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。我们研究的目的是了解导致这些分子释放的细胞内信号转导途径。在先前的研究中，我们证明了蛋白酪氨酸激酶Syk对于高亲和力免疫球蛋白E受体（FceRI）诱导的脱粒是必需的，所述脱粒导致炎症介质的释放。还鉴定了大鼠嗜碱性RBL-2 H3肥大细胞的变体，其没有可检测到的Syk，并已用于检查免疫受体聚集后Syk调节的结构基础。这些研究确定了Syk的连接区，位于第二个SH 2和激酶结构域之间，在调节这种激酶的功能中很重要。 以前，我们分离的肥大细胞特异性单克隆抗体，mAb BGD 6，识别肥大细胞前体存在于骨髓中的非常低的数字。通过使用RBL-2 H3 cDNA文库和mAb BGD 6的表达克隆，鉴定了显示与FcgRIIB（也称为CD 32）（低亲和力IgG受体）几乎完全相同的阳性克隆。然而，与GenBank中的序列相反，分离的克隆具有141 bp的插入片段，其编码先前在其他物种中报道但在大鼠细胞中未报道的该受体的同种型。这种长同种型在胞质结构域中具有额外的47个氨基酸。在RBL-2 H3细胞中，FcgRIIB的两种同种型均表达，其中较短形式的表达更高。结合研究表明，完整的mAb BGD 6与RBL-2 H3和CD 32阳性细胞均结合，但仅与F（ab？）2与RBL-2 H3细胞结合。因此，mAb BGD 6仅通过其Fc部分与FcgRIIB结合。在RBL-2 H3细胞上，抗CD 32 mAb的Fab部分抑制完整mAb BGD 6的结合，但不改变F（ab？）2 mAb BGD 6的片段。这些结果表明，mAb BDG 6的Fc部分有助于其在具有FcgRIIB的细胞上的结合。通过该模型，mAb BGD 6通过其Fab部分与细胞表面分子结合，并且Fc尾与FcgRIIB（CD 32）相互作用。 免疫受体刺激导致细胞内钙增加，激活丝氨酸磷酸酶钙调磷酸酶，然后使活化T细胞的核因子（NFAT）去磷酸化。去磷酸化的NFAT迅速易位到细胞核中并诱导各种细胞因子基因的转录。NFAT在免疫细胞如T、B和肥大细胞的活化中起重要作用。将含有与增强型绿色荧光蛋白（GFP）融合的具有三个串联NFAT结合位点的cDNA的质粒转染到RBL-2 H3细胞中。建立了克隆的细胞系，其在FceRI刺激后变得几乎完全GFP阳性，并且在未刺激的细胞中没有自发GFP表达的背景。这些细胞被用于一个项目，以比较导致核基因表达的信号通路与诱导脱粒和释放炎症介质的信号通路。 蛋白质酪氨酸磷酸化是抗原受体活化后最早可检测到的事件之一。最近，我们观察到，在培养的胎肝前B细胞，前B细胞受体刺激诱导强烈的酪氨酸磷酸化的82 kDa的蛋白质，其身份尚不清楚。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磷酸化。在肥大细胞中，这82 kDa的蛋白质的磷酸化需要Syk激酶活性，然而，这种磷酸化是独立的细胞内钙离子的上升。因此，该82 kDa蛋白的酪氨酸磷酸化与肥大细胞中的早期抗原信号传导事件直接相关。为了鉴定82 kDa磷酸化蛋白，用抗磷酸酪氨酸抗体免疫沉淀来自抗原刺激的RBL-2 H3细胞的裂解物，并对82 kDa磷酸化条带进行微测序。作为对照，还对已知含有Syk的磷酸化72 kDa条带进行微测序。82 kDa条带的主要成分是造血细胞特异性林恩底物（HS 1），而次要成分包括皮质素亚型B、肌醇多磷酸5'磷酸酶（SHIP）、FYN结合蛋白、Ga B 1和蛋白激酶C-δ。72 kDa的条带，含有src同源2，含有76 kDa的蛋白（SLP-76）、HS 1、Syk、热休克蛋白70、Btk和SHIP。在82和70 kDa条带中均检测到许多其他蛋白质的较低水平。用对照和活化的RBL-2 H3细胞进行的免疫印迹和免疫沉淀实验证实了HS 1是82 kDa的磷蛋白。因此，HS 1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.