Evidence for an intracellular pool of a migration inhibitory factor-associated, activation antigen of human mononuclear phagocytes, Mo3e.
Mo3e is a protease-sensitive Ag (p75,50) selectively expressed by human monocytic cells stimulated in vitro by exposure to various activating factors including PMA. Here, we report the existence of a large intracellular pool of Mo3e Ag in addition to that expressed on the surface of activated U-937 cells. As detected by quantitative immunofluorescence analysis, permeabilization of unstimulated and PMA-stimulated U-937 cells revealed a latent pool of Mo3e Ag that was 75-fold and 9-fold greater, respectively, than the magnitude of Mo3e Ag expressed on the surface. PMA stimulation not only induced an increase in the relative proportion of Mo3e antigen expressed on the surface membrane, but also stimulated a 1.8-fold increase in "total" Mo3e detectable in permeabilized cells. Trypsin treatment of intact PMA-stimulated U-937 cells eliminated surface Mo3e expression but had little measureable effect on the total Mo3e pool. Permeabilization also uncovered a sequestered compartment of Mo3e Ag in I-937 cells, a variant of U-937 that is surface Mo3e negative. Although the PMA-induced surface Mo3e expression of U-937 was abrogated by cycloheximide, the total pool of Mo3e detectable in permeabilized PMA-stimulated cells was only partially reduced; cycloheximide treatment caused no reduction in the intracellular Mo3e compartment of unstimulated U-937 cells. Detergent lysates of PMA-stimulated U-937 cells exhibited undiminished quantities (relative to untrypsinized cells) of p75 and p50 proteins immunoreactive with anti-Mo3e mAb as detected by Western blotting. This trypsin-sequestered intracellular Mo3e Ag may serve as a reservoir for the up-regulated surface expression of Mo3e that occurs as a result of mononuclear phagocyte activation.
Todd
R F
RF
Simpson Memorial Institute, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109.
Adams
M D
MD
Rogers
C E
CE
Tripp
L R
LR
Liu
D Y
DY
D000911:Antibodies, Monoclonal / Q000276:immunology; D000951:Antigens, Neoplasm / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis; D002463:Cell Membrane Permeability; D006801:Humans; D007424:Intracellular Fluid / Q000032:analysis; D016403:Lymphoma, Large B-Cell, Diffuse / Q000473:pathology; D009000:Monocytes / Q000187:drug effects / Q000276:immunology*; D009363:Neoplasm Proteins / Q000032:analysis; D011499:Protein Processing, Post-Translational; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000276:immunology
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D009363:Neoplasm Proteins; D014357:Trypsin; D013755:Tetradecanoylphorbol Acetate