NEUTROPHILS DEGRANULATE AND PRIME AFTER PARTIAL CHOLESTEROL DEPLETION

Abstract

Detergent resistant membranes (DRM) serve as sites for assembly for structural, signaling and functional complexes. Rationale: We hypothesized that neutrophil priming for adherence dependent oxidant production occurred through degranulation and required DRM participation. We utilized the cholesterol sequestering agent methyl-β-cyclodextrin (mβCD) to deplete cholesterol content, a common strategy for DRM function analysis. Results: MβCD, rather than disrupting and attenuating the primed state, itself served as a priming stimulus. Cholesterol-loaded MβCD had no priming effect. Flow cytometry revealed upregulation of CD11b and CD66 (specific granule marker) following MβCD treatment (not shown). These findings were confirmed and extended by confocal microscopy, which demonstrated increased spreading on fibronectin with colocalization (yellow) of the DRM marker GM1(green) and CDllb (red). Conclusions: Unstimulated neutrophils express little of the DRM marker GM1. Following priming, the GM1-containing surface area of the neutrophil expands substantially. The source of DRM markers expressed and aggregated on the surface of primed human neutrophils is the specific granule population. How altered cholesterol content causes this remains obscure. We speculate differential DRM solubility of regulatory molecules may be involved.Figure