and the associated lung injury are blunted, but not completely prevented, in mice deficient in ICAM-1. Neutrophil depletion also reduces the severity of both pancreatitis and lung injury. However, the combination of neutrophil depletion with ICAM-1 deficiency does not reduce the severity of pancreatitis or lung injury to a greater extent than either neutrophil depletion or ICAM-1 deficiency alone. Neither pancreatitis nor pancreatitis-associated lung injury are completely prevented by ICAM-1 deficiency, neutrophil depletion, or combined ICAM-1 deficiency plus neutrophil depletion. CONCLUSIONS: The observations indicate that ICAM-1 plays an important, neutrophil-mediated, proinflammatory role in pancreatitis and pancreatitis-associated lung injury. The studies also indicate that ICAM-1 and neutrophil-independent events also contribute to the evolution of pancreatitis and lung injury in these models.     

The leukocyte cell adhesion cascade and its role in myocardial ischemia-reperfusion injury

Cell-cell and cell-matrix interactions are known to be mediated by specific cell adhesion receptors expressed on the cell surface. The characterization of these cell adhesion molecules has allowed researchers to examine their roles in a variety of physiologic and pathophysiologic conditions. Numerous studies have demonstrated that myocardial ischemia-reperfusion injury is an acute inflammatory process in which leukocytes are intimately involved. In this review, we summarize the current data on the leukocyte cell adhesion cascade, focus upon studies which have demonstrated specific cell adhesion molecule interactions which mediate the leukocyte involvement in myocardial ischemia-reperfusion injury, and suggest future avenues of exploration and possible clinical implications of the studies reviewed.R. J. Gumina is a fellow in the Medical Scientist Training Program at the Medical College of Wisconsin. This work was supported in part by a predoctoral fellowship to RJG from the American Heart Association, Wisconsin Affiliate and NIH Grants HL08311 (GJG), HL40926 (PJN), and HL54280 (DCW).     

Regulation of mucosal addressin cell adhesion molecule 1 expression in human and mice by vascular adhesion protein 1 amine oxidase activity

Primary sclerosing cholangitis (PSC) and autoimmune hepatitis are hepatic complications associated with inflammatory bowel disease (IBD). The expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) on mucosal endothelium is a prerequisite for the development of IBD, and it is also detected on the hepatic vessels of patients with liver diseases associated with IBD. This aberrant hepatic expression of MAdCAM-1 results in the recruitment of effector cells initially activated in the gut to the liver, in which they drive liver injury. However, the factors responsible for the aberrant hepatic expression of MAdCAM-1 are not known. In this study, we show that deamination of methylamine (MA) by vascular adhesion protein 1 (VAP-1) [a semicarbazide-sensitive amine oxidase (SSAO) expressed in the human liver] in the presence of tumor necrosis factor α induces the expression of functional MAdCAM-1 in hepatic endothelial cells and in intact human liver tissue ex vivo. This is associated with increased adhesion of lymphocytes from patients with PSC to hepatic vessels. Feeding mice MA, a constituent of food and cigarette smoke found in portal blood, led to VAP-1/SSAO-dependent MAdCAM-1 expression in mucosal vessels in vivo. CONCLUSION: Activation of VAP-1/SSAO enzymatic activity by MA, a constituent of food and cigarette smoke, induces the expression of MAdCAM-1 in hepatic vessels and results in the enhanced recruitment of mucosal effector lymphocytes to the liver. This could be an important mechanism underlying the hepatic complications of IBD.     

Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition.

Although primarily recognized for its role in hemostasis, fibrinogen is also required for competent inflammatory reactions in vivo. It is now shown that fibrinogen promotes adhesion to and migration across an endothelial monolayer of terminally differentiated myelomonocytic cells. This process does not require chemotactic/haptotactic gradients or cytokine stimulation of the endothelium and is specific for the association of fibrinogen with intercellular adhesion molecule 1 (ICAM-1) on endothelium. Among other adhesive plasma proteins, fibronectin fails to increase the binding of leukocytes to endothelium, or transendothelial migration, whereas vitronectin promotes the binding but not the migration. The fibrinogen-mediated leukocyte adhesion and transendothelial migration could be inhibited by a peptide from the fibrinogen gamma-chain sequence N117NQKIVNL-KEKVAQLEA133, which blocks the binding of fibrinogen to ICAM-1. This interaction could also be inhibited by new anti-ICAM-1 monoclonal antibodies that did not affect the ICAM-1-CD11a/CD18 recognition, thus suggesting that the fibrinogen binding site on ICAM-1 may be structurally distinct from regions previously implicated in leukocyte-endothelium interaction. Therefore, binding of fibrinogen to vascular cell receptors is sufficient to initiate (i) increased leukocyte adhesion to endothelium and (ii) leukocyte transendothelial migration. These two processes are the earliest events of immune inflammatory responses and may also contribute to atherosclerosis.     

