Neutralization of TNF by the antibody cA2 reveals differential regulation of adhesion molecule expression on TNF-activated endothelial cells

Upregulation of adhesion proteins plays an important role in mediating inflammation. The induction of adhesive molecules has been well studied, but the reversibility of their expression has not been well characterized. A neutralizing anti-TNF monoclonal antibody (cA2) was used to study the down regulation of TNF-induced E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on cultured human umbilical vein endothelial cells (HUVECs). Addition of cA2 following TNF stimulation of HUVECs enhanced the rate of E-selectin and VCAM-1 down-regulation from the cell surface and also reduced steady state E-selectin and VCAM-1 mRNA levels. The cA2-mediated disappearance of E-selectin, but not VCAM-1 protein was microtubule and not microfilament dependent. Neutralization of TNF only slightly reduced ICAM-1 cell surface levels following initial TNF stimulation, suggesting a slower turnover of ICAM-1 compared to E-selectin and VCAM-1. Microtubule inhibition during TNF stimulation partially inhibited E-selectin, VCAM-1 and ICAM-1 mRNA upregulation. VCAM-1 and ICAM-1 cell surface expression were similarly partially inhibited, however, E-selectin levels were unaffected, presumably due to the dual, opposing effect of inhibiting protein expression and inhibiting internalization. Microfilament inhibition during protein induction specifically inhibited the maximal expression of VCAM-1 protein and mRNA, without affecting E-selectin or ICAM-1. These data support the notion that E-selectin, VCAM-1, and ICAM-1 expression are differentially regulated on HUVECs and suggest that TNF neutralizing therapies may be effective because of their ability to reduce the levels of pre-existing adhesion proteins.     

Research on the mechanism of endothelin inflammatory effects on human mesangial cells

OBJECTIVE: To investigate the mechanism of endothelin (ET) inflammatory effects on human mesangial cells (HMC). METHODS: The following experiments were performed on cultured HMC after ET-1 stimulation: (1) the expression of tumor necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelin-1 (ET-1) itself messenger ribonucleic acid (mRNA) was determined by Northern Blot analysis; (2) the TNF alpha concentration was tested with radioimmunoassay; the IL-1 activity was assayed by the enhancement of thymocyte proliferation in response to mitogen; the surface expression of ICAM-1 and VCAM-1 was measured with cell enzyme linked immunoadsorbent assay (ELISA) analysis. RESULTS: ET-1 (10(-7) mol/L) induced the following changes on HMC: (1) up-regulation of the expression of TNF alpha mRNA and protein; (2) up-regulation of the expression of ICAM-1 and VCAM-1 mRNA and protein; (3) up-regulation of the expression of ET-1 itself mRNA. However, the expression of IL-1 mRNA and protein was not changed. CONCLUSIONS: ET-1 can stimulate HMC to produce TNF alpha, ICAM-1 and VCAM-1, and thereby induce inflammatory effects. ET-1 can also stimulate HMC to up-regulate the expression of ET-1 itself, so as to amplify inflammatory effects. So, ET-1 is actually an inflammatory mediator and may play an important role in the pathogenesis of glomerulonephritis.     

Increased incidence of apoptosis in transforming growth factor alpha-deficient mouse blastocysts

Platelets, activated by various agonists, produce microparticles (MP) from the plasma membrane, which are released into the extracellular space. Although the mechanism of MP formation has been clarified, their biological importance remains ill defined. We have recently shown that platelet-derived MP influence platelet and endothelial cell function. In this study, we have further examined the mechanism of cellular activation by platelet MP. To address the possibility that they may influence monocyte-endothelial interactions, we used an in vitro assay to examine their effects on the adhesion of monocytes to human umbilical vein endothelial cells (HUVEC). Platelet MP increased the adhesion of monocytes to HUVEC in a time- and dose-dependent manner. Maximal adhesion of monocytes to resting HUVEC was observed after 24 h of stimulation with MP. Similar kinetics were observed with U-937 (human promonocytic leukemia) cells, used as a model for the blood-borne monocyte. Maximal adhesion of resting monocytes to MP-stimulated HUVEC was observed after 5 h of stimulation with MP. The EC50s for MP-induced increases in HUVEC, monocyte, and U-937 cell adhesion is 8.74, 43.41, and 10.83 microg/ml of MP protein, respectively. The induction of monocyte-endothelial adhesion was mimicked by arachidonic acid isolated from MP. The observed increased cellular adhesiveness correlated with MP-induced upregulation of cell adhesion molecules. MP-stimulated HUVEC increased intracellular cell adhesion molecule-1 (ICAM-1) but not vascular cell adhesion molecule-1 (VCAM-1), P-, or E-selectin expression. Monocyte and U-937 lymphocyte function-associated antigen-1 (CD11a/CD18) and macrophage antigen-1 (CD11b/ CD18, alpham/beta2) were both upregulated upon MP stimulation, but an increase in p150,95 (CD11c/CD18), very late antigen-1, or ICAM-1 expression was not observed. The functional importance of these changes was demonstrated with blocking antibodies. MP also induced the chemotaxis of U-937 cells in a dose-dependent manner with an EC50 of 4.40 microg/ml of MP protein. Similarly, arachidonic acid isolated from MP mimicked the chemotactic response. A role for PKC was implicated in both adhesion and chemotaxis. GF 109203X, a specific inhibitor of PKC, significantly reduced monocyte-endothelial adhesion, as well as U-937 chemotaxis. The demonstration that platelet MP may modulate important aspects of endothelial and monocyte function provides a novel mechanism by which platelets may interact with such cells in human atherosclerosis and inflammation.     

