Mast cells are potent regulators of endothelial cell adhesion molecule ICAM-1 and VCAM-1 expression

To investigate the possible role of mast cells (MC) in regulating leukocyte adhesion to vascular endothelial cells (EC), microvascular and macrovascular EC were exposed to activated MC or MC conditioned medium (MCCM). Expression of intercellular and vascular adhesion molecules (ICAM-1 and VCAM-1) on EC was monitored. Incubation of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) with activated MC or MCCM markedly increased ICAM-1 and VCAM-1 surface expression, noted as éarly as 4 hr. Maximal levels were observed at 16 hr followed by a general decline over 48 hr. A dose-dependent response was noted using incremental dilutions of MCCM or by varying the number of MC in coculture with EC. At a ratio as low as 1:1,000 of MC:EC, increased ICAM-1 was observed. The ICAM-1 upregulation by MCCM was >90% neutralized by antibody to tumor necrosis factor alpha (TNF-α), suggesting that MC release of this cytokine contributes significantly to inducing EC adhesiveness. VCAM-1 expression enhanced by MCCM was partly neutralized (70%) by antibody to TNF-α; thus other substances released by MC may contribute to VCAM-1 expression. Northern blot analysis demonstrated MCCM upregulated ICAM-1 and VCAM-1 mRNA in both HDMEC and HUVEC. To evaluate the function of MCCM-enhanced EC adhesion molecules, T cells isolated from normal human donors were used in a cell adhesion assay. T-cell binding to EC was increased significantly after exposure of EC to MCCM, and inhibited by antibodies to ICAM-1 or VCAM-1. Intradermal injection of allergen in human atopic volunteers known to develop late-phase allergic reactions led to marked expression of both ICAM-1 and VCAM-1 at 6 hr, as demonstrated by immunohistochemistry. These studies indicate that MC play a critical role in regulating the expression of EC adhesion molecules, ICAM-1 and VCAM-1, and thus augment inflammatory responses by upregulating leukocyte binding. © 1995 Wiley-Liss Inc.     

FK506 suppresses E-selectin, ICAM-1 and VCAM-1 expression on vascular endothelial cells by inhibiting tumor necrosis factor alpha secretion from peripheral blood mononuclear cells

FK506 suppresses activation of T cells; however, it down-regulates E-selectin, ICAM-1 and VCAM-1 expression in inflamed tissues. In this study, we investigated the effect of FK506 on expression of those adhesion molecules on human vascular endothelial cells (HMVEC). Culture supernatant from peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 plus anti-CD2 antibodies effectively induced the expression of E-selectin, ICAM-1 and VCAM-1 on HMVEC, and treatment with FK506 down-regulated their expression. Culture supernatant contained tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta, which effectively induced adhesion molecules, and FK506 suppressed both cytokine secretions. TNFalpha content in culture supernatant was parallel to the induction of adhesion molecules by the culture supernatant. IL-1beta content was not enough to induce those adhesion molecules. Anti-TNFalpha antibody completely inhibited those expressions. FK506 did not inhibit either TNFalpha- or IL-1beta-induced expression of adhesion molecules, or viability of HMVEC. These results indicate that FK506 suppresses migration of inflammatory cells through the inhibition of TNFalpha secretion from leukocytes.     

The effect of metronidazole on stimulation of adhesion molecule expression on the surface of vascular endothelial cells by Bacteroides fragilis endotoxins and enterotoxin]

The influence of metronidazole on the level of expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin on the surface of vascular endothelial cells activated with B. fragilis endotoxins and enterotoxin was examined. Three enterotoxigenic (ETBF) strains and one nonenterotoxigenic (NTBF) strain were used for lipopolysaccharide extraction. Enterotoxin was prepared from the culture supernatant of the reference B. fragilis ATCC 43858 strain. Expression of adhesion molecules on vascular endothelial cells (HMEC-1 cell line) was determined after their stimulation with bacterial compounds at the concentration of 10 micrograms/ml in the presence of metronidazole at the concentration of 4 micrograms/ml. Endothelial cells were activated for 4 hours (E-selectin expression) and for 24 hours (ICAM-1 and VCAM-1 expression). Adhesion molecules were detected in immunoenzymatic test (ELISA) with mouse, monoclonal antibodies against human ICAM-1, VCAM-1 and E-selectin. The results of experiments suggest, that metronidazole enhances the expression of examined adhesion molecules on endothelial cells. This antimicrobial agent causes some changes in the expression of endothelial ICAM-1, VCAM-1 and E-selectin stimulated by B. fragilis endotoxins and enterotoxin.     

