Internalization, lysosomal degradation and new synthesis of surface membrane CD4 in phorbol ester-activated T-lymphocytes and U-937 cells.
Protein kinase C activating phorbol esters downregulated membrane CD4 by endocytosis in U-937 and human T-cells. Half-time for internalization (approximately 15 min at 50 ng/ml PMA) was determined by FACS. CD4-bound 125I-labeled anti-CD4 mAb was rapidly degraded in PMA-activated cells, whereas degradation was low in resting cells. Endocytosis and/or degradation of anti-CD4 mAb was suppressed by H7, and by inhibitors of membrane traffic (Monensin) and lysosome function (methylamine, chloroquine). Immunocytochemistry localized CD4 to the surface of unstimulated T-cells. Upon PMA stimulation occasional labeling was seen in endosomes but whole cell CD4 decreased dramatically. However, methylamine-treated PMA blasts showed accumulation of CD4 in lysosomes and accordingly, pulse-chase experiments in biolabeled cell cultures suggested a manifest reduction of CD4 half-life in response to PMA. Despite their low surface CD4 density, PMA blasts exhibited uptake and accelerated degradation of anti-CD4 mAb. Also, inhibitors of protein synthesis enhanced the PMA-induced downregulation, and membrane CD4 reappeared on fully activated as well as unstimulated cells treated with trypsin. Ongoing CD4 synthesis in activated cells was further evidenced by metabolic labeling and Northern blot analysis demonstrating unaltered or slightly increased CD4 protein and mRNA levels resulting from PMA. Our findings demonstrate that phorbol esters downregulate the cellular CD4 pool by endocytosis and subsequent lysosomal degradation of membrane CD4. Transport of CD4 to the cell surface and CD4 synthesis is unaffected by activation.
Petersen
C M
CM
Department of Clinical Immunology, University Hospital of Aarhus, Denmark.
Christensen
E I
EI
Andresen
B S
BS
Møller
B K
BK
D015704:CD4 Antigens / Q000096:biosynthesis*; D002402:Cathepsin D / Q000032:analysis; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D002478:Cells, Cultured / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D015658:HIV Infections / Q000276:immunology; D006207:Half-Life; D006801:Humans; D008213:Lymphocyte Activation; D008744:Methylamines; D010703:Phorbol Esters / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology / Q000648:ultrastructure
D015704:CD4 Antigens; D008744:Methylamines; D010703:Phorbol Esters; D012333:RNA, Messenger; C027451:methylamine; D002402:Cathepsin D