ORGANIZATION AND MOBILITY OF CD11B CD18 AND TARGETING OF SUPEROXIDE ON THE SURFACE OF DEGRANULATED HUMAN NEUTROPHILS/

A monoclonal IgM, specifically recognizing both CD11b and CD18 of huma n neutrophils, was used to examine the organization and mobility of CD 11b/CD18 in the plasma membrane of human neutrophils degranulated by d ihydrocytochalasin B (dhCB) treatment and fMet-Leu-Phe (fMLF) stimulat ion. Subcellular fractionation analysis of untreated or dhCB-treated c ontrol neutrophils indicated that 20% of CD11b/CD18 cosedimented with plasma membrane and the remainder with specific granules. In contrast, fMLF stimulation of dhCB-treated cells caused a major reorganization of CD11b/CD18, in which 60-70% of CD11b/CD18 sedimented in dense plasm a membrane fractions that were also enriched in superoxide-generating NADPH oxidase activity. Similarly pretreated neutrophils were fixed, i mmunogold labeled, and examined by scanning electron microscopy. Immun ogold particles were distributed uniformly over the symmetrically ruff led surface of unstimulated neutrophils, On dhCB-treated cells, immuno gold was mostly uniformly distributed on a smooth membrane with a smal l percentage of particles lining up into linear arrays. After fMLF + d hCB stimulation, CD11b/CD18 gold label was more abundant on the cell s urface and formed large aggregates on polarized membrane protrusions. However, when cells were adhered to an albumin-coated quartz surface a nd stimulated with fMLF in the presence of dhCB, immunogold was exclud ed on the articulated and rounded cell body but concentrated on the pe riphery of adherent lamellae, Fluorescence photobleaching recovery ind icated that in unstimulated cells 38 +/- 3% of CD11b/CD18 was mobile ( R) with a diffusion constant D of 3.1 +/- 0.3 x 10(-10) cm(2)/s. Treat ment with dhCB raised R and D 24 and 74%, respectively. Stimulation us ing 1 mu M fMLF with dhCB lowered D and R to near control levels. Sinc e NADPH oxidase and CD11b/CD18 cosediment in high-density plasma membr ane domains after fMLF + dhCB stimulation, we speculate that a stimulu s-induced reorganization of CD11b/CD18 and NADPH oxidase to common mem brane domains may occur in fMLF + dhCB-degranulated neutrophils. (C) 1 998 Academic Press.