Mukherjee G, Rasmusson B, Linner J G, Quinn M T, Parkos C A, Magnusson K E, Jesaitis A J
Department of Microbiology, Montana State University, Bozeman, Montana, 59715, USA.
Arch Biochem Biophys. 1998 Sep 1;357(1):164-72. doi: 10.1006/abbi.1998.0807.
A monoclonal IgM, specifically recognizing both CD11b and CD18 of human neutrophils, was used to examine the organization and mobility of CD11b/CD18 in the plasma membrane of human neutrophils degranulated by dihydrocytochalasin B (dhCB) treatment and fMet-Leu-Phe (fMLF) stimulation. Subcellular fractionation analysis of untreated or dhCB-treated control 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 plasma membrane fractions that were also enriched in superoxide-generating NADPH oxidase activity. Similarly pretreated neutrophils were fixed, immunogold labeled, and examined by scanning electron microscopy. Immunogold particles were distributed uniformly over the symmetrically ruffled surface of unstimulated neutrophils. On dhCB-treated cells, immunogold was mostly uniformly distributed on a smooth membrane with a small percentage of particles lining up into linear arrays. After fMLF + dhCB stimulation, CD11b/CD18 gold label was more abundant on the cell surface and formed large aggregates on polarized membrane protrusions. However, when cells were adhered to an albumin-coated quartz surface and stimulated with fMLF in the presence of dhCB, immunogold was excluded on the articulated and rounded cell body but concentrated on the periphery of adherent lamellae. Fluorescence photobleaching recovery indicated 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) cm2/s. Treatment with dhCB raised R and D 24 and 74%, respectively. Stimulation using 1 microM fMLF with dhCB lowered D and R to near control levels. Since NADPH oxidase and CD11b/CD18 cosediment in high-density plasma membrane domains after fMLF + dhCB stimulation, we speculate that a stimulus-induced reorganization of CD11b/CD18 and NADPH oxidase to common membrane domains may occur in fMLF + dhCB-degranulated neutrophils.
一种特异性识别人类中性粒细胞CD11b和CD18的单克隆IgM，被用于检测经二氢细胞松弛素B（dhCB）处理和N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLF）刺激后脱颗粒的人类中性粒细胞膜中CD11b/CD18的组织和流动性。对未处理或经dhCB处理的对照中性粒细胞进行亚细胞分级分离分析表明，20%的CD11b/CD18与质膜共沉降，其余的与特异性颗粒共沉降。相比之下，fMLF对经dhCB处理的细胞的刺激导致了CD11b/CD18的重大重组，其中60 - 70%的CD11b/CD18沉降在致密质膜组分中，这些组分也富含产生超氧化物的NADPH氧化酶活性。同样，对预处理的中性粒细胞进行固定、免疫金标记，并通过扫描电子显微镜检查。免疫金颗粒均匀分布在未刺激的中性粒细胞对称起皱的表面。在经dhCB处理的细胞上，免疫金大多均匀分布在光滑的膜上，有一小部分颗粒排列成线性阵列。在fMLF + dhCB刺激后，CD11b/CD18金标记在细胞表面更丰富，并在极化的膜突起上形成大的聚集体。然而，当细胞粘附在白蛋白包被的石英表面并在dhCB存在下用fMLF刺激时，免疫金被排除在关节状和圆形的细胞体上，但集中在粘附薄片的周边。荧光光漂白恢复表明，在未刺激的细胞中，38±3%的CD11b/CD18是可移动的（R），扩散常数D为3.1±0.3×10(-10) cm2/s。用dhCB处理分别使R和D提高了24%和74%。用1μM fMLF与dhCB刺激使D和R降至接近对照水平。由于在fMLF + dhCB刺激后NADPH氧化酶和CD11b/CD18在高密度质膜结构域中共沉降，我们推测在fMLF + dhCB脱颗粒的中性粒细胞中可能发生刺激诱导的CD11b/CD18和NADPH氧化酶向共同膜结构域的重组。
