Tanaka Y, Tanowitz H, Bloom B R
Infect Immun. 1983 Sep;41(3):1322-31. doi: 10.1128/iai.41.3.1322-1331.1983.
A continuous cloned murine macrophage-like cell line, clone 16 derived from J774, has been found upon appropriate stimulation to be capable of oxidizing glucose by the hexose monophosphate shunt and producing O2- and H2O2. A variant in oxidative metabolism, clone C3C, was selected from this cell line which under similar conditions is unable to produce significant amounts of O2- and H2O2. When cells of the parental clone 16 were infected with epimastigotes of Trypanosoma cruzi, there was significant killing or growth inhibition of the parasites at 3 to 4 days after infection. In contrast, the parasites grew in the oxidative variant, clone C3C. Trypomastigote forms of T. cruzi were found to be only partially killed in the parental clone 16 but grew abundantly in the oxidative variant. Infection of the parental clone, but not the variant, was sufficient to stimulate oxygen metabolism as demonstrated by the increased reduction of nitro blue tetrazolium. Studies on the killing of T. cruzi epimastigotes in cell-free suspension by xanthine-xanthine oxidase indicated that 90% of the killing was catalase sensitive and due to H2O2, with at most 7 to 8% killing which could be inhibited by scavengers of . OH and singlet oxygen (1O2). In the in vitro experiment with H2O2 produced by glucose and glucose oxidase, the 50% lethal doses of epimastigotes and trypomastigotes were 6.0 and 8.7 nmol of H2O2 per min per ml, respectively, indicating that trypomastigotes were more resistant to killing by H2O2 than epimastigotes were. A reconstitution experiment of trypanocidal activity in clone C3C by ingestion of zymosan particles coupled with glucose oxidase showed that H2O2 was essential for this cytocidal process in the macrophage cell line. These results provide clear evidence for killing of an intracellular parasite by a continuous macrophage-like cell line and suggest the importance of the oxidative cytocidal mechanism in this process.
已发现一种连续克隆的鼠巨噬细胞样细胞系，即源自J774的克隆16，在适当刺激下能够通过磷酸己糖途径氧化葡萄糖，并产生超氧阴离子（O2-）和过氧化氢（H2O2）。从该细胞系中筛选出一种氧化代谢变体克隆C3C，在类似条件下，它无法产生大量的O2-和H2O2。当亲本克隆16的细胞感染克氏锥虫的前鞭毛体时，在感染后3至4天，寄生虫会被显著杀伤或生长受到抑制。相比之下，寄生虫在氧化变体克隆C3C中生长。发现克氏锥虫的无鞭毛体形式在亲本克隆16中仅被部分杀伤，但在氧化变体中大量生长。亲本克隆而非变体的感染足以刺激氧代谢，如硝基蓝四唑还原增加所证明的那样。关于黄嘌呤 - 黄嘌呤氧化酶在无细胞悬液中杀伤克氏锥虫前鞭毛体的研究表明，90%的杀伤对过氧化氢酶敏感且归因于H2O2，最多7%至8%的杀伤可被·OH和单线态氧（1O2）清除剂抑制。在葡萄糖和葡萄糖氧化酶产生H2O2的体外实验中，前鞭毛体和无鞭毛体的50%致死剂量分别为每分钟每毫升6.0和8.7纳摩尔H2O2，表明无鞭毛体比前鞭毛体对H2O2杀伤更具抗性。通过摄取酵母聚糖颗粒与葡萄糖氧化酶结合来恢复克隆C3C中的杀锥虫活性的实验表明，H2O2对于巨噬细胞系中的这种杀细胞过程至关重要。这些结果为连续的巨噬细胞样细胞系杀伤细胞内寄生虫提供了明确证据，并表明氧化杀细胞机制在这一过程中的重要性。
