Buchmüller-Rouiller Y, Corrandin S B, Smith J, Schneider P, Ransijn A, Jongeneel C V, Mauël J
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland.
Cell Immunol. 1995 Aug;164(1):73-80. doi: 10.1006/cimm.1995.1144.
We have examined the effects of two agents depleting the intracellular pool of glutathione (GSH) on macrophage activation induced by IFN-gamma + LPS, as measured by nitrite production and leishmanicidal activity. Diethylmaleate (DEM), which depletes intracellular GSH by conjugation via a reaction catalyzed by the GSH-S-transferase, strongly inhibited nitrite secretion and leishmanicidal activity when added before or at the time of addition of IFN-gamma + LPS; this inhibition was progressively lost when addition of DEM was delayed up to 10 hr. A close correlation was observed between levels of intracellular soluble GSH during activation and nitrite secretion. Inhibition was partially reversed by the addition of glutathione ethyl ester (GSH-Et). Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, also inhibited macrophage activation, although to a lesser extent than DEM despite a more pronounced soluble GSH depletion. This inhibition was completely reversed by the addition of GSH-Et. DEM and BSO did not alter cell viability or PMA-triggered O2- production by activated macrophages, suggesting that the inhibitory effects observed on nitrite secretion and leishmanicidal activity were not related to a general impairment of macrophage function. DEM and BSO treatment reduced iNOS specific activity and iNOS protein in cytosolic extracts. DEM also decreased iNOS mRNA expression while BSO had no effect. Although commonly used as a GSH-depleting agent, DEM may have additional effects because it can also act as a sulhydryl reagent; BSO, on the other hand, which depletes GSH by enzymatic inhibition, has no effect on protein-bound GSH. Our results suggest that both soluble and protein-bound GSH may be important for the induction of NO synthase in IFN-gamma + LPS-activated macrophages.
我们研究了两种消耗细胞内谷胱甘肽（GSH）池的试剂对IFN-γ+LPS诱导的巨噬细胞活化的影响，通过亚硝酸盐产生和杀利什曼原虫活性来衡量。马来酸二乙酯（DEM）通过谷胱甘肽S-转移酶催化的反应进行共轭作用来消耗细胞内GSH，当在添加IFN-γ+LPS之前或同时添加时，强烈抑制亚硝酸盐分泌和杀利什曼原虫活性；当DEM的添加延迟至10小时时，这种抑制作用逐渐消失。在活化过程中细胞内可溶性GSH水平与亚硝酸盐分泌之间观察到密切相关性。添加谷胱甘肽乙酯（GSH-Et）可部分逆转抑制作用。丁硫氨酸亚砜胺（BSO）是γ-谷氨酰半胱氨酸合成酶的特异性抑制剂，也抑制巨噬细胞活化，尽管其可溶性GSH消耗更明显，但抑制程度比DEM小。添加GSH-Et可完全逆转这种抑制作用。DEM和BSO不会改变活化巨噬细胞的细胞活力或PMA触发的O2-产生，这表明观察到的对亚硝酸盐分泌和杀利什曼原虫活性的抑制作用与巨噬细胞功能的一般损害无关。DEM和BSO处理降低了细胞溶质提取物中诱导型一氧化氮合酶（iNOS）的比活性和iNOS蛋白。DEM还降低了iNOS mRNA表达，而BSO没有影响。尽管DEM通常用作GSH消耗剂，但它可能还有其他作用，因为它也可以作为巯基试剂；另一方面，通过酶抑制作用消耗GSH的BSO对蛋白结合的GSH没有影响。我们的结果表明，可溶性和蛋白结合的GSH对于IFN-γ+LPS活化的巨噬细胞中一氧化氮合酶的诱导可能都很重要。
