Ghildyal N, Friend D S, Nicodemus C F, Austen K F, Stevens R L
Department of Medicine, Harvard Medical School, Boston, MA 02115.
J Immunol. 1993 Sep 15;151(6):3206-14.
BALB/cJ mouse mast cells derived by culturing bone marrow progenitor cells in WEHI-3 cell-conditioned medium (BMMCW) do not contain mouse mast cell protease 2 (mMCP-2) mRNA, but these cells can be induced to express this transcript after exposure to rIL-10. To study the translation and granule accumulation of mMCP-2 in rIL-10-treated BMMC (BMMCW+IL-10), a rabbit antibody was developed to a synthetic peptide that corresponds to the novel amino acid sequence in mMCP-2 at residues 56 to 71. After affinity purification, this antibody, anti-mMCP-2(56-71) IgG, reacted in SDS-PAGE/immunoblots against a 28-kDa protein in BMMCW+IL-10 that had the N-terminal amino acid sequence of mMCP-2. As assessed immunohistochemically, mMCP-2 protein accumulated in the secretory granules of Kirsten sarcoma virus-immortalized mouse mast cells, BMMCW+IL-10, and the mucosal mast cells present in the jejunum of Trichinella spiralis-infected BALB/cJ mice. Time course analyses of the induction of mMCP-2 mRNA and protein in BMMCW+IL-10 revealed that these cells contain a high steady-state level of mMCP-2 mRNA 24 h after their exposure to rIL-10. Although a small amount of immunodetectable mMCP-2 protein is present in the cells treated for 24 h, large amounts of this protease are not obtained until 7 days of treatment of the cells with rIL-10. Time course analyses of the loss of mMCP-2 mRNA and protein in BMMCW+IL-10 revealed that the steady-state level of mMCP-2 mRNA decreased dramatically 24 h after rIL-10 was removed from the culture medium, but that the level of mMCP-2 protein did not decline measurably until day 5 of culture. The fact that the steady-state levels of mMCP-2 mRNA and protein in BMMC can both be reversibly altered by culturing these mast cells in the presence and absence of rIL-10 suggests that the phenotype of mast cells is not fixed. Rather, it is in a dynamic state regulated by the cytokine network to which mast cells are exposed in their different microenvironments.
通过在WEHI-3细胞条件培养基（BMMCW）中培养骨髓祖细胞获得的BALB/cJ小鼠肥大细胞不含有小鼠肥大细胞蛋白酶2（mMCP-2）mRNA，但这些细胞在暴露于重组白细胞介素-10（rIL-10）后可被诱导表达该转录本。为了研究rIL-10处理的BMMC（BMMCW+IL-10）中mMCP-2的翻译和颗粒积累情况，制备了一种针对与mMCP-2中第56至71位残基的新氨基酸序列相对应的合成肽的兔抗体。经过亲和纯化后，该抗体，即抗-mMCP-2(56-71) IgG，在SDS-PAGE/免疫印迹中与BMMCW+IL-10中一种28 kDa的蛋白发生反应，该蛋白具有mMCP-2的N端氨基酸序列。免疫组织化学评估显示，mMCP-2蛋白在 Kirsten肉瘤病毒永生化的小鼠肥大细胞、BMMCW+IL-10以及旋毛虫感染的BALB/cJ小鼠空肠中的黏膜肥大细胞的分泌颗粒中积累。对BMMCW+IL-10中mMCP-2 mRNA和蛋白诱导的时间进程分析表明，这些细胞在暴露于rIL-10后24小时含有高水平的mMCP-2 mRNA稳态水平。虽然在处理24小时的细胞中存在少量可免疫检测到 的mMCP-2蛋白，但在用rIL-10处理细胞7天后才能获得大量这种蛋白酶。对BMMCW+IL-10中mMCP-2 mRNA和蛋白丢失的时间进程分析表明，从培养基中去除rIL-10后24小时，mMCP-2 mRNA的稳态水平急剧下降，但mMCP-2蛋白水平直到培养第5天才有明显下降。BMMC中mMCP-2 mRNA和蛋白的稳态水平在有和没有rIL-10的情况下培养这些肥大细胞时均可发生可逆变化，这一事实表明肥大细胞的表型不是固定不变的。相反，它处于一种动态状态，受肥大细胞在其不同微环境中所接触的细胞因子网络调节。
