Reorganization of the human neutrophil plasma membrane is associated with functional priming: implications for neutrophil preparations

Abstract

Changes in the functional and plasma membrane organizational states of human neutrophils were examined using two isolation procedures, which may simulate altered physiological states in vivo. A gelatin-based method of blood-neutrophil isolation was used to model in vivo priming, and neutrophils isolated by this method were compared with control populations prepared by a pyrogen-free, dextran-based method. Gelatin-prepared neutrophils were functionally primed for adherence and agonist-stimulated superoxide generation relative to unprimed, control neutrophils. The organizational state of the membrane cortex was examined by mapping the subcellular distribution of select cortical and transmembrane proteins by several methods, including subcellular fractionation, indirect immunofluorescence, and compositional analysis of Triton X-100-insoluble membrane skeleton preparations. Filamentous actin, fodrin, and the fodrin anchor, CD45, were largely cytoplasmic in unprimed neutrophils but translocated to plasma membranes upon priming, whereas CD43 and ezrin were exclusively surface-associated in both populations. Isopycnic sucrose density gradient analysis of N(2)-cavitated neutrophils revealed a major shift in the distribution of surface-associated transmembrane and membrane cortical components relative to the plasma membrane marker alkaline phosphatase in primed but not unprimed neutrophils. Similar results were obtained after neutrophil stimulation with known priming agents, LPS, TNF-alpha, or GM-CSF. Together, these results may suggest that priming of suspended, circulating neutrophils is associated with a large-scale reorganization of the plasma membrane and associated membrane cortex in a process that is independent of cellular adhesion and gross morphologic polarization.