Caughey G H, Lazarus S C, Viro N F, Gold W M, Nadel J A
Cardiovascular Research Institute, University of California Medical Center, San Francisco.
Immunology. 1988 Feb;63(2):339-44.
Mast cell secretory granules contain unique tryptic and chymotryptic serine proteases that differ between species and tissues. Direct comparison of these proteases in single-cell types has been hindered by the difficulty of obtaining adequate numbers of pure mast cells. In this study, we were able to compare tryptic and chymotryptic enzyme activity in cells of presumed monoclonal origin, using two stable lines ('BR' and 'G') of dog mastocytomas. The gel-filtration profiles, inhibitor susceptibilities and substrate preferences of tryptic and chymotryptic mastocytoma protease activities established their close resemblance to the tryptases and chymases of human and rodent mast cells. Striking heterogeneity was observed in the amounts and solubilities of the tryptic and chymotryptic activity in the two different mastocytoma cell lines. Incubation of cells from both lines with calcium ionophore A23187 caused non-cytotoxic release of protease activity. In contrast to chymase from rat connective tissue mast cells, protease activity that was insoluble after extraction at low ionic strength became soluble following ionophore-stimulated release. Neither tryptic nor chymotryptic activity was activated during degranulation, suggesting the absence of inactive precursors. Cells of the 'BR' line released both tryptic and chymotryptic activity in parallel with the granule marker histamine; cells of the 'G' line released a much smaller proportion of tryptic activity than of either chymotryptic activity or histamine. These differences in release of granule constituents from cells of common origin could be explained by developmental variations in the production of performed mediators or by differential regulation of preformed mediator release. We conclude that the differences in protease content, solubility and release in these mastocytoma lines are useful in evaluating the potential pathophysiological significance of the contribution of proteases to mast cell heterogeneity.
肥大细胞分泌颗粒含有独特的胰蛋白酶和糜蛋白酶样丝氨酸蛋白酶，这些蛋白酶在不同物种和组织之间存在差异。由于难以获得足够数量的纯肥大细胞，阻碍了对这些蛋白酶在单细胞类型中的直接比较。在本研究中，我们利用犬肥大细胞瘤的两个稳定细胞系（“BR”和“G”），比较了假定单克隆起源细胞中的胰蛋白酶和糜蛋白酶样酶活性。肥大细胞瘤胰蛋白酶和糜蛋白酶样蛋白酶活性的凝胶过滤图谱、抑制剂敏感性和底物偏好性表明，它们与人和啮齿动物肥大细胞的类胰蛋白酶和类糜蛋白酶极为相似。在两种不同的肥大细胞瘤细胞系中，观察到胰蛋白酶和糜蛋白酶样活性在含量和溶解性上存在显著异质性。用钙离子载体A23187孵育两个细胞系的细胞，可导致蛋白酶活性的非细胞毒性释放。与大鼠结缔组织肥大细胞的糜酶不同，低离子强度提取后不溶性的蛋白酶活性在离子载体刺激释放后变为可溶性。在脱颗粒过程中，胰蛋白酶和糜蛋白酶样活性均未被激活，提示不存在无活性前体。“BR”细胞系的细胞释放的胰蛋白酶和糜蛋白酶样活性与颗粒标志物组胺平行；“G”细胞系释放的胰蛋白酶活性比例远低于糜蛋白酶样活性或组胺。来自共同起源细胞的颗粒成分释放差异，可通过已形成介质产生的发育差异或已形成介质释放的差异调节来解释。我们得出结论，这些肥大细胞瘤系中蛋白酶含量、溶解性和释放的差异，有助于评估蛋白酶对肥大细胞异质性贡献的潜在病理生理意义。
