Cramer E M, Beesley J E, Pulford K A, Breton-Gorius J, Mason D Y
Département d'angio-hématologie, Hôpital Lariboisière, Paris, France.
Am J Pathol. 1989 Jun;134(6):1275-84.
The authors have localized elastase in human blood and bone marrow neutrophils by immunoelectron microscopy using a monoclonal anti-human elastase antibody (NP 57) and compared its distribution with myeloperoxidase (MPO) and lactoferrin (LF), which mark primary and secondary neutrophil granule, respectively. Human bone marrow and blood polymorphonuclear leukocytes (PMN), either unstimulated or after phagocytosis of latex microbeads, were fixed in 4% paraformaldehyde. Ultrathin frozen sections were immunolabeled with NP 57, followed by an immunogold probe. In bone marrow granulocyte precursors elastase appeared simultaneously in the immature first granules of myeloblasts with MPO. As these granules became denser with maturation, labeling for both enzymes became weaker and sometimes negative (possibly due to masking of immunoreactivity). The ellipsoidal primary granules were strongly labeled by NP57. LF positive granules appeared later, at the myelocyte stage, and contained neither MPO nor elastase. In mature neutrophils, immunolabeling for elastase was found together with MPO in the large electron-dense primary granules and in a different granule population from the LF-positive secondary granules. Double labeling with two different-sized gold particles was used to compare the kinetics of degranulation of secondary and primary granules. The observation and the analysis of single phagosome content was made possible by this new technique. In conclusion, immunoelectron microscopy was used to show elastase in the primary granules of neutrophils, where it appears simultaneously with MPO. This technique has also allowed comparison of the kinetics of degranulation of both types of granules, and could be applied to different experimental and pathologic conditions.
作者使用单克隆抗人弹性蛋白酶抗体（NP 57），通过免疫电子显微镜在人血液和骨髓中性粒细胞中定位了弹性蛋白酶，并将其分布与分别标记初级和次级中性粒细胞颗粒的髓过氧化物酶（MPO）和乳铁蛋白（LF）进行了比较。将未刺激的或吞噬乳胶微珠后的人骨髓和血液多形核白细胞（PMN）用4%多聚甲醛固定。超薄冰冻切片用NP 57进行免疫标记，随后用免疫金探针标记。在骨髓粒细胞前体中，弹性蛋白酶与MPO同时出现在原粒细胞未成熟的第一颗粒中。随着这些颗粒随着成熟而变得更致密，两种酶的标记变得更弱，有时呈阴性（可能是由于免疫反应性被掩盖）。椭圆形的初级颗粒被NP57强烈标记。LF阳性颗粒出现较晚，在中幼粒细胞阶段，且既不含有MPO也不含有弹性蛋白酶。在成熟中性粒细胞中，在大的电子致密初级颗粒以及与LF阳性次级颗粒不同的颗粒群体中发现了弹性蛋白酶与MPO的免疫标记。使用两种不同大小的金颗粒进行双重标记，以比较次级和初级颗粒脱颗粒的动力学。这项新技术使观察和分析单个吞噬体的内容物成为可能。总之，免疫电子显微镜用于显示中性粒细胞初级颗粒中的弹性蛋白酶，它与MPO同时出现。该技术还能够比较两种颗粒脱颗粒的动力学，并且可应用于不同的实验和病理条件。
