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Endocytosis and shedding of the decay accelerating factor on human polymorphonuclear cells
1Division of Dermatology, University of California, San Diego, School of Medicine 92103.
Abstract:
The decay-accelerating factor (DAF) is a cell membrane glycoprotein that functions in the control of C activation. We studied the modulation of membrane DAF on polymorphonuclear cells (PMN) by using anti-DAF antibodies. Fluorescence-activated cell sorter analysis showed that DAF expression was reduced by 43 +/- 7% on resting or stimulated cells that were held at 37 degrees C for 30 min when compared with those kept on ice. Most of this reduction occurred within the first 15 min, and was followed by a gradual further decrease in surface DAF. PMN that were held at 37 degrees C for varying periods of time before DAF measurement had a gradual decrease suggestive of release of DAF from the PMN membrane or endocytosis. To examine the latter, PMN were reacted with anti-DAF at 0 degree C, followed by 125I-Fab'2 secondary antibodies at either 0 degree C or 37 degrees C, and subsequently treated with pronase. Thirty +/- 11% of the 125I remained bound to cells kept at 37 degrees C compared to 2% in those held at 0 degrees C. Internalization was further confirmed by electron microscopy. In PMN that were not exposed to pronase, 26 +/- 2% of the surface-associated 125I was released at 37 degrees C compared with 7% at 0 degrees C. Immunoprecipitation and SDS-PAGE of surface-labeled PMN showed that the temperature-dependent released DAF had a lower m.w. than membrane DAF. Immunofluorescent studies revealed that 37 degrees C mediated the redistribution of DAF from a homogeneous pattern into caps. These results show that under the conditions studied DAF is partially internalized and partially released from the PMN membrane to the fluid phase; the latter may contribute to the presence of DAF in body fluids.
Insights
Temperature affects decay-accelerating factor (DAF) on polymorphonuclear cells (PMN). DAF is released or internalized from the PMN membrane at 37°C, impacting its presence in body fluids.
Area of Science:
- Immunology
- Cell Biology
Background:
- Decay-accelerating factor (DAF) is a cell membrane glycoprotein regulating complement (C) activation.
- Polymorphonuclear cells (PMN) play a crucial role in immune responses.
Purpose of the Study:
- To investigate the modulation of membrane DAF on PMN under varying temperature conditions.
- To determine the mechanisms of DAF reduction on PMN surfaces.
Main Methods:
- Fluorescence-activated cell sorter analysis to quantify DAF expression.
- Radiolabeling with 125I-Fab'2 secondary antibodies to assess internalization and release.
- Pronase treatment to differentiate surface-bound from internalized DAF.
- Electron microscopy for visualizing DAF internalization.
- Immunoprecipitation and SDS-PAGE to analyze DAF molecular weight.
- Immunofluorescent studies to observe DAF redistribution.
Main Results:
- DAF expression on PMN decreased by 43% after 30 min at 37°C compared to incubation on ice.
- DAF reduction occurred rapidly within 15 min, followed by a gradual decrease.
- 30% of surface-labeled DAF was internalized at 37°C, confirmed by pronase resistance and electron microscopy.
- 26% of surface DAF was released into the fluid phase at 37°C, with released DAF having a lower molecular weight.
- 37°C induced DAF redistribution into caps on the PMN surface.
Conclusions:
- Temperature significantly modulates DAF localization on PMN membranes.
- DAF is partially internalized and partially released from PMN membranes at physiological temperatures.
- The release of DAF into the fluid phase may explain its presence in body fluids.