Zhou M, Todd R F, van de Winkel J G, Petty H R
Department of Biological Sciences, Wayne State University, Detroit, MI 48202.
J Immunol. 1993 Apr 1;150(7):3030-41.
We have tested the possible physical interactions between the iC3b receptor (CR3), lymphocyte function-associated Ag-1, and class III Fc gamma receptor (Fc gamma RIII) at neutrophil surfaces. Cells were labeled using fluorochrome-conjugated Fab or F(ab')2 fragments of antireceptor mAb. Labeled receptors were capped using second-step F(ab')2 fragments of goat anti-mouse Fab antiserum. After 20 min at 37 degrees C, 68% of the cells capped the anti-CR3 plus second-step complex. Capping was time, temperature, and cytochalasin B sensitive. When capped cells were probed with Fab' or F(ab')2 fragments of anti-Fc gamma RIII labeled with a distinct fluorochrome, 41% of the cells cocapped Fc gamma RIII. Indistinguishable results were obtained when potential antibody combining sites within caps were blocked with a large excess of Fab or F(ab')2 fragments. When Fc gamma RIII was capped, 49% of the cells cocapped CR3. Similarly, LFA-1 cocapped with both CR3 and Fc gamma RIII. Importantly, other membrane components including HLA class I, Mo5, CD13, CR type 1, and IL-8 receptors and N-4-nitrobenzo-2-oxa-1, 3-diszole L-alpha-dimyristoyl phosphatidylethanolamine did not cocap with CR3. However, the positive control Con A did cocap with CR3 and Fc gamma RIII. We next evaluated the effect of saccharides on CR3-Fc gamma RIII cocapping and found that 0.15 M N-acetyl-D-glucosamine (NADG), alpha-methyl-D-mannoside, and D-mannose significantly inhibited cocapping by 70, 58, and 48%, respectively. No inhibition was obtained using glucose, galactose, N-acetyl-neuraminic acid, fucose, sorbitol, fructose, or sucrose. Similarly, Fc gamma RIII-lymphocyte function-associated-1 cocapping was inhibited by NADG. However, the cocapping of CR3 with lymphocyte function-associated-1 or Con A were not affected by 0.15 M NADG, which suggests that NADG inhibition of leukoadhesin-Fc gamma RIII cocapping is not due to a general effect of NADG on capping. Inasmuch as Fc gamma RIII is a glycophospholipid-linked membrane protein, we speculate that it interacts with CR3 and/or lymphocyte function-associated-1 via lectin-like interactions.
我们检测了中性粒细胞表面iC3b受体（CR3）、淋巴细胞功能相关抗原-1（LFA-1）和Ⅲ类Fcγ受体（FcγRIII）之间可能存在的物理相互作用。使用抗受体单克隆抗体的荧光染料偶联Fab或F(ab')2片段对细胞进行标记。使用山羊抗小鼠Fab抗血清的第二步F(ab')2片段对标记的受体进行封帽。在37℃孵育20分钟后，68%的细胞封帽了抗CR3加第二步复合物。封帽对时间、温度和细胞松弛素B敏感。当用另一种不同荧光染料标记的抗FcγRIII的Fab'或F(ab')2片段探测封帽细胞时，41%的细胞共封帽了FcγRIII。当用大量过量的Fab或F(ab')2片段封闭封帽内潜在的抗体结合位点时，得到了相似的结果。当FcγRIII被封帽时，49%的细胞共封帽了CR3。同样，LFA-1与CR3和FcγRIII都能共封帽。重要的是，其他膜成分，包括HLA I类分子、Mo5、CD13、CR1型分子、IL-8受体以及N-4-硝基苯-2-恶唑-1,3-二唑-L-α-二肉豆蔻酰磷脂酰乙醇胺，均不与CR3共封帽。然而，阳性对照刀豆球蛋白A（Con A）确实与CR3和FcγRIII共封帽。接下来，我们评估了糖类对CR3-FcγRIII共封帽的影响，发现0.15M的N-乙酰-D-葡萄糖胺（NADG）、α-甲基-D-甘露糖苷和D-甘露糖分别显著抑制共封帽达70%、58%和48%。使用葡萄糖、半乳糖、N-乙酰神经氨酸、岩藻糖、山梨醇、果糖或蔗糖未获得抑制作用。同样，NADG抑制了FcγRIII与淋巴细胞功能相关抗原-1的共封帽。然而，0.15M的NADG并未影响CR3与淋巴细胞功能相关抗原-1或Con A的共封帽，这表明NADG对白细胞黏附分子-FcγRIII共封帽的抑制作用并非是由于NADG对封帽的普遍影响。鉴于FcγRIII是一种糖磷脂连接的膜蛋白，我们推测它通过类似凝集素的相互作用与CR3和/或淋巴细胞功能相关抗原-1相互作用。
