Characterization of rat LECAM‐1 (L‐selectin) by the use of monoclonal antibodies and evidence for the presence of soluble LECAM‐1 in rat sera

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Abstract
We have characterized the rat LECAM‐1 (L‐selectin) by the use of newly generated hamster anti‐rat LECAM‐1 monoclonal antibodies (mAb) (HRL1, HRL2, HRL3, HRL4), with respect to the biochemistry, cellular distribution and function, and developed an ELISA system to detect the soluble form of rat LECAM‐1. In the rat, lymphocyte and neutrophil LECAM‐1 have apparent molecular masses of 65 and 62 kDa, respectively, and differential glycosylation may account for the molecular heterogeneity. Readily detectable levels of LECAM‐1 are expressed on peripheral blood lymphocytes and neutrophils, but not on thymocytes. Lymphocyte LECAM‐1 is rapidly shed from the cell surface upon cell activation with PMA, but not with interleukin (IL)‐8. In contrast, neutrophil LECAM‐1 showed rapid shedding upon stimulation with phorbol 12‐myristate 13‐acetate (PMA) or IL‐8. Concomitantly there is up‐regulated expression of Mac‐1 in PMA‐ and IL‐8‐stimulated neutrophils. Neutrophil rolling in mesenteric venules was significantly inhibited by administration of function‐blocking anti‐rat LECAM‐1 mAb HRL3, but not by non‐blocking HRL4, indicating that LECAM‐1 plays a significant role in leukocyte rolling. Given that LECAM‐1 is rapidly shed from the cell surface, we attempted to develop an ELISA system for detecting LECAM‐1 is soluble form, and measured the levels in experimental autoimmune uveitis. The circulating levels of LECAM‐1 increased from day 4, which preceded the appearance of clinical signs of uveitis and remained high until uveitis subsided, suggesting that soluble LECAM‐1 is potentially a useful parameter to monitor certain types of inflammatory or immune disorders.