Das L, Datta N, Bandyopadhyay S, Das P K
Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, Calcutta, India.
J Immunol. 2001 Mar 15;166(6):4020-8. doi: 10.4049/jimmunol.166.6.4020.
The virulence of Leishmania donovani in mammals depends at least in part on cysteine proteases because they play a key role in CD4(+) T cell differentiation. A 6-fold increase in NO production was observed with 0.5 microM chicken cystatin, a natural cysteine protease inhibitor, in IFN-gamma-activated macrophages. In a 45-day BALB/c mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by cystatin in synergistic activation with a suboptimal dose of IFN-gamma. In contrast to the case with promastigotes, cystatin and IFN-gamma inhibited the growth of amastigotes in macrophages. Although in vitro cystatin treatment of macrophages did not induce any NO generation, significantly enhanced amounts of NO were generated by macrophages of cystatin-treated animals. Their splenocytes secreted soluble factors required for the induction of NO biosynthesis, and the increased NO production was paralleled by a concomitant increase in antileishmanial activity. Moreover, splenocyte supernatants treated with anti-IFN-gamma or anti-TNF-alpha Abs suppressed inducible NO generation, whereas i.v. administration of these anticytokine Abs along with combined therapy reversed protection against infection. mRNA expression and flow cytometric analysis of infected spleen cells suggested that cystatin and IFN-gamma treatment, in addition to greatly reducing parasite numbers, resulted in reduced levels of IL-4 but increased levels of IL-12 and inducible NO synthase. Not only was this treatment curative when administered 15 days postinfection, but it also imparted resistance to reinfection. These studies provide a promising alternative for protection against leishmaniasis with a switch of CD4(+) differentiation from Th2 to Th1, indicative of long-term resistance.
杜氏利什曼原虫在哺乳动物中的毒力至少部分取决于半胱氨酸蛋白酶，因为它们在CD4(+) T细胞分化中起关键作用。在IFN-γ激活的巨噬细胞中，用0.5微摩尔的鸡半胱氨酸蛋白酶抑制剂（一种天然的半胱氨酸蛋白酶抑制剂）可使一氧化氮（NO）的产生增加6倍。在内脏利什曼病的45天BALB/c小鼠模型中，半胱氨酸蛋白酶与次优剂量的IFN-γ协同激活可完全消除脾脏中的寄生虫负荷。与前鞭毛体的情况相反，半胱氨酸蛋白酶和IFN-γ可抑制巨噬细胞中无鞭毛体的生长。虽然体外对半胱氨酸蛋白酶处理巨噬细胞未诱导产生任何NO，但经半胱氨酸蛋白酶处理的动物的巨噬细胞产生的NO量显著增加。它们的脾细胞分泌诱导NO生物合成所需的可溶性因子，NO产生的增加与抗利什曼活性的相应增加平行。此外，用抗IFN-γ或抗TNF-α抗体处理的脾细胞上清液可抑制诱导型NO的产生，而静脉注射这些抗细胞因子抗体与联合治疗可逆转对感染的保护作用。对感染的脾细胞进行mRNA表达和流式细胞术分析表明，半胱氨酸蛋白酶和IFN-γ处理除了大大减少寄生虫数量外，还导致IL-4水平降低，但IL-12和诱导型NO合酶水平升高。这种治疗不仅在感染后15天给药时具有治愈作用，而且还赋予了对再感染的抵抗力。这些研究为通过将CD4(+)分化从Th2转换为Th1来预防利什曼病提供了一种有前景的替代方法，这表明具有长期抵抗力。