Ibuprofen inhibits pyrogen-dependent expression of VCAM-1 and ICAM-1 on human endothelial cells

Leukocyte adhesion and transmigration through the endothelial cell (EC) layer plays a crucial role in inflammation. IL-1 alpha and TNF alpha increase EC-adhesiveness for leukocytes by stimulating surface expression of ICAM-1 (intercellular adhesion molecule 1, CD54), VCAM-1 (vascular cell adhesion molecule 1, CD106) and E-selectin (CD62E). In this study, the effects of ibuprofen on IL-1 alpha and TNF alpha-induced expression of ICAM-1, VCAM-1 and E-selectin on cultured human umbilical vein EC (HUVEC) were analyzed. Exposure to IL-1 alpha or TNF alpha resulted in an increased expression of VCAM-1, ICAM-1, and E-selectin. Ibuprofen was identified as a potent inhibitor of IL-1 alpha and TNF alpha-induced surface expression of VCAM-1 and a less potent inhibitor of pyrogen-induced expression of ICAM-1, whereas no effect on E-selectin was found. The effects of ibuprofen on VCAM-1 expression were dose-dependent (IC50 [IL-1 alpha]: 0.5 mM; IC50 [TNF alpha]: 0.5 mM) and time-dependent with maximum responses observed after 18 h. Moreover, ibuprofen abrogated pyrogen-dependent adhesion of leukocytes to HUVEC. Ibuprofen also inhibited VCAM-1 mRNA expression in pyrogen activated EC. VCAM-1-downregulation on EC by ibuprofen may contribute to the anti-inflammatory actions of the drug.     

Expression of cell adhesion molecules in placentae from pregnancies complicated by pre-eclampsia and intrauterine growth retardation

Platelets and neutrophils are involved in maternal placental vascular damage in pre-eclampsia. Recruitment of these cells is probably mediated by cell adhesion molecules expressed at the uteroplacental bed. It remains controversial as to whether platelets and neutrophils mediate damage to trophoblast or villous vasculature. The purpose of this study was to determine the expression of cell adhesion molecules in placentae from normal pregnancies and pregnancies complicated by pre-eclampsia and intrauterine growth retardation (IUGR). Immunostaining for platelet endothelial cell adhesion molecule (PECAM) and intercellular adhesion molecule-1 (ICAM-1) was localized mainly to the endothelium of stem villi, intermediate villi, terminal villi and decidual vessels. Scattered staining for ICAM-1 was also evident in the stroma and fetal membranes. The endothelium of stem villi, intermediate villi and terminal villi were all negative for vascular cell adhesion molecule-1 (VCAM-1) and E-Selectin. PECAM, ICAM-1 and ICAM-2 mRNA were all detectable in normal placentae using northern blotting analysis whereas mRNA for E-Selectin and VCAM-1 were both undetectable. There were no differences in cell adhesion molecule immunostaining or mRNA expression in placentae from pregnancies complicated by pre-eclampsia and IUGR inconclusion, expression of cell adhesion molecules in placentae from pre-eclampsia and IUGR are consistent with a normal physiological role in vascular function.     

Circadian rhythms of atrioventricular conduction properties in chronic atrial fibrillation with and without heart failure

Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are members of the immunoglobulin superfamily adhesion molecules on vascular endothelium and important in the development of eosinophil (EOS) accumulation in allergic inflammation. To define the role of these adhesion proteins in EOS inflammation, peripheral blood EOS from allergic donors were incubated in either buffer (control)-, recombinant human (rh)-VCAM-1-, or rh-ICAM-1-coated plates, and the effects of these adhesion proteins on EOS effector functions were determined. VCAM-1 induced spontaneous EOS adhesion whereas EOS adhesion to ICAM-1 required a second signal, such as granulocyte macrophage colony-stimulating factor (GM-CSF). Although only VCAM-1 stimulated EOS superoxide anion (O2-) generation, the addition of GM-CSF (100 pM) to the reactions resulted in a greater and equivalent production of O2- with VCAM-1 and ICAM-1. In the presence of GM-CSF, ICAM-1 and VCAM-1 caused significant release of EOS-derived neurotoxin (EDN). Moreover, only ICAM-1 (no GM-CSF) promoted calcium ionophore A23187 (0.2 microM)-induced EOS leukotriene C4 (LTC4). Enhanced O2- generation, EDN release, and LTC4 generation observed with ICAM-1 and VCAM-1 were significantly inhibited by anti-beta2-integrin antibody. These results suggest that ICAM-1 and VCAM-1 are important in determining the eventual function of airway EOS.     

Rickettsia conorii infection enhances vascular cell adhesion molecule-1- and intercellular adhesion molecule-1-dependent mononuclear cell adherence to endothelial cells

Leukocyte adherence to the endothelium is an essential component of the inflammatory response during rickettsial infection. In vitro, Rickettsia conorii infection of endothelial cells enhances the expression of adhesive molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in a time- and dose-dependent manner. Rickettsial lipopolysaccharide does not seem to be involved, because polymyxin B does not reduce their expression. The intracellular presence of the organism and de novo host protein synthesis are required for expression of cell adhesive molecules, since rickettsial inactivation by formol and pretreatment of cells with cycloheximide inhibits an increase in expression. The contribution of interleukin-1alpha (IL-1alpha) to this endothelial adhesive phenotype was shown by inhibitory experiments 8 and 24 h after infection with IL-1 receptor antagonist and IL-1alpha blocking antibodies. Enhanced adherence of mononuclear cells to infected endothelial cells involved VCAM-1- and ICAM-1-dependent mechanisms at the late phase of the inflammatory response. This endothelial adhesive phenotype may constitute a key pathophysiologic mechanism in R. conorii-induced vascular injury.