A FLUORESCENT CELLULAR ADHESION ASSAY USING INSECT-CELL PRODUCED HUMAN VCAM1

Activated endothelium and some dendritic cells express the adhesion mo lecule VCAM1, a member of the immunoglobulin gene superfamily. Mononuc lear leukocytes display the integrin VLA4 that functions as a counter- receptor for VCAM1. The interaction of VCAM1 with VLA4 mediates cell t o cell adhesion events believed to be important regulators of inflamma tion, cancer cell metastasis, and atherosclerosis. This report describ es the development of a fluorescent adhesion assay that specifically m easures T cell adhesion to recombinant human VCAM1 (rVCAM1) expressed in a baculovirus expression vector system (BEVS). We describe a simple and rapid protocol to partially purify non-denatured rVCAM1 from inse ct cell membrane preparations (VCAM1 infected Sf9 cells). Jurkat cells , a T cell line expressing VLA4, specifically adhered to the rVCAM1 me mbrane preparations coated onto 96-well plates. Jurkat cells did not a dhere to control membrane preparations that lacked rVCAM1 protein. Bot h unstimulated and IL-2 stimulated Jurkat cells displayed functional V LA4 capable of binding to immobilized rVCAM1. Monoclonal antibodies re cognizing either VCAM1 (E1/6, BBA6) or VLA4 (HP2/1) blocked specific V CAM1/VLA4 adhesion, whereas a monoclonal antibody to the alpha chain o f LFA1 did not block adhesion. The methods described here could be app lied to develop similar functional assays for other cell surface recep tors/counterreceptors expressed in a BEVS.