Analysis of IgE-independ activation of mast cells and its physiologilay importance

我们开发了一种新的培养系统，在该系统中，小鼠骨髓来源的，未成熟的肥大细胞（BMMC）与瑞士3 T3成纤维细胞在干细胞因子的存在下共培养，迅速经历了形态和功能成熟的结缔组织肥大细胞（CTMC）样表型。因此，共培养的BMMC含有更多的组胺、肝素蛋白多糖和丝氨酸蛋白酶MMCP-4，产生更多的PGD_2以响应Fc_εRI交联，更重要的是，响应于P物质和化合物48/80，众所周知的多阳离子促分泌剂，其引起IgE非依赖性，G蛋白-进行cDNA消减策略以鉴定一系列基因，其表达水平仅与CTMC表型依赖性激活有关。在体外肥大细胞成熟过程中发生了巨大的变化。在大约100个测序的克隆中，近50%是14号染色体相关的丝氨酸蛋白酶，包括MMCP-4和诱导细胞凋亡的蛋白酶颗粒酶B。 ...更多信息 已报道在分化或应激反应期间在几种细胞类型中诱导的胞质蛋白。在BMMC中，Ndr-1的诱导先于CTMC样细胞的成熟，并伴随着IgE依赖性和非依赖性胞吐反应的增强。将Ndr-1强行导入大鼠肥大细胞瘤RBL-2 H3细胞导致IgE依赖性和非依赖性脱粒增强。在Ndr-1的C-末端附近的三个亲水性串联重复序列的缺失废除了去颗粒化增强功能。这些结果表明，Ndr-1是肥大细胞体外成熟过程中的一个强诱导基因，可能在刺激诱导的脱颗粒过程中起重要作用，而AVRL-2编码一个100 kDa的蛋白质，其C端与血管紧张素/血管加压素受体样（AVRL）有较弱的同源性。AVRL-2包含几个推测参与蛋白质-蛋白质相互作用的基序，如螺旋-环-螺旋和亮氨酸拉链，以及独特的C-末端三个串联重复序列。至少有三个选择性剪接变异体被确定，其中每一个缺乏一个或多个串联重复。全长AVRL-2及其剪接变体在共培养过程中在BMMC中显示出不同的诱导特征。少

We developed a novel culture system in which mouse bone marrow-derived, immature, mast cells (BMMC) coclultured with Swiss 3T3 fibroblasts in the presence of stem cell factor quickly underwent morphological and functional maturation toward a connective tissue mast cell (CTMC)-like phenotype. Thus, cocultured BMMC contained more histamine, heparin proteoglycan and serine protease MMCP-4, produced more PGD_2 in response to Fc_εRI crosslinking, and importantly, reponded to substance P and compound 48/80, well-known polycationic secretagogues that elicit IgE-independent, G protein-dependent activation of CTMC phenotype only.The cDNA subtraction strategy was conducted to identify a series of genes whose expression levels were dramatically changed during this in vitro mast cell maturation process. Of approximately 100 sequenced clones, nearly 50% were chromosome 14-associated serine proteases, including MMCP-4 and apoptosis-inducing protease granzyme B.Approximately 14% encoded Ndr-1, a 43-k … More Da cytosolic protein that has been reported to be induced in several cell types during differentiation or stress responses. Induction of Ndr-1 in BMMC preceded the maturation into CTMC-like cells and was accompanied by enhancement of the IgE-dependent and -independent exocytotic responses. Forcible introduction of Ndr-1 into rat mastocytoma RBL-2H3 cells led to augmentation of IgE-dependent and -independent degranulation. Deletion of three hydrophilic tandem repeats near the C-terminus in Ndr-1 abrogated the degranulation-enhancing function. These results suggest that Ndr-1, a strongly inducible gene during in vitro maturation of mast cells, is a novel component that may play a pivotal role in the regulation of stimulus-induced degranulation.Another novel gene, AVRL-2, encoded a 100-kDa protein, the C-terminal half of which showed a weak homology with angiotensin/vasopression receptor-like (AVRL). AVRL-2 contained several motifs that are presumed to participate in protein-protein interaction, such as helix-loop-helix and leucine zipper, and unique C-terminal three tandem repeats. At least three alternative splicing variants were identified, each of which lacked one or more of the tandem repeats. Full-length AVRL-2 and its splicing variants showed distinct induction profiles in BMMC during coculture. Less