Fedyk E R, Phipps R P
Cancer Center, University of Rochester School of Medicine and Dentistry, NY 14642.
Int J Immunopharmacol. 1994 Jul;16(7):533-46. doi: 10.1016/0192-0561(94)90105-8.
A potential role for lipoxygenase (LO) products and reactive oxygen species (ROS) in mouse B-lymphocyte activation and differentiation was investigated. Previously published investigations with the nonspecific 5-LO (EC 1.13.11.34) and 12-LO (EC 1.13.11.31) inhibitors such as nordihydroguaiaretic acid (NDGA) and 6,7-dihydroxycoumarin (Esculetin), are misleading in that they suggest lymphocyte LO activity is required for activation and differentiation of these cells. In initial support of this concept, we report that NDGA and Esculetin completely inhibited B-lymphocyte activation mediated by either membrane immunoglobulin (mIg), or the lipopolysaccharide (LPS) receptor. NDGA and Esculetin completely inhibited cell enlargement and proliferation, exhibiting half maximal inhibitory concentrations (IC50S) of approximately 1 x 10(-6) M. In contrast, the highly specific 5-LO inhibitors BAY X 1005, MK-886 and Wy 50,295 did not inhibit cell enlargement or proliferation. Moreover, 5,8,11-eicosatriynoic acid (ETI) which inhibits 5- and 12-LO, and 5, 8, 11, 14-eicosatetraynoic acid (ETYA) which inhibits all known LOs did not affect B-lymphocyte proliferation. Interestingly, NDGA and Esculetin are antioxidants, unlike BAY X 1005, MK-886, Wy 50,295, ETI and ETYA. Our hypothesis was that the antioxidant activities of NDGA and Esculetin were reponsible for inhibiting B-lymphocyte activation and proliferation and we speculated that ROS and not LO activity was required for both processes. Additional antioxidants such as butylated hydroxy toluene, o-phenanthroline, thiourea, and alpha-tocopherol (vitamin E), also inhibited B-lymphocyte proliferation induced by either the LPS or mIg receptors. These agents exhibited IC50S of 1 x 10(-8) M, 5 x 10(-10) M, 6 x 10(-3) M and 5 x 10(-5) M, respectively. When resting B-lymphocytes were treated with a source of ROS (1 x 10(-5) M H2O2), cells enlarged in a temperature-sensitive manner, which is similar to LPS-induced enlargement. Both NDGA and Esculetin completely inhibited H2O2-induced enlargement. These results further indicate that ROS are required for B-lymphocyte activation and proliferation. Similar results were obtained for B-lymphocyte differentiation. NDGA and Esculetin completely inhibited the development of plasma cells and displayed IC50S of 5 x 10(-6) M. Conversely, BAY X 1005, MK-886, Wy 50,295, ETI, and ETYA did not block the formation of plasma cells. Therefore, ROS are also crucial for differentiation into plasma cells. These experiments are the first to directly illustrate that intracellular ROS mediate B-lymphocyte activation, proliferation and differentiation and that LO products are not required for these processes.(ABSTRACT TRUNCATED AT 400 WORDS)
研究了脂氧合酶（LO）产物和活性氧（ROS）在小鼠B淋巴细胞活化和分化中的潜在作用。先前发表的使用非特异性5-LO（EC 1.13.11.34）和12-LO（EC 1.13.11.31）抑制剂如去甲二氢愈创木酸（NDGA）和6,7-二羟基香豆素（七叶亭）的研究具有误导性，因为它们表明淋巴细胞LO活性是这些细胞活化和分化所必需的。作为这一概念的初步支持，我们报告NDGA和七叶亭完全抑制由膜免疫球蛋白（mIg）或脂多糖（LPS）受体介导的B淋巴细胞活化。NDGA和七叶亭完全抑制细胞增大和增殖，半数最大抑制浓度（IC50）约为1×10（-6）M。相比之下，高度特异性的5-LO抑制剂BAY X 1005、MK-886和Wy 50,295不抑制细胞增大或增殖。此外，抑制5-LO和12-LO的5,8,11-二十碳三烯酸（ETI）以及抑制所有已知LO的5,8,11,14-二十碳四烯酸（ETYA）不影响B淋巴细胞增殖。有趣的是，与BAY X 1005、MK-886、Wy 50,295、ETI和ETYA不同，NDGA和七叶亭是抗氧化剂。我们的假设是NDGA和七叶亭的抗氧化活性是抑制B淋巴细胞活化和增殖的原因，并且我们推测这两个过程都需要ROS而不是LO活性。其他抗氧化剂如丁基羟基甲苯、邻菲罗啉、硫脲和α-生育酚（维生素E）也抑制由LPS或mIg受体诱导的B淋巴细胞增殖。这些试剂的IC50分别为1×10（-8）M、5×10（-10）M、6×10（-3）M和5×10（-5）M。当静止的B淋巴细胞用ROS源（1×10（-5）M H2O2）处理时，细胞以温度敏感的方式增大，这与LPS诱导的增大相似。NDGA和七叶亭都完全抑制H2O2诱导的增大。这些结果进一步表明B淋巴细胞活化和增殖需要ROS。B淋巴细胞分化也得到了类似的结果。NDGA和七叶亭完全抑制浆细胞的发育，IC50为5×10（-6）M。相反，BAY X 1005、MK-886、Wy 50,295、ETI和ETYA不阻断浆细胞的形成。因此，ROS对于分化为浆细胞也至关重要。这些实验首次直接表明细胞内ROS介导B淋巴细胞的活化、增殖和分化，并且这些过程不需要LO产物。（摘要截于400字）
