Modulation of the plasma membrane domain structure of human neutrophils

Abstract

Eukaryotic cell plasma membranes form an interface between cells and their environment and function to detect and interpret environmental cues. The work described in this dissertation examines the changes that occur in membrane structure during plasma membrane function in human neutrophils and a fungal opportunist. The body of this work examines how circulating neutrophils can remain functionally inactive in the presence of perturbing influences inherent in the blood circulation, and yet rapidly activate upon exposure to proinflammatory agents. It is hypothesized that the regulated modulation of plasma membrane domain structure determines the activation of blood-leukocytes, in vivo. Experimentation is based the isolation of blood-neutrophils in either nonactivated or activated (primed) cellular states using dextran- or gelatin-based preparative methods, respectively. Analysis of plasma membrane cortical components actin, fodrin, ezrin, CD45 and CD43 by sucrose density sedimentation, flow cytometry and indirect immunofluorescence microscopy indicated significant differences in the plasma membrane structure of both neutrophil populations. In nonactivated neutrophils, cortical actin and fodrin were cytosolic, thus indicating the absence of cortical structure in this population.

However, cortical actin and fodrin were membrane-associated in activated neutrophils showing the existence of a cortex. Fodrin, actin, ezrin and their respective anchors, CD45 and CD43 did not codistribute with the plasma membrane marker, alkaline phosphatase, in sucrose density gradients made with primed neutrophils. These latter results suggested the lateral compartmentalization of the plasma membrane cortex into compositionally distinct surface domains. Additional studies were performed to examine the surface-association of hCap, a soluble microbicidal component of neutrophil specific granules. Results indicated association of hCap with primed and degranulated but not nonactivated plasma membranes. This interaction was resistant to 1M salt but labile to 10 mM NaOH, indicating a high affinity association. In support of this, hCap also co-partitioned with detergent in Triton X-114 phase experiments. In separate studies, marked alterations in the plasma membrane lipid metabolism of isolates from Candida glabrata are correlated with an ability to survive and grow in vivo. Altogether, this work provides insight into structure-function relationships at the plasma membrane level.