Tausk F, Fey M, Gigli I
Division of Dermatology, University of California, San Diego, School of Medicine 92103.
J Immunol. 1989 Nov 15;143(10):3295-302.
The decay-accelerating factor (DAF) is a cell membrane glycoprotein that functions in the control of C activation. We studied the modulation of membrane DAF on polymorphonuclear cells (PMN) by using anti-DAF antibodies. Fluorescence-activated cell sorter analysis showed that DAF expression was reduced by 43 +/- 7% on resting or stimulated cells that were held at 37 degrees C for 30 min when compared with those kept on ice. Most of this reduction occurred within the first 15 min, and was followed by a gradual further decrease in surface DAF. PMN that were held at 37 degrees C for varying periods of time before DAF measurement had a gradual decrease suggestive of release of DAF from the PMN membrane or endocytosis. To examine the latter, PMN were reacted with anti-DAF at 0 degree C, followed by 125I-Fab'2 secondary antibodies at either 0 degree C or 37 degrees C, and subsequently treated with pronase. Thirty +/- 11% of the 125I remained bound to cells kept at 37 degrees C compared to 2% in those held at 0 degrees C. Internalization was further confirmed by electron microscopy. In PMN that were not exposed to pronase, 26 +/- 2% of the surface-associated 125I was released at 37 degrees C compared with 7% at 0 degrees C. Immunoprecipitation and SDS-PAGE of surface-labeled PMN showed that the temperature-dependent released DAF had a lower m.w. than membrane DAF. Immunofluorescent studies revealed that 37 degrees C mediated the redistribution of DAF from a homogeneous pattern into caps. These results show that under the conditions studied DAF is partially internalized and partially released from the PMN membrane to the fluid phase; the latter may contribute to the presence of DAF in body fluids.
衰变加速因子（DAF）是一种细胞膜糖蛋白，在补体（C）激活的调控中发挥作用。我们使用抗DAF抗体研究了多形核白细胞（PMN）上膜DAF的调节情况。荧光激活细胞分选分析显示，与保存在冰上的细胞相比，在37℃保持30分钟的静息或刺激细胞上，DAF表达降低了43±7%。这种降低大部分发生在最初的15分钟内，随后表面DAF逐渐进一步减少。在测量DAF之前在37℃保持不同时间的PMN呈现出逐渐下降的趋势，提示DAF从PMN膜释放或发生内吞作用。为了研究后者，PMN在0℃与抗DAF反应，随后在0℃或37℃与125I-Fab'2二抗反应，随后用链霉蛋白酶处理。与保存在0℃的细胞相比，30±11%的125I仍与保存在37℃的细胞结合。电子显微镜进一步证实了内吞作用。在未暴露于链霉蛋白酶的PMN中，与0℃时的7%相比，37℃时26±2%的表面相关125I被释放。表面标记的PMN的免疫沉淀和SDS-PAGE显示，温度依赖性释放的DAF分子量低于膜DAF。免疫荧光研究显示，37℃介导DAF从均匀模式重新分布成帽状。这些结果表明，在所研究的条件下，DAF部分从PMN膜内化并部分释放到液相中；后者可能导致DAF在体液中的存在。
