Erz W, Carrle D, Klessen C
Institute of Anatomy, University of Tübingen, Germany.
Acta Histochem. 1993 Feb;94(1):46-53. doi: 10.1016/S0065-1281(11)80338-7.
Polymorphonuclear neutrophils (PMN) contain neutral proteases which are cytochemically detectable on blood smears. The activity of these proteases can be visualized by pericellular lysis, i.e. through a disk like degradation of plasma and erythrocytes around centrally located neutrophils. This degradation is inhibited by protease inhibitors in a concentration-dependent manner. Striking differences in halo formation were found depending on the type of buffer and salt used and change of temperature. A halo formation was obtained in Tris-HCl buffer, pH 7.5 without salt, at room temperature (22 degrees C), and with short incubation periods (30 min). Borate buffer, pH 8.5, showed a comparable halo formation only at 37 degrees C, after addition of salts, and after longer incubation times (180 min). When different alkali chlorides were used, halo size increased with molecular mass. Optimum halo size was achieved at a concentration of 250 mM. Addition of MgCl2 and CaCl2 (250 mM) resulted in a small halo formation at 22 degrees C, but inhibited halo formation at 37 degrees C. The present study shows that the pericellular lysis caused around neutrophils by the release of proteases is affected by a complex interaction of salts, temperature, buffers and thermodynamic aspects, all of which should be evaluated carefully in these kinds of experiments.
多形核中性粒细胞（PMN）含有中性蛋白酶，这些蛋白酶在血液涂片上可通过细胞化学方法检测到。这些蛋白酶的活性可通过细胞周围溶解来显现，即通过位于中央的中性粒细胞周围血浆和红细胞的圆盘状降解来实现。这种降解受到蛋白酶抑制剂的浓度依赖性抑制。根据所用缓冲液和盐的类型以及温度变化，在晕圈形成方面发现了显著差异。在室温（22摄氏度）下，于无盐的pH 7.5 Tris-HCl缓冲液中，经过短时间孵育（30分钟）可获得晕圈形成。pH 8.5的硼酸盐缓冲液仅在37摄氏度、添加盐后以及较长孵育时间（180分钟）后显示出类似的晕圈形成。当使用不同的碱金属氯化物时，晕圈大小随分子量增加。在浓度为250 mM时可达到最佳晕圈大小。添加MgCl2和CaCl2（250 mM）在22摄氏度时导致形成小晕圈，但在37摄氏度时抑制晕圈形成。本研究表明，蛋白酶释放导致的中性粒细胞周围的细胞周围溶解受到盐、温度、缓冲液和热力学因素复杂相互作用的影响，在这类实验中所有这些因素都应仔细评估。
