Jaso-Friedmann L, Leary J H, Evans D L
Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, USA.
Exp Parasitol. 2000 Oct;96(2):75-88. doi: 10.1006/expr.2000.4561.
Numerous different species of parasites and pathogenic microorganisms produce programmed cell death (PCD) and apoptosis in eukaryotic targets. How ever, only a few studies have demonstrated that effector cells, cytokines, growth factors, or soluble apoptosis-inducing factors are capable of initiating apoptosis in protozoan parasites. Certain Tetrahymena spp. in teleosts are opportunistic pathogens. In the present study these pathogenic protozoans were developed as a model system to describe the potential role of the Fas ligand (FasL)-Fas receptor (FasR) system as a means of innate immunity in teleosts. Nonspecific cytotoxic cells (NCC) constitutively express soluble FasL (sFasL). Binding of the antigen receptor (i.e., NCCRP-1) on NCC to target cells caused the release of sFasL into the milieu. The presence of functional sFasL in these supernatants was determined by Western blot analysis and by demonstrating the lysis of FasR(+) HL-60 but not IM-9 (FasR(-)) targets. Soluble FasL containing supernatants generated by tumor cell-activated NCC also produced a reduction in 2 N DNA (i.e., DNA hypoploidy) of T. furgasoni. The induction of DNA hypoploidy by NCC supernatants could be neutralized by adsorption of the supernatants with anti-FasL antibody (but not with an isotype control). Experiments were next done to determine the expression of FasR on Tetrahymena and study the effects of anti-FasR monoclonal crosslinkage and treatment with soluble human recombinant FasL (huFasL) on initiation of PCD in Tetrahymena. Cell cycle analysis revealed that both crosslinkage and soluble huFasL binding to Tetrahymena produced DNA hypoploidy. The reduction in diploid DNA was confirmed by observing oligonucleosome fragmentation (DNA laddering) following anti-FasR treatment. Additional evidence for FasR expression on Tetrahymena was obtained using fluorescence microscopy and flow cytometry. Both methods showed that all Tetrahymena examined (three species consisting of four isolates) expressed membrane FasR. These studies demonstrated the potential of the FasL-FasR system in teleosts for initiation of antiparasite innate immunity. Effector NCC may initiate PCD of Tetrahymena that express a FasR-like protein. Induction of apoptosis may be a major mechanism of homeostatic control of protozoan parasite infestations/infections.
许多不同种类的寄生虫和病原微生物会在真核生物靶标中引发程序性细胞死亡（PCD）和凋亡。然而，仅有少数研究表明效应细胞、细胞因子、生长因子或可溶性凋亡诱导因子能够引发原生动物寄生虫的凋亡。硬骨鱼中的某些四膜虫属物种是机会性病原体。在本研究中，这些致病原生动物被开发为一个模型系统，以描述Fas配体（FasL）-Fas受体（FasR）系统作为硬骨鱼先天免疫手段的潜在作用。非特异性细胞毒性细胞（NCC）组成性表达可溶性FasL（sFasL）。NCC上的抗原受体（即NCCRP-1）与靶细胞结合会导致sFasL释放到周围环境中。通过蛋白质免疫印迹分析以及证明FasR(+) HL-60细胞（而非IM-9（FasR(-)）靶细胞）的裂解，确定了这些上清液中功能性sFasL的存在。肿瘤细胞激活的NCC产生的含有可溶性FasL的上清液也使弗氏四膜虫的2N DNA（即DNA亚二倍体）减少。NCC上清液诱导的DNA亚二倍体可通过用抗FasL抗体（而非同型对照）吸附上清液来中和。接下来进行实验以确定四膜虫上FasR的表达，并研究抗FasR单克隆交联以及用可溶性人重组FasL（huFasL）处理对四膜虫中PCD起始的影响。细胞周期分析表明，交联以及可溶性huFasL与四膜虫结合均产生了DNA亚二倍体。通过观察抗FasR处理后的寡核小体片段化（DNA梯状条带），证实了二倍体DNA的减少。使用荧光显微镜和流式细胞术获得了四膜虫上FasR表达的额外证据。这两种方法均表明，所有检测的四膜虫（由四个分离株组成的三个物种）均表达膜FasR。这些研究证明了硬骨鱼中FasL-FasR系统在启动抗寄生虫先天免疫方面的潜力。效应性NCC可能引发表达FasR样蛋白的四膜虫的PCD。凋亡诱导可能是原生动物寄生虫侵扰/感染稳态控制的主要机制。
