title: Quantitative fluorescence analysis of cyclosporine binding to human leukocytes.
abstract: The purpose of this investigation was to estimate the binding of cyclosporine at the single-cell level on human peripheral lymphocytes, and to test possible identity of the cyclosporine-binding site with a common receptor of T cell activation. A dansyl-coupled derivative (Dans cyclosporine) was used as a fluorescent probe. The histograms of unseparated, labeled peripheral leukocytes obtained by a fluorescence-activated cell sorter (FACS) showed that Dans cyclosporine stained all leukocytes--but two distinct populations could be separated based on the intensity of fluorescence. The more brightly labeled cells consisted mainly of granulocytes and monocytes, whereas the less-bright cells represented the lymphocyte compartment. Fluorescence microscopy revealed binding on the membrane for both cell populations; the label was, however, rapidly internalized in phagocytes. For both populations binding was saturable, time and temperature dependent, and reversible. Half-saturation occurred at approximately 5 X 10(-7) M (Kd). With respect to lymphocyte subpopulations, no difference of cellular fluorescence was found between unseparated lymphocytes and T cell subsets. In addition, mitogens such as concanavalin A, phytohemagglutinin, phorbol 12-myristate 13-acetate, or OKT3 antibody did not inhibit Dans cyclosporine binding. These results clearly indicate that cyclosporine binds to all peripheral blood lymphocytes, and no preferential binding on T cell subsets can be detected.
mesh_terms: D000328:Adult; D001667:Binding, Competitive; D003524:Cyclosporins / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006098:Granulocytes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000145:classification / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism; D011971:Receptors, Immunologic / Q000032:analysis*
chemical_list: D003524:Cyclosporins; D011971:Receptors, Immunologic; C028752:cyclosporin receptor