Sármay G, Koncz G, Gergely J
Lorand Eotvos University, Department of Immunology, God, Hungary.
Immunol Lett. 1996 Dec;54(2-3):93-100. doi: 10.1016/s0165-2478(96)02655-7.
Fc gamma receptors type IIb1 (Fc gammaRIIb1) inhibit B-cell activation when co-ligated with B-cell antigen receptors (BCR) by immune complexes. In murine B-cells the inhibition is mediated by the interaction of the phosphorylated immunoreceptor tyrosine-based inhibitory motif (P-ITIM) of Fc gammaRIIb1 with the SH2 domain containing protein tyrosine phosphatase. SHP1. To clarify the mechanism of Fc gammaRIIb mediated inhibition of human B-cells we have studied the association of signaling molecules with human Fc gammaRIIb1 after co-ligating with BCR. Fc gammaRIIb1 were affinity purified from the Burkitt lymphoma cell line, BL41. Several tyrosine phosphorylated proteins were co-isolated with Fc gammaRIIb1 at 145, 110, and 50 60 kDa, which were not present in Fc gammaRIIb1 free immune complexes. Among these molecules we have identified the p52 Shc adaptor protein. Furthermore, we have shown that the insolubilised synthetic peptide corresponding P-ITIM bound Shc, Lyn and the p75 and p 10 unidentified tyrosine phosphorylated proteins. Here we describe that the cell membrane associated Shc is partially dephosphorylated in BCR-Fc gammaRIIb1 co-ligated samples, suggesting that its function in regulating p21ras monomeric G protein is impaired. Indeed, we have detected a lower p21ras activity in BCR-Fc gammaRIIb1 co-crosslinked samples. These data indicate that co-ligation of BCR and Fc gammaRIIb1 interrupts signal transduction between protein tyrosine kinase activation and p21ras mediated activation pathway. Since in contrast to the mouse B-cells both Fc gammaRIIb1 and Fc gammaRIIb2 are expressed in human B-cells, we have investigated the inhibitory function of the two receptors in Fc gammaRIIb negative Burkitt lymphoma cell line ST486 transfected with Fc gammaRIIb1 and Fc gammaRIIb2, respectively. Both Fc gammaRIIb1 and Fc gammaRIIb2 inhibited the rise of intracellular Ca2+ induced by the crosslinking of BCR. The rate of the inhibition depended on the ratio of the co-crosslinked receptors (BCR-Fc gammaRIIb1) to the crosslinked BCR (BCR-BCR). Co-crosslinking of the two receptors inhibited not only the capacitive Ca2+ entry but rather the total Ca2+ response in both Fc gammaRIIb1 and Fc gammaRIIb2 transfected human B-cells. CD19 represents the signal transduction unit of complement receptor, CR2 (CD21), and is responsible for the complement activating IgM-immune complex induced enhancement of B-cell activation. Co-crosslinking of CD19 and BCR was shown to enhance B-cell activation due to the recruitment of further signaling molecules to the activator complex by the phosphorylated tyrosine residues of CD19. Here we show a novel finding that co-ligation of CD19 with Fc gammaRIIb1 inhibits the CD19-induced upregulation of Ca2+ response. The results indicate that IgG plus complement containing immune complexes may inhibit B-cell activation in vivo, due to the Fc gammaRIIb1-mediated interruption of signal transduction via both BCR and CD19.
Fcγ受体IIb1（FcγRIIb1）在与免疫复合物中的B细胞抗原受体（BCR）共连接时可抑制B细胞活化。在鼠B细胞中，这种抑制作用是由FcγRIIb1的磷酸化免疫受体酪氨酸抑制基序（P-ITIM）与含SH2结构域的蛋白酪氨酸磷酸酶SHP1相互作用介导的。为阐明FcγRIIb介导的人B细胞抑制机制，我们研究了与BCR共连接后信号分子与人FcγRIIb1的结合情况。FcγRIIb1从伯基特淋巴瘤细胞系BL41中亲和纯化得到。有几种酪氨酸磷酸化蛋白与FcγRIIb1在145、110和50 - 60 kDa处共分离，而在不含FcγRIIb1的免疫复合物中不存在。在这些分子中，我们鉴定出了p52 Shc衔接蛋白。此外，我们还表明，对应于P-ITIM的不溶性合成肽与Shc、Lyn以及p75和p10未鉴定的酪氨酸磷酸化蛋白结合。在此我们描述，在BCR-FcγRIIb1共连接的样品中，细胞膜相关的Shc部分去磷酸化，这表明其在调节p21ras单体G蛋白中的功能受损。实际上，我们在BCR-FcγRIIb1共交联的样品中检测到较低的p21ras活性。这些数据表明，BCR和FcγRIIb1的共连接中断了蛋白酪氨酸激酶激活与p21ras介导的激活途径之间的信号转导。由于与鼠B细胞不同，FcγRIIb1和FcγRIIb2在人B细胞中均有表达，我们分别研究了在FcγRIIb阴性的伯基特淋巴瘤细胞系ST486中转入FcγRIIb1和FcγRIIb2后这两种受体的抑制功能。FcγRIIb1和FcγRIIb2均抑制BCR交联诱导的细胞内Ca2+升高。抑制速率取决于共交联受体（BCR-FcγRIIb1）与交联BCR（BCR-BCR）的比例。两种受体的共交联不仅抑制了FcγRIIb1和FcγRIIb2转染的人B细胞中的电容性Ca2+内流，还抑制了总Ca2+反应。CD19代表补体受体CR2（CD21）的信号转导单位，负责补体激活的IgM免疫复合物诱导的B细胞活化增强。CD19和BCR的共交联由于CD19的磷酸化酪氨酸残基将更多信号分子募集到激活复合物中而增强了B细胞活化。在此我们展示了一个新发现，即CD19与FcγRIIb1的共连接抑制了CD19诱导的Ca2+反应上调。结果表明，含IgG加补体的免疫复合物可能在体内抑制B细胞活化，这是由于FcγRIIb1介导的通过BCR和CD19的信号转导中断。
