Distinct ICAM-1 forms and expression pathways in synovial microvascular endothelial cells.

Human synovial endothelial cell (HSE) intracellular adhesion molecule-1 (ICAM-1) is upregulated maximally by synergy of tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma). Such synergy is not as pronounced in human umbilical vein endothelium (HUVE). ICAM surface staining and ELISA detection reflected similar levels on HUVE and HSE cells, yet mRNA levels were much higher in HSE cells in response to TNF alpha/IFN gamma. To correlate protein and mRNA levels of ICAM-1, both cell types were permeabilized and stained with a monoclonal antibody against ICAM-1. HSE cells displayed a distinct vesicular cytoplasmic staining for ICAM while HUVE cells were devoid of such stained vesicles upon staining with the antibody. ICAM-1 immunostaining of HSE cytoplasmic vesicles appeared enhanced in cells treated with TNF alpha/IFN gamma and monensin, an endosomal processing inhibitor. Monensin inhibited HSE cell surface expression of ICAM-1 routinely up to 70%, while HUVE cell expression was unaffected. In addition, monensin also inhibited soluble ICAM-1 release from HSE cells while not effecting HUVE cells. Immunoprecipitation of ICAM-1 followed by gel electrophoresis indicated that HUVE and HSE cell ICAMs are expressed in cell-specific forms. These results define distinct forms and distinct secretory pathways for ICAM-1 in HSE cells and HUVE cells that indicate functional differences between these human endothelia.

Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression.

Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha-induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking.

Regulation of the expression of intercellular adhesion molecule 1 in cultured human endothelial cells derived from rheumatoid synovium.

OBJECTIVE: To examine the regulation of intercellular adhesion molecule 1 (ICAM-1) in human synovial microvascular endothelial cells (HSE) and human umbilical vein endothelial cells (HUVE) upon exposure to a variety of agents. METHODS: Cultured endothelial cells were treated with various cytokines alone and in combination. The expression of ICAM-1 was evaluated at several levels, including an investigation of messenger RNA (mRNA) and surface protein expression. RESULTS: Treatment of HSE with interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF alpha) resulted in minimal increases in ICAM-1 expression, in contrast to findings with HUVE. Incubation of HUVE or HSE with IL-1 or TNF in combination with interferon-gamma (IFN gamma) greatly potentiated the increase in ICAM-1 surface expression. The synergistic effect of IFN gamma and TNF was confirmed by several methods, including a cell-based enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, immunofluorescence staining, and determination of mRNA levels. IFN gamma also augmented the actions of several other agonists on HSE, i.e., IL-4, lipopolysaccharide, and TNF beta/lymphotoxin. Immunoprecipitation of TNF alpha + IFN gamma-stimulated, 125I-labeled HSE cells with anti-ICAM-1 revealed a single 90-kd band, similar in size to ICAM-1 from HUVE treated in an identical manner. Unexpectedly, IFN gamma alone was a potent stimulus for HSE ICAM-1 mRNA synthesis, but was relatively ineffective in HUVE. CONCLUSION: These studies indicate that IFN gamma plays a critical synergistic role in the regulation of ICAM-1 expression in human synovial endothelial cells.