1,4-dihydroxyxanthone modulates the adhesive property of endothelial cells by inhibiting intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin

Cell adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1) and E-selectin, play important roles in the recruitment of leukocytes to the site of inflammation. Blocking the expression of these molecules or preventing their interaction with the receptors has been shown to be important in controlling various inflammatory diseases. These cell adhesion molecules are induced on endothelial cells by various proinflammatory cytokines like IL-1beta and TNF-alpha and also by bacterial LPS. We demonstrate here that 1,4-Dihydroxyxanthone (1,4 DHX) inhibits the expression of cell adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin, on endothelial cells in a concentration and time dependent manner. The inhibition by 1,4 DHX is reversible. On further analysis, our results also show that 1,4 DHX inhibits the adhesion of peripheral neutrophils to the endothelial cell monolayers. 1,4 DHX, therefore, could be used as a novel target for controlling various pathological conditions associated with upregulation of endothelial leukocyte adhesion molecules.     

Inhibition of THP-1 cell adhesion to endothelial cells by alpha-tocopherol and alpha-tocotrienol is dependent on intracellular concentration of the antioxidants

Vitamin E analogs such as alpha-tocopherol and alpha-tocotrienol have been shown to reduce endothelial expression of adhesion molecules. The reactivity of alpha-tocopherol and alpha-tocotrienol in inhibiting lipid peroxidation in vitro was essentially identical but the inhibition of adhesion of THP-1 cells, a monocytic-"like" cell line, to endothelial cells differs substantially. To determine the mechanism underlying this response, human umbilical vein endothelial cells (HUVECs) were assessed for their ability to accumulate vitamin E analogs. alpha-Tocotrienol accumulated in HUVECs to levels approximately 10-fold greater than that of alpha-tocopherol. The decrease in expression of vascular cell adhesion molecule-1 (VCAM-1) and the adhesion of THP-1 cells to HUVECs by alpha-tocopherol and alpha-tocotrienol was also determined. Both alpha-tocopherol and alpha-tocotrienol suppressed VCAM-1 expression and adhesion of THP-1 cells to HUVECs in a concentration-dependent manner. The efficacy of tocotrienol for reduction of VCAM-1 expression and adhesion of THP-1 cells to HUVECs was also 10-fold higher than that of tocopherol. The inhibitory effects of vitamin E analogs on the adhesiveness of endothelial cells clearly correlated with their intracellular concentrations. The data demonstrated that, in assessing the biological responses of antioxidants, intracellular accumulation and metabolism were additional important factors that must be considered.     

The influence of garlic (Allium sativum) extract on interleukin 1alpha-induced expression of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1.

Inflammation plays an important role in both the initiation of atherosclerosis and development of atherothrombotic events. The adherence of leukocytes/monocytes to the endothelium is an early event in atherogenesis. Phytotherapeutica as garlic and garlic extracts were shown to have beneficial modulating effects in patients with atherosclerotic disease. The aim of this study was to evaluate in vitro the influence of water-soluble garlic (Allium sativum) extract on the cytokine-induced expression of endothelial leukocyte adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1, CD54) and vascular cell adhesion molecule-1 (VCAM-1, CD106). Cytokine-induced expression of cellular adhesion molecules was measured on primary human coronary artery endothelial cell (HCAEC) cultures. HCAEC were cultured in microvascular endothelial cell growth medium and preincubated with garlic extract at various concentrations (0.25-4.0 mg/ml), after which human interleukin-1alpha (IL-1alpha, 10 ng/ml) was added for 1 day. Fluorescein isothiocyanate (FITC)-labeled anti-ICAM-1 and FITC-labeled anti-VCAM-1 were used to analyze the IL-1alpha-induced expression of ICAM-1 and VCAM-1 by flow cytometry. Incubation of HCAEC with garlic extract significantly decreased ICAM-1 and VCAM-1 expression induced by IL-1alpha. In addition, we examined the effects of garlic extract on the adhesion of monocytes to endothelial cells, using the monocytic U937 cell line. The presence of garlic extract significantly inhibited the adhesion of monocytes to IL-1alpha-stimulated endothelial cells. These results indicate that garlic extract modulates the expression of ICAM-1 and VCAM-1, thus potentially contributing to the beneficial effects traditionally attributed to garlic.     