Platelet-endothelial cell adhesion molecule-1 (PECAM-1)-deficient mice demonstrate a transient and cytokine-specific role for PECAM-1 in leukocyte migration through the perivascular basement membrane

Studies with neutralizing antibodies have indicated roles for platelet-endothelial cell adhesion molecule-1 (PECAM-1) in leukocyte migration through the endothelium and the perivascular basement membrane. Because some of these findings have been contentious, this study aimed to explore the role of PECAM-1 in leukocyte migration by analyzing leukocyte responses in interleukin 1beta (IL-1beta)- and tumor necrosis factor-alpha (TNFalpha)-activated cremasteric venules of PECAM-1-deficient mice using intravital and electron microscopy. Although no differences in levels of leukocyte rolling flux or firm adhesion were observed, a delay in leukocyte transmigration in response to IL-1beta, but not TNFalpha, was detected in PECAM-1-deficient mice. Electron microscopy indicated that this delay occurred at the level of perivascular basement membrane. To address the cytokine specificity of PECAM-1 dependence, in vitro experiments demonstrated that TNFalpha, but not IL-1beta, could induce rapid adhesion of murine neutrophils to protein-coated surfaces, suggesting that TNFalpha elicited leukocyte transmigration in wild-type mice via direct stimulation of leukocytes. In summary, the results suggest a regulatory role for PECAM-1 in leukocyte migration through the perivascular basement membrane, a role that appears to be cytokine-specific and associated with the ability of the cytokine to stimulate rapid neutrophil adhesion.     

Role of intercellular adhesion molecule 1 in acute lung injury induced by candidemia

Candidemia, a complication often affecting immunocompromised patients, is a common cause of acute lung injury. Yeast-phase Candida albicans has been shown to express a protein that is antigenically and structurally related to Mac-1. C. albicans is reported to stimulate intercellular adhesion molecule 1 (ICAM-1) expression on endothelial cells. In this study, the authors examined the role of ICAM-1 in acute lung injury induced by candidemia. The authors cultured rat pulmonary artery endothelial cells (RPAEC) and investigated the effect of anti-ICAM-1 antibodies on adhesion of C. albicans to RPAEC. In addition, the authors administered anti-ICAM-1 antibodies to rats to examine the effect of the antibodies on experimentally induced candidemia. Survival rates, lung wet-to-dry (W/D) weight ratios, bronchoalveolar lavage (BAL) fluid, histopathological findings, and colony-forming units (CFUs) of lung C. albicans were examined. The adherence of C. albicans to RPAEC was significantly decreased by anti-ICAM-1 antibodies. Anti-ICAM-1 antibodies significantly increased survival, decreased lung W/D weight ratios, decreased neutrophil counts in the BAL fluid, reduced microscopic lung injury, and decreased the quantity of lung C. albicans. These results indicate that ICAM-1 plays a role in adherence of C. albicans to pulmonary vascular endothelial cells, which likely leads to invasion of lung tissue by the organism.     

Growth hormone increases vascular cell adhesion molecule 1 expression: in vivo and in vitro evidence

We investigated the impact of GH administration on endothelial adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, in vivo and in vitro. Soluble VCAM-1, E-selectin, and C-reactive protein concentrations were measured before and after treatment in 25 healthy subjects and 25 adult GH-deficient (GHD) patients randomized to GH treatment or placebo. Furthermore, we studied the direct effect of GH and IGF-I and serum from GH-treated subjects on basal and TNF alpha-stimulated expression of VCAM-1 and E-selectin on cultured human umbilical vein endothelial cells. Baseline levels of VCAM-1, but not E-selectin, were significantly lower in GHD patients than in healthy subjects (362 +/- 15 microg/liter vs. 516 +/- 21 microg/liter, P < 0.001) and increased in GHD patients during GH treatment, compared with placebo [net difference between groups 151.8 microg/liter (95% confidence interval: 95.0-208.7 microg/liter); P < 0.0001]. In human umbilical vein endothelial cells, there was no direct stimulatory effect of either GH or IGF-I on the expression of VCAM-1 and E-selectin, but serum from GH-treated healthy subjects significantly increased the expression of VCAM-1 (P < 0.01). Our findings are compatible with the notion that GH may stimulate the expression of VCAM-1 indirectly through modulation of circulating factors. VCAM-1-mediated leukocyte extravasation is implicated in several illnesses including atherosclerosis and multiple-organ failure in sepsis, and we hypothesize that enhanced expression of VCAM-1 may contribute to the detrimental effects of GH in critically ill patients.     

