Petty H R, Francis J W, Todd R F, Petrequin P, Boxer L A
Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202.
J Cell Physiol. 1987 Nov;133(2):235-42, 256. doi: 10.1002/jcp.1041330206.
Video-intensification fluorescence microscopy has been used to study the cell surface distribution of the complement receptor (CR) for C3bi (CR3) on human neutrophils. Fluorescein- or rhodamine-labeled monoclonal IgG or Fab fragments of antireceptor antibody were used as probes of receptor localization. C3bi receptors are uniformly distributed on untreated cells. Glass coverslips were coated with lipopolysaccharide (LPS) and serum was added; the serum deposits complement components, including C3bi, on the surface. When neutrophils were adherent to these coverslips, receptors were found in large clusters, and a fraction of the fluorescence remained uniform. Double-labeling studies were conducted by first labeling with anti-CR3 followed by attachment to LPS/serum-treated slides. This, in turn, was followed by labeling with the antibody conjugated to a second fluorophore. These studies revealed that the CR3 clusters were predominantly new antigenic sites exposed after attachment to the LPS/serum-treated slides. To determine the contribution of granule-associated CR3, we have studied neutrophils defective in receptor up-regulation, neutrophil cytoplasts, and a stimulator of granule release, A23187. Neutrophils from a patient with specific granule deficiency were found to be defective in granular CR3 and did not form clusters on C3-modified surfaces. The patient's neutrophils were defective in CR3 up-regulation and enzyme release as shown by fluorescence flow cytometry and gelatinase release, respectively. Cytoplasts also failed to show CR3 clusters on LPS/serum-treated coverslips. Furthermore, neutrophils treated with A23187 demonstrated numerous CR3 clusters. We suggest that formation of CR3 membrane domains during immune recognition requires the participation of intracellular granules. We speculate that these domains are formed by fusion of CR3-bearing granules at local sites of adhesion.
视频增强荧光显微镜已被用于研究人中性粒细胞上C3bi补体受体（CR3）的细胞表面分布。用荧光素或罗丹明标记的抗受体抗体的单克隆IgG或Fab片段作为受体定位的探针。C3bi受体均匀分布在未处理的细胞上。用脂多糖（LPS）包被玻璃盖玻片并加入血清；血清在表面沉积补体成分，包括C3bi。当中性粒细胞粘附于这些盖玻片时，发现受体形成大的簇，且一部分荧光保持均匀。通过先用抗CR3标记，然后附着于LPS/血清处理的玻片上进行双标记研究。接着，再用与第二种荧光团偶联的抗体进行标记。这些研究表明，CR3簇主要是在附着于LPS/血清处理的玻片后暴露的新抗原位点。为了确定颗粒相关CR3的作用，我们研究了受体上调缺陷的中性粒细胞、中性粒细胞胞质体以及颗粒释放刺激剂A23187。发现一名患有特异性颗粒缺乏症患者的中性粒细胞在颗粒CR3方面存在缺陷，并且在C3修饰的表面上不形成簇。分别通过荧光流式细胞术和明胶酶释放显示，该患者的中性粒细胞在CR3上调和酶释放方面存在缺陷。胞质体在LPS/血清处理的盖玻片上也未显示出CR3簇。此外，用A23187处理的中性粒细胞显示出大量CR3簇。我们认为在免疫识别过程中CR3膜结构域的形成需要细胞内颗粒的参与。我们推测这些结构域是由携带CR3的颗粒在局部粘附位点融合形成的。