Expression of adhesion molecules in lungs of mice infected with Paracoccidioides brasiliensis conidia

In infected tissues, leukocyte recruitment is mediated by interactions between adhesion molecules, expressed on activated vascular endothelial cells, and ligands present on circulating cells. We evaluated the inflammatory response and the expression of cellular adhesion molecules (ICAM-1, VCAM-1, CD18, LFA-1 and Mac-1) in lungs of BALB/c mice infected with Paracoccidioides brasiliensis conidia. When compared with uninfected animals, infected mice had a significant increase in the inflammatory response during the first 4 days, peaking 2-3 days post-challenge, 40.3% vs. 0.0% and 41.8% vs. 0.7%, respectively. This inflammatory infiltrate was composed mainly of neutrophils and macrophages with a few eosinophils and lymphocytes. An increase in the intensity of immunofluorescence (IF) for ICAM-1 was also observed during days 1-4. ICAM-1 was present in bronchiolar epithelium, type II pneumocytes, and macrophages, as well as on vascular endothelium. The control animals presented ICAM-1 constitutively. In infected mice, VCAM-1 was only observed on vascular endothelium during the first 2 days, with some macrophages expressing this molecule throughout the study periods. CD18 and Mac-1 but not LFA-1 were expressed with a high intensity on neutrophils and macrophages present in the inflammatory infiltrate. In addition, we observed a significant decrease in Colony forming units (CFUs) after the first 2 days post-challenge. These findings suggest that during these early stages, up-regulation of ICAM-1, VCAM-1, CD18 and Mac-1 expression occurs, participating in the inflammatory process and as such, in the pathogenesis of paracoccidioidomycosis (PCM).     

Ionizing radiation increases adhesiveness of human aortic endothelial cells via a chemokine-dependent mechanism

Exposure to radiation from a variety of sources is associated with increased risk of heart disease and stroke. Since radiation also induces inflammation, a possible mechanism is a change in the adhesiveness of vascular endothelial cells, triggering pro-atherogenic accumulation of leukocytes. To investigate this mechanism at the cellular level, the effect of X rays on adhesiveness of cultured human aortic endothelial cells (HAECs) was determined. HAECs were grown as monolayers and exposed to 0 to 30 Gy X rays, followed by measurement of adhesiveness under physiological shear stress using a flow chamber adhesion assay. Twenty-four hours after irradiation, HAEC adhesiveness was increased, with a peak effect at 15 Gy. Radiation had no significant effect on surface expression of the endothelial adhesion molecules ICAM-1 and VCAM-1. Antibody blockade of the leukocyte integrin receptors for ICAM-1 and VCAM-1, however, abolished the radiation-induced adhesiveness. Since these leukocyte integrins can be activated by chemokines presented on the endothelial cell surface, the effect of pertussis toxin (PTX), an inhibitor of chemokine-mediated integrin activation, was tested. PTX specifically inhibited radiation-induced adhesiveness, with no significant effect on nonirradiated cells. Therefore, radiation induces increased adhesiveness of aortic endothelial cells through chemokine-dependent signaling from endothelial cells to leukocytes, even in the absence of increased expression of the adhesion molecules involved.     

Omega-3 fatty acids suppress monocyte adhesion to human endothelial cells: role of endothelial PAF generation

Monocyte-endothelium interaction is a fundamental process in many acute and chronic inflammatory diseases. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are fish oil-derived alternative (omega-3) precursor fatty acids implicated in the suppression of inflammatory events. We investigated their influence on rolling and adhesion of monocytes to human umbilical vein endothelial cells (HUVEC) under laminar flow conditions in vitro. Exposure of HUVEC to tumor necrosis factor (TNF-alpha) strongly increased 1) surface expression of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and E-selectin, 2) platelet-activating factor (PAF) synthesis as assessed by thrombin challenge, and 3) rate of rolling and adhesion of monocytes. Preincubation of HUVEC with EPA or DHA markedly suppressed PAF synthesis, monocyte rolling, and adherence, whereas expression of endothelial adhesion molecules was unchanged. Also, PAF receptor antagonists markedly suppressed the adhesion rate of monocytes, and EPA or DHA revealed no additional inhibitory capacity. In contrast, arachidonic acid partially reversed the effect of the antagonist. We conclude that omega-3 fatty acids suppress rolling and adherence of monocytes on activated endothelial cells in vitro by affecting endothelial PAF generation.     

Induction of VCAM-1 and ICAM-1 on human neural cells and mechanisms of mononuclear leukocyte adherence.

