An endothelial cell adhesion protein for monocytes recognized by monoclonal antibody IG9. Expression in vivo in inflamed human vessels and atherosclerotic human and Watanabe rabbit vessels.
BACKGROUND
Monocyte adhesion to the vascular endothelium, an important component of an inflammatory response, is one of the earliest detected events in the pathogenesis of atherosclerosis. We have identified a monocyte adhesion molecule, recognized by monoclonal antibody (mAb) IG9, on the cell surface of human umbilical vein endothelial cells (HUVEC) 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 characterized.


METHODS
The kinetics of cytokine-induced IG9 protein expression on HUVEC were evaluated by enzyme-linked immunosorbent assay. TNF-alpha-treated HUVEC surface proteins, labeled with [125I]Na, were solubilized in NP-40 detergent and immunoprecipitated with mAb IG9 to determine the molecular weight of the IG9 protein. The functional role of the IG9 protein in monocyte binding in vitro to cytokine-activated endothelial cells was established in adhesion assays utilizing U937 cells (human promyelomonocytic cell line) and human peripheral blood monocytes. Minimally oxidized or modified low density lipoproteins (MM-LDL) have previously been shown to induce monocyte adhesion 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 cells 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-monocyte adhesion assays. To assess IG9 protein expression in vivo, formalin-fixed, paraffin-embedded sections of inflamed human tissues obtained from lung and healing myocardial infarctions, in addition to sections of human atherosclerotic coronary arteries, were analyzed by immunohistochemistry. Tissue sections from atherosclerotic Watanabe heritable hyperlipidemic rabbit aortas were also included in these studies.


RESULTS
The IG9 protein, undetected on untreated HUVEC, was expressed on their 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 enzyme-linked immunosorbent assay. A similar kinetic profile was elicited by interleukin-1 and lipopolysaccharide, whereas interferon-gamma (IFN-gamma) had minimal effect on IG9 expression. The IG9 protein has a molecular weight of 105,000 as determined by immunoprecipitation studies with TNF-alpha-treated HUVEC protein lysates. mAb IG9 significantly inhibited the binding of U937 cells and human peripheral blood monocytes to TNF-alpha-treated HUVEC and had no effect on peripheral blood lymphocyte 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 antibodies 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 analysis demonstrated that the endothelial cell lining of vessels in human lung and heart with evidence of inflammation characterized by an extensive mononuclear cell infiltration exhibited reactivity with mAb IG9, whereas vessels with no evidence of inflammation in the same sections as well as in sections from normal lung and heart were nonreactive.(ABSTRACT TRUNCATED AT 250 WORDS)
Calderon
T M
TM
Department of Pathology, Albert Einstein College of Medicine, Bronx, New York.
Factor
S M
SM
Hatcher
V B
VB
Berliner
J A
JA
Berman
J W
JW
D000818:Animals; D000911:Antibodies, Monoclonal; D001011:Aorta; D001161:Arteriosclerosis / Q000378:metabolism* / Q000473:pathology*; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000096:biosynthesis*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000378:metabolism* / Q000473:pathology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007249:Inflammation; D015473:Leukemia, Promyelocytic, Acute; D008168:Lung / Q000378:metabolism; D009000:Monocytes / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology; D009206:Myocardium / Q000378:metabolism; D011817:Rabbits; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D014409:Tumor Necrosis Factor-alpha