Signal Transduction In Mast Cells

肥大细胞通过释放一系列介质，在许多炎症和免疫反应中发挥重要作用。我们研究的目标是了解导致这些分子释放的细胞内信号转导途径。在以前的研究中，我们证明了蛋白酪氨酸激酶Syk在高亲和力免疫球蛋白E受体(FceRI)诱导的脱颗粒过程中是必不可少的，该脱颗粒导致炎症介质的释放。还发现了大鼠嗜碱性RBL-2H3肥大细胞的一个变体，它没有检测到Syk，并已用于研究免疫受体聚集后Syk调节的结构基础。这些研究发现，Syk的连接区位于第二个SH2和激酶结构域之间，在调节该激酶的功能方面起着重要的作用。 在此之前，我们分离了一种肥大细胞特异性的单抗，mAbBGD6，它识别存在于骨髓中的肥大细胞前体，数量非常少。用RBL-2H3文库和单抗BGD6进行表达克隆，获得了一个与低亲和力的Ig G受体FcgRIIB(又称CD32)几乎完全一致的阳性克隆。然而，与GenBank中的序列相反，分离的克隆有一个141bp的插入片段，它编码了以前在其他物种中报道的这种受体的异构体，但在大鼠细胞中没有。这个长的异构体在细胞质区域中有额外的47个氨基酸。在RBL-2H3细胞中，FcgRIIB的两种异构体都有表达，其中较短的FcgRIIB表达较高。结合研究表明，完整的单抗BGD6可与RBL-2H3和CD32阳性细胞结合，但只与RBL-2H3细胞结合F(ab？)2。因此，mAbBGD6仅通过其Fc部分与FcgRIIB结合。在RBL-2H3细胞上，抗CD32单抗的Fab部分抑制了完整单抗BGD6的结合，但不改变单抗BGD6的F(ab？)2片段的结合。这些结果表明，单抗BDG6的Fc部分有助于其与具有FcgRIIB的细胞的结合。通过该模型，单抗BGD6通过其Fab部分与细胞表面分子结合，Fc尾部与FcgRIIB(CD32)相互作用。 免疫受体刺激导致细胞内钙的增加，激活丝氨酸磷酸酶钙调神经磷酸酶，然后去磷酸化激活的T细胞的核因子(NFAT)。去磷酸化的NFAT迅速移位到细胞核，并诱导各种细胞因子基因的转录。NFAT在T、B、肥大细胞等免疫细胞的激活中起重要作用。将含有三个串联NFAT结合位点的cDNA与增强型绿色荧光蛋白(GFP)融合的表达载体导入RBL-2H3细胞。建立了一个经FceRI刺激后几乎完全呈GFP阳性的克隆细胞系，在未刺激的细胞中没有自发表达GFP的背景。这些细胞正被用于一个项目，以比较导致核基因表达的信号通路与那些诱导脱颗粒和释放炎症介质的信号通路。 蛋白酪氨酸磷酸化是抗原受体激活后最早可检测到的事件之一。最近，我们观察到在培养的胎肝Pre-B细胞中，Pre-B细胞受体刺激诱导了一个82 kDa蛋白的强烈酪氨酸磷酸化，其特征尚不清楚。在抗原受体刺激的脾B细胞和肥大细胞中也观察到该蛋白的强烈磷酸化。在肥大细胞中，这个82 kDa蛋白的磷酸化需要Syk激酶的活性；然而，这种磷酸化不依赖于细胞内钙的升高。因此，这个82 kDa蛋白的酪氨酸磷酸化与肥大细胞早期的抗原信号事件直接相关。用抗磷酸酪氨酸抗体免疫沉淀RBL-2H3细胞的裂解产物，并对82 kDa的磷酸化条带进行微量测序，以鉴定82 kDa的磷酸化蛋白。作为对照，已知含有Syk的磷酸化72 kDa条带也进行了微测序。82 kDa条带的主要成分是造血细胞特异性LYN底物(HS1)，次要成分包括皮质蛋白亚型B、肌醇多聚磷酸酶(SHIP)、FYN结合蛋白、Gab1和蛋白激酶C-Delta。72 kDa的条带包括Src同源2，包括76 kDa的SLP-76蛋白、HS1、Syk、热休克70蛋白、BTK和SHIP。在82 kDa和70 kDa条带中都检测到了较低水平的其他一些蛋白质。免疫印迹和免疫沉淀实验证实HS1为82 kDa的磷酸化蛋白。因此，HS1的酪氨酸磷酸化可以作为早期抗原信号通路功能状态的标志。

Mast cells play an important role in many inflammatory and immunological reactions by releasing an array of mediators. The goal of our studies is to understand the intracellular signal transduction pathways that lead to the release of these molecules. In previous studies, we demonstrated that the protein tyrosine kinase Syk is essential for the high affinity immunoglobulin E receptor (FceRI)-induced degranulation that results in the release of inflammatory mediators. A variant of the rat basophilic RBL-2H3 mast cells that has no detectable Syk was also identified and has been used to examine the structural basis of the regulation of Syk after immune receptor aggregation. These studies identified the linker region of Syk, located between the second SH2 and the kinase domain, as important in regulating the function of this kinase. Previously we isolated a mast cell specific monoclonal antibody, mAb BGD6, that recognizes a mast cell precursor present in the bone marrow at very low numbers. By expression cloning using an RBL-2H3 cDNA library and mAb BGD6 a positive clone was identified that showed almost complete identity with FcgRIIB (also called CD32), the low affinity IgG receptor. However, in contrast to the sequence in GenBank, the isolated clone had an insert of 141 bp which codes for an isoform of this receptor previously reported in other species but not in rat cells. This long isoform has an extra 47 amino acids in the cytoplasmic domain. In RBL-2H3 cells both isoforms of FcgRIIB were expressed, with higher expression of the shorter form. Binding studies demonstrated that intact mAb BGD6 bound to both RBL-2H3 and