AN ENDOTHELIAL-CELL ADHESION PROTEIN FOR MONOCYTES RECOGNIZED BY MONOCLONAL-ANTIBODY IG9 - EXPRESSION IN-VIVO IN INFLAMED HUMAN VESSELS ANDATHEROSCLEROTIC HUMAN AND WATANABE RABBIT VESSELS

BACKGROUND: Monocyte adhesion to the vascular endothelium, an importan t component of an inflammatory response, is one of the earliest detect ed events in the pathogenesis of atherosclerosis. We have identified a monocyte adhesion molecule, recognized by monoclonal antibody (mAb) I G9, on the cell surface of human umbilical vein endothelial cells (HUV EC) treated with tumor necrosis factor-alpha (TNF-alpha), interleukin- 1, or lipopolysaccharide. Endothelial cell expression in vitro and in vivo of the protein recognized by mAb IG9 (IG9 protein) was further ch aracterized. EXPERIMENTAL DESIGN: The kinetics of cytokine-induced IG9 protein expression on HUVEC were evaluated by enzyme-linked immunosor bent assay. TNF-alpha-treated HUVEC surface proteins, labeled with [I- 125]Na, were solubilized in NP-40 detergent and immunoprecipitated wit h mAb IG9 to determine the molecular weight of the IG9 protein. The fu nctional role of the IG9 protein in monocyte binding in vitro to cytok ine-activated endothelial cells was established in adhesion assays uti lizing U937 cells (human promyelomonocytic cell line) and human periph eral blood monocytes. Minimally oxidized or modified low density lipop roteins (MM-LDL) have previously been shown to induce monocyte adhesio n to endothelial cells for up to 48 hours after exposure. In order to characterize the adhesion molecule(s) contributing to this increase in monocyte binding, MM-LDL-treated HUVEC and human aortic endothelial c ells were assayed for monocyte adhesion molecule expression by enzyme- linked immunosorbent assay. In addition, mAb IG9-mediated alterations in MM-LDL-induced monocyte binding were studied in endothelial-monocyt e adhesion assays. To assess IG9 protein expression in vivo, formalin- fixed, paraffin-embedded sections of inflamed human tissues obtained f rom lung and healing myocardial infarctions, in addition to sections o f human atherosclerotic coronary arteries, were analyzed by immunohist ochemistry. Tissue sections from atherosclerotic Watanabe heritable hy perlipidemic rabbit aortas were also included in these studies. RESULT S: The IG9 protein, undetected on untreated HUVEC, was expressed on th eir cell surface within 3 hours of treatment with TNF-alpha, peaked at 4 to 9 hours, and persisted for up to 48 hours as determined by enzym e-linked immunosorbent assay. A similar kinetic profile was elicited b y interleukin-1 and lipopolysaccharide, whereas interferon-gamma (IFN- gamma) had minimal effect on IG9 expression. The IG9 protein has a mol ecular weight of 105,000 as determined by immunoprecipitation studies with TNF-alpha-treated HUVEC protein lysates. mAb IG9 significantly in hibited the binding of U937 cells and human peripheral blood monocytes to TNF-alpha-treated HUVEC and had no effect on peripheral blood lymp hocyte or granulocyte adhesion. Treatment of human aortic endothelial cells or HUVEC with MM-LDL for 24 hours induced IG9 protein expression 3-fold above background with no concomitant increase in binding of an tibodies to intercellular adhesion molecule-1 (ICAM-1), E-selectin, or vascular cell adhesion molecule-1 (VCAM-1), endothelial cell adhesion proteins involved in monocyte binding. mAb IG9 F(ab')(2) inhibited MM -LDL-induced monocyte adhesion to HAEC by 23%. Immunohistochemical ana lysis demonstrated that the endothelial cell lining of vessels in huma n lung and heart with evidence of inflammation characterized by an ext ensive mononuclear cell infiltration exhibited reactivity with mAb IG9 , whereas vessels with no evidence of inflammation in the same section s as well as in sections from normal lung and heart were nonreactive. Human coronary arteries representing a range of atherosclerotic lesion involvement (fibrous and complex) also exhibited endothelial cell spe cific reactivity with mAb IG9. The IG9 monocyte adhesion protein was d etected in paraffin sections of 43 vessels from 23 cases, with complex plaques exhibiting a higher level of antibody reactivity both on the arterial endothelial cell surface and within areas of the plaque when compared with the reactivity in vessels with fibrous lesions. Aortic e ndothelium overlying atherosclerotic plaques in Watanabe heritable hyp erlipidemic rabbits was also reactive with mAb IG9. The endothelial ce ll lining of human atherosclerotic coronary arteries have only previou sly been reported to express increased levels of ICAM-1.