We propose that leukocyte-derived cytokines induce the expression of adhesion molecules on the surface of neural cells that facilitates the subsequent attachment of leukocytes. Leukocyte adherence may contribute to some of the neural cell injury seen with various inflammatory diseases of the nervous system. With an in vitro model system, we have shown that mononuclear leukocytes bind to human neuroblastoma and cortical neuron cells only after the neural cells are stimulated with TNF-alpha. TNF-alpha stimulates expression of vascular cell adhesion molecule-1 (VCAM-1) in both of these neural cell lines. VCAM-1 mRNA is increased and VCAM-1 protein can be identified on the neural cell membranes with a new VCAM-1-specific mAb, CL40/2 F8. TNF-alpha also induces ICAM-1 in both of these neural cell lines. Leukocyte beta 1 (CD29) and beta 2 (CD18) integrins and their respective ligands, ICAM-1 and VCAM-1, on neural cells appear to be the dominant ligands mediating MNL:neural cell adhesive interactions. mAb to CD18 block 32 to 57% of the MNL binding to neural cells; similar inhibition is seen with mAb to ICAM-1. mAb to CD29 block 16 to 17% of the MNL binding to the neural cells suggesting that leukocyte beta 1 integrins and neural VCAM-1 may be a second route for MNL:neural cell interactions. Addition of both anti-CD18 and anti-CD29 mAb have an additive blocking effect; both ligand pairs may participate in MNL adhesion to neural cells, reminiscent of the multiplicity of ligands used by MNL when binding to endothelium.     

A novel role for interleukin-18 in adhesion molecule induction through NF kappa B and phosphatidylinositol (PI) 3-kinase-dependent signal transduction pathways

Interleukin-18 (IL-18) is a novel proinflammatory cytokine found in serum and joints of patients with rheumatoid arthritis (RA). We studied a novel role for IL-18 in mediating cell adhesion, a vital component of the inflammation found in RA and other inflammatory diseases. We examined the expression of cellular cell adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and RA synovial fibroblasts using flow cytometry. Adhesion of the monocyte-like cell line HL-60 to endothelial cells was determined by immunofluorescence. IL-18 significantly enhanced ICAM-1 and VCAM-1 expression on endothelial cells and RA synovial fibroblasts. In addition, IL-18 induced E-selectin expression on endothelial cells and promoted the adhesion of HL-60 cells to IL-18-stimulated endothelial cells. Neutralizing anti-VCAM-1 and anti-E-selectin could completely inhibit HL-60 adherence to endothelial cells. IL-18-induced adhesion molecule expression appears to be mediated through nuclear factor kappa B (NF kappa B) and phosphatidyl-inositol 3 kinase (PI 3-kinase) since addition of inhibitors to either NF kappa B (pyrrolidine dithiocarbamate and N-acetyl-l-cysteine) or PI 3-kinase (LY294002) inhibited RA synovial fibroblast VCAM-1 expression by 50 to 60%. Addition of both inhibitors resulted in inhibition of VCAM-1 expression by 85%. In conclusion, the ability of IL-18 to induce adhesion molecule expression on endothelial cells and RA synovial fibroblasts indicates that IL-18 may contribute to RA joint inflammation by enhancing the recruitment of leukocytes into the joint. IL-18 requires NF kappa B as well as PI 3-kinase to induce VCAM-1 on RA synovial fibroblasts, suggesting that there may be two distinct pathways in IL-18-induced adhesion molecule expression.     

Expression of cell adhesion molecules and lymphocyte-endothelium interaction under simulated hypogravity in vitro

Using histochemical staining and FACS-analysis we have studied the basal and TNF-alpha induced expression of E-selectin, ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (ECs) exposed to simulated hypogravity. Control ECs did not contain detectable amounts of E-selectin or VCAM-1 but were ICAM-1 positive. As soon as after 6-8 hrs of clinorotation at 5 RPM the cellular content of ICAM- 1 increased. Moreover, hypogravity potentiated the effect of inflammatory cytokines (TNF-alpha and IL-1) on ICAM-1 expression. No increase in E-selectin or VCAM-1 expression was observed in ECs exposed to hypogravity itself. However, hypogravity reduced E-selectin and VCAM-1 expression in cell cultures activated by cytokines, more visible at their low (5-10 U/ml) concentrations. Both, control and clinorotated ECs poorly supported spontaneous lymphocyte adhesion; the adhesion of PMA-activated leukocytes was 15-20-fold higher. The interaction of unstimulated lymphocytes with cytokine-activated endothelium was more noticeable but significantly lower in cultures exposed to hypogravity. Activated blood cells interacted with endothelium more effectively, particularly, under hypogravity. Obtained results suggest that EC adhesion molecule expression and endothelium-lymphocyte interaction are altered under simulated hypogravity conditions in direction of increase of endotlielial adhesiveness for activated blood cells.     

The VLA-4/VCAM-1 molecules participate in gamma delta cell interaction with endothelial cells.

