Ultrastructural localization of the Charcot-Leyden crystal protein (lysophospholipase) to a distinct crystalloid-free granule population in mature human eosinophils.
Ultrastructural localization of the Charcot-Leyden crystal protein (lysophospholipase) to a distinct crystalloid-free granule population in mature human eosinophils.
复制标题
DOI：
10.1182/blood.v72.1.150.150
复制
发表时间：
1988-07
期刊：
影响因子：
20.3
通讯作者：
Ann M. Dvorak;L. Letourneau;G. Login;Peter F. Weller;Steven J. Ackerman
中科院分区：
文献类型：
--
作者：
Ann M. Dvorak;L. Letourneau;G. Login;Peter F. Weller;Steven J. Ackerman
Charcot-Leyden晶体（CLC）蛋白是嗜酸性粒细胞和嗜碱性粒细胞的独特成分。该蛋白形成在组织中嗜酸性粒细胞聚集部位观察到的六边形双锥体晶体，具有溶血磷脂酶活性（溶血磷脂酰水解酶E.C.3.1.1.5），估计占总嗜酸性粒细胞蛋白的7%至10%。我们研究了正常人和特发性嗜酸性粒细胞增多综合征患者成熟外周血嗜酸性粒细胞中CLC蛋白的超微结构定位。亚细胞定位进行了评估，通过免疫电子显微镜使用嗜酸性粒细胞，无论是从血沉棕黄层和纯化的细胞悬液，通过各种方法固定。免疫化学检测CLC蛋白采用兔抗血清嗜酸性粒细胞CLC蛋白，亲和层析纯化的单特异性IgG抗体，和植入后的免疫金技术。特异性对照包括（1）省略CLC蛋白的一抗和（2）用非免疫预免疫血清、蛋白A纯化的非免疫IgG或从固相CLC蛋白-琼脂糖凝胶吸附的抗CLC抗血清制备的蛋白A纯化的非反应性IgG替代一抗。CLC蛋白定位于嗜酸性粒细胞颗粒的次要（约5%）亚群。这些膜结合的细胞质颗粒大（大于0.5亩），没有晶体夹杂物，并与持续的嗜酸性粒细胞初级颗粒形态兼容。含有晶体的大的特异性颗粒不对CLC蛋白染色。小的致密颗粒，脂质体和囊泡管状结构的数量不足，以充分评估其作为CLC蛋白亚细胞定位的额外位点的潜力。嗜酸性粒细胞中CLC蛋白的免疫金定位的细胞特异性通过在相同切片中存在的中性粒细胞和淋巴细胞中不存在染色来确认。CLC蛋白（溶血磷脂酶）在成熟循环嗜酸性粒细胞中的大的无晶体颗粒的超微结构免疫金定位支持作为该酶的主要胞质内储存库的独特的“初级”颗粒群体的持久性。
The Charcot-Leyden crystal (CLC) protein is a unique constituent of eosinophils and basophils. This protein forms the hexagonal bipyramidal crystals observed in tissues at sites of eosinophil accumulations, possesses lysophospholipase activity (lysolecithin acylhydrolase E.C.3.1.1.5), and comprises an estimated 7% to 10% of total eosinophil protein. The ultrastructural localization of CLC protein was studied in mature peripheral blood eosinophils from normal donors and from patients with the idiopathic hypereosinophilic syndrome. Subcellular localization was evaluated by immunoelectron microscopy using eosinophils, both from buffy coat and purified cell suspensions, that were fixed by a variety of methods. Immunochemical detection of CLC protein employed rabbit antiserum to eosinophil CLC protein, affinity chromatography-purified monospecific IgG antibodies, and postembedding immunogold techniques. Controls for specificity included (1) omission of the primary antibody to CLC protein and (2) substitution of primary antibody with a nonimmune preimmunization serum, a protein A-purified nonimmune IgG, or a protein A-purified nonreactive IgG prepared from solid-phase CLC protein-Sepharose-absorbed anti-CLC antiserum. CLC protein was localized to a minor (approximately 5%) subpopulation of eosinophil granules. These membrane-bound cytoplasmic granules were large (greater than 0.5 mu), were devoid of crystalloid inclusions, and were morphologically compatible with persisting eosinophil primary granules. The crystalloid-containing, large, specific granules did not stain for CLC protein. Insufficient numbers of small dense granules, lipid bodies, and vesiculotubular structures were present to adequately evaluate their potential as additional sites for the subcellular localization of CLC protein. The cellular specificity of the immunogold localization of CLC protein in the eosinophil was affirmed by the absence of staining in neutrophils and lymphocytes present in the same sections. The ultrastructural immunogold localization of CLC protein (lysophospholipase) to a large, crystalloid-free granule in mature circulating eosinophils supports the persistence of a distinct "primary" granule population that serves as a major intracytoplasmic repository for this enzyme.