Localization of ligands for L-selectin in mouse peripheral lymph node high endothelial cells by colloidal gold conjugates

Abstract

L-selectin, a Ca(2+)-dependent lectin-like receptor, mediates lymphocyte attachment to high endothelial venules (HEV) of peripheral lymph nodes (PLN) during the process of lymphocyte homing. Two endothelial-derived ligands for L-selectin, known as GlyCAM-1 (Sgp50) and CD34 (Sgp90), have been identified by affinity precipitation of lymph node extracts with a chimeric molecule that combines the extracellular domains of L-selectin with the human IgG1 Fc region (L-selectin-IgG) (J Cell Biol 110:2221, 1990). Here, using a histologic probe based on colloidal gold conjugated to L-selectin-IgG (LS-Ig), we performed morphologic mapping of the HEV ligands in PLN at both the light and electron microscopic levels. With a postembedding labeling method, intense LS-Ig-gold staining of PLN HEV was observed, while the HEV of Peyer's patches (PP) were negative. The specificity of LS-Ig-gold staining was established by pretreatment of sections with sialidase and coincubation of sections with EGTA, fucoidin, or L-selectin-IgG itself. In ultrastructural studies of high endothelial cells(HEC), gold particles were bound to the trans-Golgi network(TGN) and to peripheral vesicles in the cytoplasm. Gold labeling was also detected in a patchy distribution on the entire luminal vascular surface of HEC. Although the perivascular fibroreticular sheath of HEV was frequently labeled limited labeling was observed on the basolateral surfaces of the HEC. In most cases, the HEC membrane surrounding migrating lymphocytes was negative. These results show that L-selectin ligands pass through the Golgi apparatus during their biosynthesis, are stored in secretory granules, and are expressed on the vascular luminal surface of the HEC. A polyclonal antiserum to GlyCAM-1 intensely stained intracellular organelles in the biosynthetic pathway including cytoplasmic vesicles, but failed to stain the cell surface of HEC. Given its presence in serum as a soluble factor, GlyCAM-1 is likely to be a secretory product.