Leukocyte adhesion to endothelial cells (EC) is a central process in the pathogenesis of inflammatory disease. Specialized receptors for initial attachment enable blood-borne cells to initiate slow rolling venules. Subsequently, activating stimuli trigger engagement of activation- dependent integrins which, in turn, mediate firm attachment. This project is aimed at defining the role of transmembrane and intracellular (TM/IC) segments of leukocyte adhesion molecules in the early steps of this cascade. Using immunoelectron microscopy, it was found that many adhesion molecules display a distinct surface topography. L-selectin (CD62L), a specialized receptor for contact initiation, is clustered on microvilli. In contrast, CD44, an adhesion molecule with undefined intravascular function, is restricted to the planar cell body. Based on preliminary findings, it is proposed that the conspicuous topographies of L-selectin and CD44 are differentially regulated by their TM/IC domains and that topography itself is an important determinant of adhesion under flow. Microvillous receptors preferentially initiate attachment, whereas exclusion from microvilli limits a molecule's availability to situations when the cell has already been tethered. Following binding, TM/IC domains of L-selectin and CD44 may trigger differential activation pathways to signal ligation of ectodomains to the leukocyte. To investigate this concept, a panel of nine L1-2 lymphoma cell lines has been stably transfected with microvillous L-selectin, cell body CD44, E- selectin, and CD31 (both randomly distributed), and with chimeric genes encoding the TM/IC domains of one receptor and the ectodomain of another (aim 1). Transfectants will be used to study the importance of TM/IC domains in receptor topography using immunogold staining and scanning electron microscopy (aim 2). Topography-function relationships (aim 3) will be assessed in static and flow assays of cell binding to soluble and immobilized ligands. Cytochalasin-treated transfectants will also be used to study the general role of the cytoskeleton, and ezrin and moesin, two cytoskeletal proteins required for microvillous assembly, will be inhibited by antisense treatment and cytoplasmic loading with antibodies to determine the importance of microvilli in particular. Using intravital microscopy the in vivo relevance of topographic distribution for leukocyte trafficking in acute inflammation and in physiologic homing to high endothelial venules will be defined. Finally, the role of TM/IC domains in differential cytoplasmic Ca2 plus/minus mobilization by crosslinked receptors will be assessed, and additional mutant transfectants will be generated to identify distinct regions within TM/IC domains that are important for their function in topography and Ca2+ signaling (aim 4). Together, these studies will dissect the complex interplay of biophysical, molecular, biochemical, and cellular mechanisms that determine the earliest prerequisite steps in microvascular leukocyte adhesion.
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