Issn Print: 1073-2322
Publication Date: 2006/06/01
NEUTROPHILS ARE DEGRANULATED AND PRIMED AFTER PARTIAL CHOLESTEROL DEPLETION: INVOLVEMENT OF PHOSPHATASE SHP-1
CM Cave; CT Robinson; AB Lentsch; JS Solomkin
Excerpt
Neutrophil (PMN) priming is in part characterized by fusion of plasma (PM) and specific granule (SG) membranes as a means of delivering the components of focal adhesions (i.e.integrin CD11b) and the NADPH oxidase. In recent years the role of detergent resistant membranes (DRM), which can serve as sites of assembly for structural and signaling molecules, has received renewed attention. We sought to confirm the importance of DRM molecules by altering the cholesterol content and therefore DRM solubility. We utilized the cholesterol sequestering agent methyl-β-cyclodextrin (mβCD) to partially deplete cholesterol content. Plasma and granule membrane fractions of PMNs were obtained by N2 cavitation and Percoll gradient centrifugation. DRMs were isolated from these fractions and examined for the presence of tyrosine-phosphorylated lyn and the phosphatase SHP-1. Our results indicate that MβCD induced degranulation as evidenced by increased CD11b surface expression (A). Immunoblot analysis showed decreased phosphorylation of lyn in the DRMs of PM after mβCD treatment (B), while these fractions showed an increase in SHP-1(C). Our data suggest that one mechanism for this might be the differential solubility of kinases and phosphatases in the altered DRM produced by limited removal of cholesterol with MβCD.
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