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Shedding as a mechanism of down-modulation of CD14 on stimulated human monocytes
1Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Abstract:
CD14, expressed on the surface of monocytes as a phospholipid-linked protein, is a receptor for serum LPS binding protein/LPS complex. It was specifically down-modulated after stimulation of monocytes by physiologic activating/differentiating agents such as bacterial LPS and IFN-gamma, by the pharmacologic agents PMA and calcium ionophore A23187, and by anti-CD14 antibodies. The down-modulation was almost totally blocked at 4 degrees C or at pH 4.5 and markedly inhibited by the protease inhibitors diisopropylfluorophosphate and PMSF. A soluble labeled CD14 was isolated from culture supernatant of surface iodinated monocytes after their activation, indicating that CD14 is shed from the cell surface rather than internalized. The size of the soluble CD14 shed from the monocytes in vitro was smaller than that of either the membrane-bound form or a soluble CD14 cleaved from the cell surface by phosphatidyl inositol-specific phospholipase C, but identical to the size of one of the two major soluble CD14 forms normally found in human serum. These data suggest that CD14 shedding induced by monocyte stimulation may play an important role in the regulation of surface CD14 expression.
Insights
Monocyte stimulation causes CD14 shedding, a process regulated by temperature, pH, and proteases. This shedding, releasing soluble CD14, may control surface CD14 expression levels.
Area of Science:
- Immunology
- Cell Biology
Background:
- CD14 is a monocyte surface receptor for LPS binding protein/LPS complexes.
- Monocyte activation involves complex signaling pathways affecting surface protein expression.
Purpose of the Study:
- To investigate the mechanism of CD14 down-modulation on monocytes.
- To determine if CD14 is internalized or shed upon monocyte activation.
Main Methods:
- Monocyte stimulation with various agents (LPS, IFN-gamma, PMA, A23187, anti-CD14 antibodies).
- Analysis of CD14 expression under different conditions (temperature, pH, protease inhibitors).
- Isolation and characterization of soluble CD14 from cell supernatants.
Main Results:
- CD14 expression was down-modulated by multiple stimuli.
- Down-modulation was inhibited by low temperature, acidic pH, and protease inhibitors.
- Soluble CD14 was detected in supernatants, indicating shedding rather than internalization.
- The shed soluble CD14 size matched a form found in human serum.
Conclusions:
- Monocyte activation induces CD14 shedding.
- This shedding mechanism is temperature, pH, and protease-sensitive.
- CD14 shedding likely plays a role in regulating surface CD14 expression during monocyte activation.