title: Identification by analytical flow cytometry of prolactin receptors on immunocompetent cell populations in the mouse.
abstract: Five monoclonal antibodies (T1, T6, U5, U6, and E21) made to the external portion of the rat PRL receptor (PRL-R) were conjugated to fluorescein isothyrocynate and used to examine the presence of PRL-R on mouse lymphocytes and macrophages using analytical flow cytometry. The monoclonals were initially evaluated using Nb2 cells, a cloned line from a rat lymphoma, and NOG-8 cells, a cloned line from normal mouse mammary gland tissue, which are known to have PRL-R. All monoclonal antibodies bound to these cells, but the U5 monoclonal gave the best separation from unstained cells. CTLL-2 cells, a mouse lymphoma cell line containing interleukin-2 receptors, did not bind to any of the monoclonals. Isolated thioglycolate-induced peritoneal macrophages contained PRL-R, and the PRL-R monoclonal U5 gave the best separation from unstained cells. Eighty-five percent of macrophages constitutively had PRL-R using this monoclonal. In vivo stimulation of the popliteal lymph node by the injection of Concanavalin-A (Con-A) into the right foot pad of intact and ovariectomized (OVX) BALB/c mice induced, at the end of 10-12 h, a marked increase in interleukin-2 receptor (IL-2R) expression on CD4, CD8, and B-cells compared to the unstimulated left popliteal lymph node. The number of CD4 and CD8 cells from OVX animals with IL-2R was twice that from intact animals, whereas no difference in the percentage of B-cells with IL-2R was evident. PRL-R were constitutively expressed on 5% of the CD4 cells and 20% of the CD8 cells and were increased in the Con-A-stimulated lymph node when examined with the U5 PRL-R monoclonal. A higher percentage of CD4 and CD8 cells from OVX animals constitutively expressed PRL-R, and when stimulated with Con-A, a further increase was observed compared to the level in intact animals. Using the U5 monoclonal, over 80% of the B220 cells constitutively expressed PRL-R; however, when T1, T6, and U6 monoclonals were examined, the percentage was considerably below (20%) than that observed for U5. Con-A stimulation did not alter the percentage of B220 cells expressing PRL-R. These results show the importance of identifying lymphocyte subsets and examining a number of PRL-R monoclonals in determining lymphocyte PRL-R expression on the surface of the cell.
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201.
authors.forename: R R
authors.initials: RR
authors.lastname: Gala
authors.forename: E M
authors.initials: EM
authors.lastname: Shevach
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D015704:CD4 Antigens / Q000032:analysis; D016827:CD8 Antigens / Q000032:analysis; D002460:Cell Line; D003208:Concanavalin A / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D007121:Immunocompetence; D016131:Lymphocyte Subsets / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism* / Q000502:physiology; D008264:Macrophages / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D010052:Ovariectomy; D011981:Receptors, Prolactin / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D016827:CD8 Antigens; D011981:Receptors, Prolactin; D003208:Concanavalin A; D016650:Fluorescein-5-isothiocyanate