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Upregulation of human neutrophil CD59, a regulator of the membrane attack complex of complement, following cell
D L Gordon1, H Papazaharoudakis, T A Sadlon
1Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, Australia.
Abstract:
CD59 is a membrane glycoprotein that regulates the membrane attack complex of complement and protects cells from autologous complement damage. Human polymorphonuclear leucocyte (PMN) expression of CD59 was confirmed by flow cytometry following staining with mAb 1F5, and western blotting revealed staining of a 19-23 kDa band. Warming of PMN from 4 to 37 degrees C resulted in spontaneous CD59 upregulation. A dose-dependent increase in expression following PMN stimulation with FMLP was observed and occurred within minutes, indicating that new protein synthesis was not required. Treatment of PMN with calcium ionophore A23187 resulted in similar increases in CD59 expression. This occurred in the presence or absence of extracellular calcium, indicating that upregulation was dependent on release of calcium from intracellular stores. Evidence for a mobilizable intracellular pool of CD59 was obtained by detection of increased binding of 1F5 following PMN permeabilization; CD59 could also be re-expressed after stripping by phosphatidylinositol specific phospholipase C (PI-PLC) by treatment with FMLP or A23187. There was a correlation between CD59 upregulation and lactoferrin release, suggesting that stores of CD59 may be associated with secondary granules. These studies indicate that PMN expression of CD59 is enhanced by cell activation and suggest the presence of an intracellular pool of CD59 which can be translocated to the cell membrane upon PMN stimulation.
Insights
Cell activation enhances CD59 expression on polymorphonuclear leucocytes (PMNs) through a calcium-dependent mechanism. An intracellular pool of CD59 is mobilized to the cell membrane upon stimulation.
Area of Science:
- Immunology
- Cell Biology
Background:
- CD59 is a crucial complement regulatory protein protecting cells from damage.
- Polymorphonuclear leucocytes (PMNs) are key immune cells involved in inflammatory responses.
Purpose of the Study:
- To investigate the regulation of CD59 expression on human PMNs.
- To explore the mechanisms underlying CD59 mobilization and upregulation.
Main Methods:
- Flow cytometry and western blotting to confirm CD59 expression.
- Stimulation of PMNs with FMLP and calcium ionophore A23187.
- Analysis of intracellular CD59 pools and re-expression after PI-PLC stripping.
Main Results:
- CD59 expression on PMNs increased with temperature and FMLP stimulation.
- Upregulation was rapid, not requiring new protein synthesis, and dependent on intracellular calcium release.
- Evidence suggests an intracellular CD59 pool associated with secondary granules, mobilized upon activation.
Conclusions:
- PMN activation enhances CD59 expression via mobilization of an intracellular pool.
- This mechanism contributes to complement regulation during inflammatory processes.