Ducker T P, Skubitz K M
Department of Medicine, University of Minnesota Medical School, Minneapolis.
J Leukoc Biol. 1992 Jul;52(1):11-6. doi: 10.1002/jlb.52.1.11.
CD66 and CD67 are granulocyte-specific activation antigens; their surface expression is up-regulated when neutrophils are activated. CD66 antibodies recognize an approximately 180-kd neutrophil surface protein that is also recognized by anti-carcinoembryonic antigen (CEA) antibodies and is therefore a nonspecific cross-reacting antigen (NCA). CD67 antibodies recognize an approximately 100-kd neutrophil surface protein that is attached to the membrane via a glycosyl-phosphatidylinositol anchor. To identify an intracellular pool from which CD66 and CD67 could be up-regulated, the subcellular distribution of proteins recognized by CD66 and CD67 monoclonal antibodies and polyclonal anti-CEA was studied. Neutrophil plasma membranes, granules, and cytoplasm were prepared by nitrogen cavitation and differential centrifugation and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Most of the 180-kd protein recognized by CD66 antibodies and the 100-kd protein recognized by CD67 antibodies were located in the secondary granule fraction, with lesser amounts detectable in the plasma membrane fraction. Several NCA species ranging from approximately 40 to 200 kd were identified, and the distribution of these NCAs was different in the primary granules, secondary granules, and plasma membrane fractions. The major NCAs in the plasma membrane fraction were of approximately 95 to 100 and approximately 180 to 200 kd; the secondary granule fraction contained major NCAs of approximately 42, 85, 95 to 100, and 180 to 200 kd. NCAs were also detected in the primary granule fraction, the most prominent being of approximately 90-100 kd; no NCA of approximately 180 to 200 kd was detected in the primary granules. The presence of CD66, CD67, and NCAs in the secondary granules suggests secondary granules as a likely source from which these antigens could be recruited to the cell surface with activation. The potential role for NCAs in the primary granules is unknown.
CD66和CD67是粒细胞特异性激活抗原；当中性粒细胞被激活时，它们的表面表达上调。CD66抗体识别一种约180kd的中性粒细胞表面蛋白，该蛋白也可被抗癌胚抗原（CEA）抗体识别，因此是一种非特异性交叉反应抗原（NCA）。CD67抗体识别一种约100kd的中性粒细胞表面蛋白，该蛋白通过糖基磷脂酰肌醇锚定附着于细胞膜。为了确定CD66和CD67可能上调的细胞内池，研究了CD66和CD67单克隆抗体以及多克隆抗CEA识别的蛋白质的亚细胞分布。通过氮气空化和差速离心制备中性粒细胞膜、颗粒和细胞质，然后通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹进行分析。CD66抗体识别的180kd蛋白和CD67抗体识别的100kd蛋白大部分位于次级颗粒部分，在细胞膜部分可检测到的量较少。鉴定出几种大小约为40至200kd的NCA，这些NCA在初级颗粒、次级颗粒和细胞膜部分的分布不同。细胞膜部分的主要NCA约为95至100kd和约180至200kd；次级颗粒部分包含约42、85、95至100kd和约180至200kd的主要NCA。在初级颗粒部分也检测到了NCA，最突出的约为90 - 100kd；在初级颗粒中未检测到约180至200kd的NCA。次级颗粒中存在CD66、CD67和NCA，表明次级颗粒可能是这些抗原在激活时被募集到细胞表面的来源。NCA在初级颗粒中的潜在作用尚不清楚。
