Nagineni C N, Pardhasaradhi K, Martins M C, Detrick B, Hooks J J
Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Infect Immun. 1996 Oct;64(10):4188-96. doi: 10.1128/iai.64.10.4188-4196.1996.
Inflammation associated with retinochoroiditis is a major complication of ocular toxoplasmosis in infants and immunocompetent individuals. Moreover, Toxoplasma gondii-induced retinal disease causes serious complications in patients with AIDS and transplant patients. The retinal pigment epithelial (RPE) cell is an important regulatory cell within the retina and is one of the cells infected with T. gondii in in vivo. We have developed a human RPE (HRPE) cell in vitro model system to evaluate T. gondii replication and the regulation of this replication by cytokines. T. gondii replication was quantitated by counting the foci of infection (plaque formation) and the numbers of tachyzoites released into the supernatant fluids. Pretreatment of cultures with recombinant human tumor necrosis factor alpha, alpha interferon (IFN-alpha), IFN-beta, or IFN-gamma for 24 h prior to inoculation inhibited T. gondii replication in a dose-dependent manner. Of these cytokines, IFN-gamma was the most potent, and T. gondii replication was completely inhibited at a concentration of 100 U/ml. The anti-toxoplasmotic activity of IFN-gamma was significantly blocked by monoclonal antibody to IFN-gamma. Treatment of the cultures with IFN-gamma from day 1 or 2 postinoculation with T. gondii also offered protection against the parasite. The anti-toxoplasmotic activity of tumor necrosis factor alpha or IFN-alpha, -beta, or -gamma in these cultures was found to be independent of the nitric oxide (NO) pathway, since NO production was not found in HRPE cells treated with these cytokines. However, addition of tryptophan to IFN-gamma-treated cells significantly reversed the inhibitory effects of IFN-gamma, suggesting that IFN-gamma acts by depleting cellular tryptophan. This effect was further confirmed by reverse transcription-PCR and Northern (RNA) blot analysis, which indicated induction of indoleamine 2,3-dioxygenase (IDO), an enzyme that converts tryptophan to kynurenine. These results indicated that interferons inhibited T. gondii replication in HRPE by NO-independent but IDO-dependent mechanisms. This in vitro model of T. gondii replication in HRPE may be useful in evaluating the effects of cytokines and drugs on T. gondii replication within the retina.
与视网膜脉络膜炎相关的炎症是婴儿和免疫功能正常个体眼部弓形虫病的主要并发症。此外，弓形虫引起的视网膜疾病在艾滋病患者和移植患者中会导致严重并发症。视网膜色素上皮（RPE）细胞是视网膜内一种重要的调节细胞，也是体内受弓形虫感染的细胞之一。我们建立了一种人RPE（HRPE）细胞体外模型系统，以评估弓形虫的复制以及细胞因子对这种复制的调节作用。通过计数感染灶（噬斑形成）和释放到上清液中的速殖子数量来定量弓形虫的复制。在接种前用重组人肿瘤坏死因子α、α干扰素（IFN-α）、IFN-β或IFN-γ对培养物预处理24小时，可剂量依赖性地抑制弓形虫的复制。在这些细胞因子中，IFN-γ最有效，在浓度为100 U/ml时可完全抑制弓形虫的复制。IFN-γ的抗弓形虫活性被抗IFN-γ单克隆抗体显著阻断。在接种弓形虫后第1天或第2天用IFN-γ处理培养物也能提供对该寄生虫的保护。在这些培养物中发现肿瘤坏死因子α或IFN-α、-β或-γ的抗弓形虫活性与一氧化氮（NO）途径无关，因为在用这些细胞因子处理的HRPE细胞中未发现NO的产生。然而，向IFN-γ处理的细胞中添加色氨酸可显著逆转IFN-γ的抑制作用，表明IFN-γ通过消耗细胞色氨酸发挥作用。逆转录-PCR和Northern（RNA）印迹分析进一步证实了这一效应，这表明诱导了吲哚胺2,3-双加氧酶（IDO），一种将色氨酸转化为犬尿氨酸的酶。这些结果表明，干扰素通过不依赖NO但依赖IDO的机制抑制HRPE中弓形虫的复制。这种HRPE中弓形虫复制的体外模型可能有助于评估细胞因子和药物对视网膜内弓形虫复制的影响。