CD32 positive cells, but only the F(ab?)2 bound to the RBL-2H3 cells. Therefore, mAb BGD6 binds to the FcgRIIB only through its Fc portion. On RBL-2H3 cells, the Fab of an anti-CD32 mAb partially inhibited the binding of intact mAb BGD6, but it did not change the binding of the F(ab?)2 fragment of mAb BGD6. These results suggest that the Fc portion of mAb BDG6 contributes to its binding on cells that have FcgRIIB. By this model mAb BGD6 binds through its Fab portion to a cell-surface molecule and the Fc tail interacts with FcgRIIB (CD32). Immune receptor stimulation results in an increase in intracellular calcium that activates the serine phosphatase calcineurin, which then dephosphorylates the nuclear factor of activated T cells (NFAT). The dephosphorylated NFAT rapidly translocates into the nucleus and induces the transcription of various cytokine genes. NFAT plays an important role in the activation of immune cells such as T, B and mast cells. A plasmid containing the cDNA with three tandem NFAT-binding sites fused to enhanced green fluorescent protein (GFP) was transfected into the RBL-2H3 cells. A cloned cell line was established that became almost totally GFP positive upon FceRI stimulation and had no background of spontaneous GFP expression in non-stimulated cells. These cells are being used in a project to compare the signaling pathways that result in nuclear gene expression from those that induce degranulation and the release of inflammatory mediators. Protein tyrosine phosphorylation is one of the earliest detectable events after antigen receptor activation. Recently, we observed that in cultured fetal liver pre-B cells, pre-B cell receptor stimulation induced the strong tyrosine phosphorylation of an 82 kDa protein, whose identity is unclear. The strong phosphorylation of this protein was also observed in antigen-receptor stimulated spleen B cells and mast cells. In mast cells, the phosphorylation of this 82 kDa protein required Syk kinase activity; however, this phosphorylation was independent of the rise of intracellular calcium. Therefore, the tyrosine phosphorylation of this 82 kDa protein directly correlated with early antigen signaling events in mast cells. To identify the 82 kDa phosphorylated protein, lysates from antigen stimulated RBL-2H3 cells were immunoprecipitated with anti-phospho-tyrosine antibody and the 82 kDa phosphorylated band was micro-sequenced. As a control, the phosphorylated 72 kDa band, that is known to contain Syk, was also micro-sequenced. The major component of the 82 kDa band was the hematopoietic cell specific Lyn substrate (HS1), while minor components included cortactin isoform B, inositol polyphosphate 5' phosphatase (SHIP), FYN binding protein, Gab 1, and protein kinase C-delta. The 72 kDa band, contained src homology 2 containing protein of 76 kDa (SLP-76), HS1, Syk, heat shock 70 protein, Btk and SHIP. There were a number of other proteins detected at lower levels in both the 82 and 70 kDa bands. Immunoblotting and immunoprecipitation experiments with control and activated RBL-2H3 cells confirmed that HS1 was the 82 kDa phospho-protein. Therefore, tyrosine phosphorylation of HS1 can serve as a marker for the functional status of early antigen signaling pathways.