Identification by analytical flow cytometry of prolactin receptors on immunocompetent cell populations in the mouse.
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.
Gala
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RR
Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201.
Shevach
E M
EM
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*
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D016827:CD8 Antigens; D011981:Receptors, Prolactin; D003208:Concanavalin A; D016650:Fluorescein-5-isothiocyanate