The accumulation of T lymphocytes at the site of chronic inflammation depends on a number of factors including adherence of T cells to vascular endothelial cells (EC) and endothelial permeability. We examined the effects of human gamma delta + T lymphocytes on the permeability of EC to macromolecules and characterized the cell surface molecules that are involved in these interactions. In this model, the flux of [125I]albumin was measured across the EC monolayer after a short-term culture with cloned gamma delta cells. Our results show that coculture of activated, but not resting, gamma delta cells with EC enhances endothelial permeability by a cytolytic process. Pretreating gamma delta cells with monoclonal antibodies directed at either LFA-1 or VLA-4 molecules or pretreating EC with monoclonal antibodies directed against either ICAM-1 or VCAM-1 molecules significantly inhibited gamma delta cell-mediated enhancement in endothelial permeability. This indicated that VLA-4/VCAM-1 and LFA-1/ICAM-1 adhesion pathways participate in gamma delta cell-EC interaction.     

Regulation of immune response and inflammatory reactions against viral infection by VCAM-1

The migration of activated antigen-specific immune cells to the target tissues of virus replication is controlled by the expression of adhesion molecules on the vascular endothelium that bind to ligands on circulating lymphocytes. Here, we demonstrate that the adhesion pathway mediated by vascular cell adhesion molecule 1 (VCAM-1) plays a role in regulating T-cell-mediated inflammation and pathology in nonlymphoid tissues, including the central nervous system (CNS) during viral infection. The ablation of VCAM-1 expression from endothelial and hematopoietic cells using a loxP-Cre recombination strategy had no major effect on the induction or overall tissue distribution of antigen-specific T cells during a systemic infection with lymphocytic choriomeningitis virus (LCMV), except in the case of lung tissue. However, enhanced resistance to lethal LCM and the significantly reduced magnitude and duration of footpad swelling observed in VCAM-1 mutant mice compared to B6 controls suggest a significant role for VCAM-1 in promoting successful local inflammatory reactions associated with efficient viral clearance and even life-threatening immunopathology under particular infection conditions. Interestingly, analysis of the infiltrating populations in the brains of intracerebrally infected mice revealed that VCAM-1 deletion significantly delayed migration into the CNS of antigen-presenting cells (macrophages and dendritic cells), which are critical for optimal stimulation of migrating virus-specific CD8⁺ T cells initiating a pathological cascade. We propose that the impaired migration of these accessory cells in the brain may explain the improved clinical outcome of infection in VCAM-1 mutant mice. Thus, these results underscore the potential role of VCAM-1 in regulating the immune response and inflammatory reactions against viral infections.     

Butyrate inhibits cytokine-induced VCAM-1 and ICAM-1 expression in cultured endothelial cells: the role of NF-kappaB and PPARalpha

Adhesion and migration of leukocytes into the surrounding tissues is a crucial step in inflammation, immunity, and atherogenesis. Expression of cell adhesion molecules by endothelial cells plays a leading role in this process. Butyrate, a natural short-chain fatty acid produced by bacterial fermentation of dietary fiber, has been attributed with anti-inflammatory activity in inflammatory bowel disease. Butyrate in vitro is active in colonocytes and several other cell types. We have studied the effect of butyrate on expression of endothelial leukocyte adhesion molecules by cytokine-stimulated human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with butyrate-inhibited tumor necrosis factor-alpha (TNFalpha)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) in a time and concentration-dependent manner. Butyrate at 10 mM/L inhibited interleukin-1 (IL-1)-stimulated VCAM-1 and ICAM-1 expression. The effect of butyrate on cytokine-stimulated VCAM-1 expression was more pronounced than in the case of ICAM-1. Butyrate decreased TNFalpha-induced expression of mRNA for VCAM-1 and ICAM-1. Suppressed expression of VCAM-1 and ICAM-1 was associated with reduced adherence of monocytes and lymphocytes to cytokine-stimulated HUVEC. Butyrate inhibited TNFalpha-induced activation of nuclear factor-kappaB (NF-kappaB) in HUVEC. Finally, butyrate enhanced peroxisome proliferator-activated receptor-alpha (PPARalpha) expression in HUVEC. These results demonstrate that butyrate may have anti-inflammatory properties not only in colonocytes but also in endothelial cells. The anti-inflammatory and (perhaps) antiatherogenic properties of butyrate may partly be attributed to an effect on activation of NF-kappaB and PPARalpha and to the associated expression of VCAM-1 and ICAM-1. The present findings support further investigations on the therapeutic benefits of butyrate in several pathological events involving leukocyte recruitment.