Mukherjee Piyali, Sen Parimal C, Ghose Asoke C
Department of Microbiology, Bose Institute, P-1/12 CIT Scheme VII M, Kolkata, 700 054, India.
Apoptosis. 2006 Nov;11(11):2013-29. doi: 10.1007/s10495-006-0088-7.
Visceral leishmaniasis (VL) produced in BALB/c mice through intracardial administration of Leishmania donovani amastigotes was accompanied by hepatosplenomegaly with high organ parasite load and lymphadenopathy when followed up to 4-months or so. To elucidate the mechanism of immunosuppression associated with VL, we report here progressive impairment of the proliferative response of lymph node cells (lymphocytes) from infected animals (I-LNC) to in vitro stimulation with the combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io) that could be related to the downregulation of PKC and MAP kinase (ERK 1/2) activation process. Further, pretreatment of I-LNC with the protein phosphatase inhibitor okadaic acid (OA), but not with calyculin A or sodium orthovanadate, significantly restored their proliferative response as well as PMA-induced activation of PKC. A population of LNC (primarily T-lymphocytes) from chronically infected animals was shown to undergo apoptosis, the number of which increased considerably following PMA+ Io stimulation. The apoptotic pathway, which was followed through binding of cells to Annexin V, activation of caspase-3 and fragmentation of DNA, involved destabilization of mitochondria, probably as a result of downregulation of PKC and Bcl-2. Interestingly, prior incubation of I-LNC with OA reversed the state of cell cycle arrest (anergy) and apoptosis through progression of cells from G0/G1 to S and G2/M phases with transcriptional activation of IL-2 and IL-2R genes. Our results suggest that the cellular (immune) dysfunction in VL could be attributed to dephosphorylation of key molecules in the T-lymphocyte signaling pathway by Ser/Thr phosphatase leading to their inactivation.
通过向BALB/c小鼠心内注射杜氏利什曼原虫无鞭毛体所产生的内脏利什曼病（VL），在随访约4个月时，伴有肝脾肿大、器官寄生虫负荷高以及淋巴结病。为阐明与VL相关的免疫抑制机制，我们在此报告，感染动物的淋巴结细胞（淋巴细胞）（I-LNC）对佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）和离子霉素（Io）联合体外刺激的增殖反应逐渐受损，这可能与蛋白激酶C（PKC）和丝裂原活化蛋白激酶（ERK 1/2）激活过程的下调有关。此外，用蛋白磷酸酶抑制剂冈田酸（OA）预处理I-LNC，但不用花萼海绵诱癌素A或原钒酸钠预处理，可显著恢复其增殖反应以及PMA诱导的PKC激活。来自慢性感染动物的一群淋巴结细胞（主要是T淋巴细胞）显示发生凋亡，在PMA + Io刺激后凋亡细胞数量显著增加。通过细胞与膜联蛋白V结合、半胱天冬酶-3激活和DNA片段化所追踪的凋亡途径，涉及线粒体的不稳定，这可能是PKC和Bcl-2下调的结果。有趣的是，用OA预先孵育I-LNC可通过细胞从G0/G1期进展到S期和G2/M期以及IL-2和IL-2R基因的转录激活来逆转细胞周期停滞（无反应性）和凋亡状态。我们的结果表明，VL中的细胞（免疫）功能障碍可能归因于丝氨酸/苏氨酸磷酸酶导致T淋巴细胞信号通路中关键分子去磷酸化，从而使其失活。
