Wagner J M, Franco M, Kephart G M, Gleich G J
Department of Immunology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
Am J Trop Med Hyg. 1998 Jul;59(1):66-72. doi: 10.4269/ajtmh.1998.59.66.
Paracoccidioidomycosis is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Although eosinophils have long been associated with the immune defense against helminths, the role of eosinophils in the immune response to fungal diseases is not as well studied. The eosinophil granule major basic protein is toxic to helminths and mammalian cells in vitro, and its release has been used as a marker of eosinophil localization and degranulation. To determine whether eosinophil infiltration and degranulation, as evidenced by the deposition of major basic protein, occur in lesions of P. brasiliensis, we used an immunofluorescence technique to localize the P. brasiliensis organisms and eosinophils and major basic protein. Initially, all tissues were stained with polyclonal antibody to major basic protein; subsequently, colocalization of major basic protein and P. brasiliensis by double staining with mouse and rabbit antibodies, respectively, was performed. Nine biopsy tissues from seven patients were analyzed. All nine biopsies showed infiltration of intact eosinophils using both the monoclonal and the polyclonal anti-major basic protein antibodies, along with the presence of P. brasiliensis. Furthermore, using the polyclonal anti-major basic protein antibody, nine of nine tissues showed extracellular major basic protein deposition (granular or diffuse fluorescence staining outside of intact eosinophils). The double staining procedure using the anti-major basic protein monoclonal antibody showed extracellular deposition in five of eight biopsies; in these five biopsies, approximately 60% of the areas containing P. brasiliensis had extracellular major basic protein deposited on the organisms. These observations support the hypothesis that the eosinophil, through toxic granule proteins such as major basic protein, participates in the pathophysiology of paracoccidioidomycosis.
副球孢子菌病是一种由巴西副球孢子菌引起的慢性肉芽肿性疾病。尽管嗜酸性粒细胞长期以来一直与针对蠕虫的免疫防御相关，但嗜酸性粒细胞在真菌疾病免疫反应中的作用尚未得到充分研究。嗜酸性粒细胞颗粒主要碱性蛋白在体外对蠕虫和哺乳动物细胞有毒性，其释放已被用作嗜酸性粒细胞定位和脱颗粒的标志物。为了确定嗜酸性粒细胞浸润和脱颗粒（以主要碱性蛋白沉积为证据）是否发生在巴西副球孢子菌病变中，我们使用免疫荧光技术来定位巴西副球孢子菌、嗜酸性粒细胞和主要碱性蛋白。最初，所有组织都用针对主要碱性蛋白的多克隆抗体染色；随后，分别用小鼠和兔抗体进行双重染色，以确定主要碱性蛋白和巴西副球孢子菌的共定位。对7名患者的9份活检组织进行了分析。使用单克隆和多克隆抗主要碱性蛋白抗体，所有9份活检均显示有完整嗜酸性粒细胞浸润，同时存在巴西副球孢子菌。此外，使用多克隆抗主要碱性蛋白抗体，9份组织中有9份显示细胞外主要碱性蛋白沉积（完整嗜酸性粒细胞外的颗粒状或弥漫性荧光染色）。使用抗主要碱性蛋白单克隆抗体的双重染色程序在8份活检中的5份显示有细胞外沉积；在这5份活检中，约60%含有巴西副球孢子菌的区域有细胞外主要碱性蛋白沉积在病原体上。这些观察结果支持以下假设：嗜酸性粒细胞通过主要碱性蛋白等有毒颗粒蛋白参与副球孢子菌病的病理生理过程。
