Studies of binding and internalization of human recombinant monocyte chemotactic and activating factor (MCAF) by monocytic cells.
Recombinant human monocyte chemotactic and activating factor (MCAF) was iodinated and specific binding sites for this cytokine were detected on human peripheral blood monocytes, the monocytic leukemia cell line THP-1, and on PMA-differentiated HL60 and U937 cell lines. The binding sites were specific for MCAF since other polypeptide cytokines and the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) failed to compete for 125I-rhMCAF binding. Steady-state binding experiments at 4 degrees C revealed the presence of 13,000 and 18,000 receptor sites/cell on monocytes and THP-1 cells with Kd values of 22.5 nM and 25.7 nM, respectively. Compared to a human natural MCAF, rhMCAF was less potent in inducing maximal monocyte migration. Human natural MCAF similarly competed more efficiently for 125I-rhMCAF binding than unlabelled rhMCAF. The ligand-receptor association was highly temperature-dependent, with maximal ligand uptake at 37 degrees C accompanied by internalization of the ligand-receptor complexes. The internalized 125I-MCAF was progressively degraded and released into the culture medium starting at 30 min. Lysosomotropic ammonium chloride could inhibit the degradation of this ligand suggesting the involvement of lysosomal enzymes in the proteolytic digestion. Incubation with cycloheximide did not block the rapid reappearance of MCAF receptors within 20 min on the cell surface indicative of receptor recycling rather than new protein synthesis. These data indicate that monocytic cells express specific receptors for rhMCAF which can be dynamically regulated by MCAF.
Wang
J M
JM
Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick Cancer Research and Development Center, Maryland 21702.
Hishinuma
A
A
Oppenheim
J J
JJ
Matsushima
K
K
D001692:Biological Transport; D002460:Cell Line; D018932:Chemokine CCL2; D002630:Chemotactic Factors / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D007948:Leukemia, Monocytic, Acute; D009000:Monocytes / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism*; D054390:Receptors, CCR2; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D018932:Chemokine CCL2; D002630:Chemotactic Factors; D016207:Cytokines; D054390:Receptors, CCR2; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine; D011994:Recombinant Proteins; D003513:Cycloheximide; D013755:Tetradecanoylphorbol Acetate