Attenuated liver fibrosis after bile duct ligation and defective hepatic stellate cell activation in neural cell adhesion molecule knockout mice

Aim: Neural cell adhesion molecule (N‐CAM) is expressed by activated hepatic stellate cells (HSC), portal fibroblasts, cholangiocytes and hepatic progenitor cells during liver injury. Its functional role in liver disease and fibrogenesis is unknown. The aim of this study was to investigate the role of N‐CAM in liver fibrogenesis. Methods: To induce fibrosis, N‐CAM knockout mice and wild‐type controls were subjected to bile duct ligation (BDL) or repeated carbon tetrachloride (CCl₄) injections. Fibrosis was quantified by hydroxyproline, immunhistochemistry staining and image analysis. Protein levels were determined with immunoblotting. HSCs were isolated by ultracentrifugation in a Larcoll gradient and thereafter in vitro stimulated with recombinant transforming growth factor (TGF)‐β1. Results: Two weeks after BDL, wild‐type mice had developed pronounced liver fibrosis while N‐CAM?/? mice had less such alterations. N‐CAM?/? mice had less deposition of collagen and fibronectin seen in immunhistochemistry. The protein levels of fibronectin were higher in the liver from the wild type, while laminin were unaltered. CCl₄‐treated N‐CAM?/? and wild‐type mice showed no significant difference in the extent of liver fibrosis or the expression levels of the above‐mentioned genes. HSC isolated from N‐CAM?/? mice showed declined levels of smooth muscle actin and desmin after stimulation in vitro with TGF‐β1. Conclusions: Loss of N‐CAM results in decreased hepatic collagen and fibronectin deposition in mice subjected to BDL, but not in animals exposed to repeated CCl₄ injections. HSC isolated from N‐CAM null mice show impaired activation in vitro. This indicates a role of N‐CAM in cholestatic liver disease and HSC activation.     

L5, the most electronegative subfraction of plasma LDL, induces endothelial vascular cell adhesion molecule 1 and CXC chemokines, which mediate mononuclear leukocyte adhesion

We have previously reported that L5, the most negatively charged subfraction of plasma low-density lipoprotein (LDL), induces mononuclear leukocyte (MNC) adhesion under flow conditions in vitro when endothelial cells are incubated with L5. The present study was undertaken to identify responsible adhesion molecules and chemokines. LDL isolated from patients with homozygous familial hypercholesterolemia was separated into five distinct subfractions by high-capacity ion-exchange chromatography. Differentially expressed mRNA between human umbilical vein endothelial cells (HUVEC) incubated (for 22h) with the earliest subfraction (L1: 20 microg/ml) and the latest and most negatively charged subfraction (L5: 20 microg/ml) was identified by DNA microarray analysis using three independent sets of RNA. mRNA consistently upregulated by L5 included VCAM-1 (2.3-fold) and CXC chemokines GRO-alpha (2.3), GRO-beta (4.6), IL-8 (2.5), ENA-78 (2.3), GRO-gamma (1.6) and GCP-2 (1.5). These results were validated by Northern analysis, semi-quantitative RT-PCR or ELISA. Blocking studies using monoclonal antibodies revealed that both primary capture and stable adhesion of MNC to HUVEC and human aortic endothelial cells (HAEC) incubated with L5 was mediated by VCAM-l/alpha4 integrin, whereas GRO and its receptor CXCR2 were involved in the stable adhesion of MNC to L5-treated HAEC.     

The role of vascular cell adhesion molecule 1/ very late activation antigen 4 in endothelial progenitor cell recruitment to rheumatoid arthritis synovium

