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Evidence for an intracellular pool of a migration inhibitory factor-associated, activation antigen of human
R F Todd1, M D Adams, C E Rogers
1Simpson Memorial Institute, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109.
Abstract:
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.
Insights
A large intracellular pool of Mo3e antigen exists in human monocytic cells, serving as a reservoir for surface expression upon activation. This finding sheds light on mononuclear phagocyte activation and Mo3e regulation.
Area of Science:
- Immunology
- Cell Biology
Background:
- Mo3e antigen (p75,50) is a protease-sensitive marker expressed on activated human monocytic cells.
- U-937 cells, a monocytic cell line, express Mo3e upon stimulation with phorbol 12-myristate 13-acetate (PMA).
Purpose of the Study:
- To investigate the presence and characteristics of intracellular Mo3e antigen in U-937 cells.
- To determine the relationship between intracellular and surface Mo3e pools during cellular activation.
Main Methods:
- Quantitative immunofluorescence analysis to detect Mo3e antigen.
- Permeabilization of cells to access intracellular compartments.
- Trypsin treatment to assess surface-bound antigen.
- Cycloheximide treatment to evaluate protein synthesis dependence.
- Western blotting to confirm protein presence.
Main Results:
- A significant intracellular pool of Mo3e antigen was identified, substantially larger than the surface pool in both unstimulated (75-fold) and PMA-stimulated (9-fold) U-937 cells.
- PMA stimulation increased surface Mo3e expression and the total detectable Mo3e pool by 1.8-fold.
- Trypsin treatment removed surface Mo3e but minimally affected the total pool, indicating intracellular sequestration.
- A variant cell line (I-937) negative for surface Mo3e still contained an intracellular Mo3e compartment.
- While PMA-induced surface expression was blocked by cycloheximide, the total intracellular pool was only partially reduced, suggesting a stable intracellular reservoir.
Conclusions:
- Human monocytic cells possess a substantial intracellular reservoir of Mo3e antigen.
- This intracellular pool serves as a source for the increased surface expression of Mo3e observed during mononuclear phagocyte activation.
- Mo3e regulation involves both synthesis and the mobilization of a pre-existing intracellular pool.