Kaji K, Takeshita S, Miyake K, Takai T, Kudo A
Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan.
J Immunol. 2001 Mar 1;166(5):3256-65. doi: 10.4049/jimmunol.166.5.3256.
CD9, a member of the tetraspan family of proteins, is highly expressed on macrophages. Although a clear function for the molecule has yet to be described, we have found that the anti-CD9 mAb activates mouse macrophages. The rat anti-CD9 mAb, KMC8.8, but not the F(ab')(2), induced tyrosine phosphorylation of proteins including syk and cbl and induced cell aggregation in the mouse macrophage cell line, J774, suggesting that co-cross-linking of CD9 and Fc gamma R was required for the signal. Co-cross-linking of CD9-Fc gamma R with KMC8.8 on macrophages from three different FcR-deficient mice, FcR gamma-chain(-/-), Fc gamma RIIB(-/-), and Fc gamma RIII(-/-), revealed that Fc gamma RIII is specific and crucial for syk phosphorylation. Although both KMC8.8 and the anti-Fc gamma RIIB/III mAb, 2.4G2, evoked similar phosphorylation patterns, only KMC8.8 induced cell aggregation. Additionally, KMC8.8 treatment led to reduce levels of TNF-alpha production and p42/44 extracellular signal-related kinase phosphorylation relative to 2.4G2 stimulation. Immunofluorescence staining showed that co-cross-linking of CD9-Fc gamma R with KMC8.8 induced filopodium extension before cell aggregation, which was followed by simultaneous colocalization of CD9, Fc gamma RIIB/III, Mac-1, ICAM-1, and F-actin at the cell-cell adhesion site. Moreover, KMC8.8 treatment of Fc gamma R-deficient macrophages revealed that the colocalization of CD9, Fc gamma RIII, Mac-1, and F-actin requires co-cross-linking of CD9-Fc gamma RIII, whereas co-cross-linking of CD9-Fc gamma RIIB induced the colocalization of only CD9 and Fc gamma RIIB. Our results demonstrate that co-cross-linking of CD9 and Fc gamma Rs activates macrophages; therefore, CD9 may collaborate with FcRs functioning in infection and inflammation on macrophages.
CD9是四跨膜蛋白家族的成员之一，在巨噬细胞上高表达。尽管该分子的明确功能尚未明确，但我们发现抗CD9单克隆抗体可激活小鼠巨噬细胞。大鼠抗CD9单克隆抗体KMC8.8可诱导包括syk和cbl在内的蛋白质酪氨酸磷酸化，并在小鼠巨噬细胞系J774中诱导细胞聚集，而其F(ab')(2)片段则无此作用，这表明CD9和FcγR的共同交联是信号传导所必需的。在来自三种不同FcR缺陷小鼠（FcRγ链(-/-)、FcγRIIB(-/-)和FcγRIII(-/-)）的巨噬细胞上，用KMC8.8使CD9-FcγR共同交联，结果显示FcγRIII对syk磷酸化具有特异性且至关重要。尽管KMC8.8和抗FcγRIIB/III单克隆抗体2.4G2引发了相似的磷酸化模式，但只有KMC8.8诱导细胞聚集。此外，与2.4G2刺激相比，KMC8.8处理导致肿瘤坏死因子-α（TNF-α）产生水平和p42/44细胞外信号调节激酶磷酸化水平降低。免疫荧光染色显示，用KMC8.8使CD9-FcγR共同交联在细胞聚集前诱导丝状伪足伸展，随后CD9、FcγRIIB/III、Mac-1、细胞间黏附分子-1（ICAM-1）和F-肌动蛋白在细胞间黏附位点同时共定位。此外，用KMC8.8处理FcγR缺陷巨噬细胞表明，CD9、FcγRIII、Mac-1和F-肌动蛋白的共定位需要CD9-FcγRIII的共同交联，而CD9-FcγRIIB的共同交联仅诱导CD9和FcγRIIB的共定位。我们的数据表明，CD9和FcγR的共同交联可激活巨噬细胞；因此，CD9可能与FcR协同作用，在巨噬细胞的感染和炎症中发挥功能。
