Lee S C, Kress Y, Zhao M L, Dickson D W, Casadevall A
Department of Pathology (Neuropathology), Albert Einstein College of Medicine, Bronx, New York, USA.
Lab Invest. 1995 Dec;73(6):871-9.
Cryptococcus neoformans (CN) is an opportunistic pathogen that can cause fatal meningoencephalitis in patients with immune deficiency. Among the central nervous system (CNS) cells that are infected by CN are perivascular microglia and macrophages. Little is known about the interaction of CN and CNS phagocytes at the cellular level.
To better understand the fate of CN in microglia, we followed Ab-opsonized CN in human fetal microglial culture by phase-contrast microscopy, combined lighted microscopy and transmission electron microscopy of plastic-embedded monolayers, and immunocytochemistry for localization of capsular Ag.
Phase-contrast microscopy revealed that microglia initially internalized and contained Ab-opsonized yeast cells within phagolysosomes. However, CN escaped from microglia and resumed extracellular growth 16 to 24 hours after being phagocytosed. Transmission electron microscopy/1-mu epoxy sections revealed that intracellular CN were localized in two types of phagosomes in microglia: spacious phagosomes (SP) and close-fitting phagosomes (CP). Three lines of evidence indicate that SP are the primary sites for intracellular CN survival and replication: (a) SP contained multiple, budding yeast cells, whereas CP contained only single yeast cells within a tightly bound phagosomal membrane; (b) the number of SP and the number of CN within SP increased considerably at 24 hours compared with at 2 hours; and (c) microglial cultures challenged with heat- or amphotericin B-treated CN had significantly fewer SP than those challenged with live CN. Both SP and CP phagosomes fused with lysosomes, suggesting that CN survival in SP was not due to failure of phagolysosomal fusion. In SP, there was attenuation and diffusion of capsular polysaccharide within the phagosome, whereas in CP the fungal capsules remained compact and homogeneous. Immunocytochemistry with an mAb directed to capsular glucoronoxylomannan supported continued synthesis of polysaccharide within SP.
The results suggest that, in human microglia, CN survive and replicate within SP. Modification of CN capsular polysaccharide within SP may be a contributing factor to this aberrant microglial-CN interaction.
新型隐球菌（CN）是一种机会性致病病原体，可在免疫缺陷患者中引起致命的脑膜脑炎。被CN感染的中枢神经系统（CNS）细胞包括血管周围的小胶质细胞和巨噬细胞。关于CN与CNS吞噬细胞在细胞水平上的相互作用知之甚少。
为了更好地了解CN在小胶质细胞中的命运，我们通过相差显微镜观察了人胎儿小胶质细胞培养物中经抗体调理的CN，结合了塑料包埋单层的光学显微镜和透射电子显微镜，以及用于荚膜抗原定位的免疫细胞化学方法。
相差显微镜显示，小胶质细胞最初将经抗体调理的酵母细胞内化并包含在吞噬溶酶体内。然而，CN在被吞噬16至24小时后从小胶质细胞中逸出并恢复细胞外生长。透射电子显微镜/1微米环氧树脂切片显示，细胞内的CN定位于小胶质细胞中的两种吞噬体：宽敞吞噬体（SP）和紧密贴合吞噬体（CP）。有三条证据表明SP是细胞内CN存活和复制的主要部位：（a）SP包含多个出芽的酵母细胞，而CP在紧密结合的吞噬体膜内仅包含单个酵母细胞；（b）与2小时相比，24小时时SP的数量和SP内CN的数量显著增加；（c）用热或两性霉素B处理的CN攻击的小胶质细胞培养物中的SP比用活CN攻击的培养物中的SP明显更少。SP和CP吞噬体均与溶酶体融合，这表明CN在SP中的存活不是由于吞噬溶酶体融合失败。在SP中，吞噬体内的荚膜多糖有衰减和扩散，而在CP中真菌荚膜保持紧密和均匀。用针对荚膜葡糖醛酸木甘露聚糖的单克隆抗体进行免疫细胞化学支持SP内多糖的持续合成。
结果表明，在人小胶质细胞中，CN在SP内存活和复制。SP内CN荚膜多糖的修饰可能是这种异常的小胶质细胞-CN相互作用的一个促成因素。
