Molecular mechanisms underlying lymphocyte recirculation. III. Characterization of the LECAM-1 (L-selectin)-dependent adhesion pathway in rats

Abstract

LECAM-1 (L-selectin) is thought to play an important role in the binding of lymphocytes to high endothelial venules (HEV) of peripheral lymph nodes (LN), which is an essential process in lymphocyte recirculation. Previously we cloned the rat LECAM-1 cDNA. In this study, by using this probe we have sought to characterize a LECAM-1-dependent adhesion pathway in the rat. We have constructed a cDNA for rat LECAM-1-IgG chimera (rLEC-IgG), expressed it, purified the secreted recombinant chimera molecules, and produced mAb reactive with the rat homologue of LECAM-1 by using the chimera molecules. The use of rLEC-IgG revealed that ligands for LECAM-1 are selectively accumulated in high endothelial (HE) cells in LN, the white matter, neurons, cerebellar Purkinje cells, and choroid plexus of the central nervous system and also distal tubules and capillary blood vessels of the kidney. Binding of lymphocytes to LN HEV on frozen sections was blocked by either rLEC-IgG or the anti-rat LECAM-1 mAb. An HEV-derived cell line, Ax, specifically bound to rLEC-IgG fixed on plastic plate. Consistent with the presence of a C-type lectin domain in the ligand-binding region of LECAM-1, the binding was Ca2+ dependent and inhibitable by either the mannose-6-phosphate-rich polysaccharide polyphosphomannan ester or the anti-rat LECAM-1 mAb. These results indicate that the specific ligand for rat LECAM-1 is expressed on the Ax cells. rLEC-IgG precipitated 55-, 65-, 120-, 190-, and > 250-kDa sulfated glycoproteins from LN lysates and 190-, > 250-, and > 500-kDa proteins from Ax cell lysate. The precipitation was Ca2+ dependent and LECAM-1 specific. These results suggest that a carbohydrate structure on HE cells recognized by LECAM-1 is borne possibly on a limited number of cell surface-sulfated glycoproteins. The ligands were also found to be secreted in LN culture supernatants. rLEC-IgG and Ax cells should prove valuable for studying further the role of LECAM-1 in dynamic interactions between lymphocytes and HE cells.