OBJECTIVE: Marrow-derived endothelial progenitor cells (EPCs) are important in the neovascularization that occurs in diverse conditions such as cardiovascular disorders, inflammatory diseases, and neoplasms. In rheumatoid arthritis (RA), synovial neovascularization propels disease by nourishing the inflamed and hyperproliferative synovium. This study was undertaken to investigate the hypothesis that EPCs selectively home to inflamed joint tissue and may perpetuate synovial neovascularization. METHODS: In a collagen-induced arthritis (CIA) model, neovascularization and EPC accumulation in mouse ankle synovium was measured. In an antibody-induced arthritis model, EPC recruitment to inflamed synovium was evaluated. In a chimeric SCID mouse/human synovial tissue (ST) model, mice were engrafted subcutaneously with human ST, and EPC homing to grafts was assessed 2 days later. EPC adhesion to RA fibroblasts and RA ST was evaluated in vitro. RESULTS: In mice with CIA, cells bearing EPC markers were significantly increased in peripheral blood and accumulated in inflamed synovial pannus. EPCs were 4-fold more numerous in inflamed synovium from mice with anti-type II collagen antibody-induced arthritis versus controls. In SCID mice, EPC homing to RA ST was 3-fold greater than to normal synovium. Antibody neutralization of vascular cell adhesion molecule 1 (VCAM-1) and its ligand component alpha4 integrin potently inhibited EPC adhesion to RA fibroblasts and RA ST cryosections. CONCLUSION: These data demonstrate the selective recruitment of EPCs to inflamed joint tissue. The VCAM-1/very late activation antigen 4 adhesive system critically mediates EPC adhesion to cultured RA fibroblasts and to RA ST cryosections. These findings provide evidence of a possible role of EPCs in the synovial neovascularization that is critical to RA pathogenesis.     

Anti-inflammatory drugs and endothelial cell adhesion molecule expression in murine vascular beds

Background—Inflammatory boweldiseases (IBD) are characterised by an intense infiltration ofleucocytes that is mediated by adhesion molecules expressed on thesurface of activated endothelial cells.
Aims—To determine whether drugsused in the treatment of IBD, specifically dexamethasone (DEX),5-aminosalicylic acid (5-ASA), methotrexate (MTX), and 6-mercaptopurine(6-MP), alter the expression of endothelial cell adhesion molecules (ECAMs).
Methods—The expression ofP-selectin, E-selectin, intercellular adhesion molecule 1 (ICAM-1), andvascular CAM 1(VCAM-1) in different vascular beds of C57Bl/6J mice wasmeasured using the dual radiolabelled monoclonal antibody technique.
Results—Lipopolysaccharide (LPS)elicited a profound increase in the expression of all ECAMs in themesentery, small intestine, caecum, and distal colon. The LPS inducedincrease in CAM expression was not significantly affected by priortreatment with either MTX or 6-MP. However, pretreatment with eitherDEX or 5-ASA significantly attenuated LPS induced increases inexpression of P- and E-selectin, and VCAM-1 in the majority of tissuesevaluated. DEX also blunted the LPS induced increase in ICAM-1expression in the caecum and distal colon. DEX, but not 5-ASA, largelyabolished the rise in plasma tumour necrosis factor α elicited by LPS.
Conclusions—These findings suggestthat DEX and 5-ASA may exert their beneficial therapeutic action inIBD, at least in part, by inhibiting the expression of ECAMs whichmediate leucocyte adhesion and transmigration in the microvasculature.

     

ICAM-1 mediates lung leukocyte recruitment but not pulmonary fibrosis in a murine model of bleomycin-induced lung injury.

Bleomycin-induced lung injury has been extensively used as a model of interstitial pneumonia and pulmonary fibrosis. Intercellular adhesion molecule (ICAM)-1 is a ligand for lymphocyte function-associated antigen (LFA)-1alpha and has been shown to be required for leukocyte migration into inflamed areas. The purpose of this report was to investigate the role of the ICAM-1/LFA-1alpha pathway in a murine model of bleomycin-induced lung injury. Animals received 75 mg x kg(-1) bleomycin (BLM) i.v. followed by treatment with phosphate-buffered saline (BLM group), anti-ICAM-1 and LFA-1alpha monoclonal antibodies (mAb) (BLM+mAb group). Inflammatory cell counts of bronchoalveolar lavage (BAL) fluid, hydroxyproline content and histological findings were compared between these groups. In the BLM group, significant increases in total cell count, macrophage count and neutrophil count of BAL fluid were observed on days 7 and 14. In the BLM+mAb group, bleomycin-induced accumulation of neutrophils was significantly reduced on days 7 and 14 (p<0.01). However, the administration of mAb to ICAM-1 and LFA-1alpha did not decrease the lung hydroxyproline content or the histopathological fibrosis grading score, indicating that the antagonism of ICAM-I and LFA-1alpha did not attenuate bleomycin-induced pulmonary fibrosis. This study suggests that the intercellular adhesion molecule-1/lymphocyte function-associated antigen-1alpha pathway mediates the accumulation of inflammatory cells in the injured lung caused by bleomycin; however, other mechanisms are important for the subsequent development of pulmonary fibrosis.