Ehrenwald E, Fox P L
Department of Cell Biology, Cleveland Clinic Research Institute, Ohio 44195, USA.
J Clin Invest. 1996 Feb 1;97(3):884-90. doi: 10.1172/JCI118491.
Oxidation of lipids and lipoproteins by macrophages is an important event during atherogenesis. Activation of monocytic cells by zymosan and other agonists results in the release of multiple oxidant species and consequent oxidation of LDL. We now show evidence that ceruloplasmin, a copper-containing acute phase reactant, is secreted by zymosan-activated U937 monocytic cells, and that the protein has an important role in LDL oxidation by these cells. In one approach, ceruloplasmin has been shown to exhibit oxidant activity under the appropriate conditions. Exogenous addition of purified human ceruloplasmin stimulates U937 cell oxidation of LDL to nearly the same extent as activation by zymosan. In contrast to previous cell-free experiments (Ehrenwald, E., G.M. Chisom, and P.L. Fox. 1994. Intact human ceruloplasmin oxidatively modifies low density lipoprotein. J. Clin. Invest. 93:1493-1501.) in which ceruloplasmin by itself (in PBS) oxidizes LDL, under the conditions of the current experiments (in RPMI 1640 medium) ceruloplasmin only oxidizes LDL in the presence of cells; the mechanism by which cells overcome the inhibition by medium components has not been ascertained. As further evidence for a role of ceruloplasmin, activation of U937 cells with zymosan induces ceruloplasmin mRNA and ceruloplasmin protein synthesis after a 5-6 h lag that is consistent with that preceding LDL oxidation. Finally, neutralization by a highly specific polyclonal antibody to human ceruloplasmin inhibits LDL oxidation by at least 65%. Moreover, multiple antisense oligodeoxynucleotides targeted to different regions of the ceruloplasmin mRNA block LDL oxidation by up to 95%. The specific action of the antisense oligonucleotides has been verified by showing inhibition of ceruloplasmin synthesis and by the ability of exogenous ceruloplasmin to overcome the inhibition. In summary, these results are consistent with a mechanism in which cell-derived ceruloplasmin participates in oxidation of LDL by U937 monocytic cells. The data also show that cellular factors in addition to ceruloplasmin, possibly active oxygen species and/or lipoxygenases, are essential and act synergistically with ceruloplasmin to oxidize LDL.
巨噬细胞对脂质和脂蛋白的氧化是动脉粥样硬化形成过程中的一个重要事件。酵母聚糖和其他激动剂激活单核细胞会导致多种氧化剂的释放，进而使低密度脂蛋白（LDL）发生氧化。我们现在有证据表明，铜蓝蛋白（一种含铜的急性期反应物）由酵母聚糖激活的U937单核细胞分泌，并且该蛋白在这些细胞对LDL的氧化过程中起重要作用。一方面，已证明铜蓝蛋白在适当条件下具有氧化活性。外源性添加纯化的人铜蓝蛋白刺激U937细胞对LDL的氧化，其程度与酵母聚糖激活的程度几乎相同。与先前的无细胞实验（Ehrenwald, E., G.M. Chisom, and P.L. Fox. 1994. Intact human ceruloplasmin oxidatively modifies low density lipoprotein. J. Clin. Invest. 93:1493 - 1501.）不同，在先前的实验中铜蓝蛋白自身（在磷酸盐缓冲液中）就能氧化LDL，而在当前实验条件下（在RPMI 1640培养基中），铜蓝蛋白只有在细胞存在时才能氧化LDL；细胞克服培养基成分抑制作用的机制尚未确定。作为铜蓝蛋白发挥作用的进一步证据，用酵母聚糖激活U937细胞会在延迟5 - 6小时后诱导铜蓝蛋白mRNA和铜蓝蛋白的合成，这与LDL氧化之前的情况一致。最后，用人铜蓝蛋白的高度特异性多克隆抗体进行中和可使LDL氧化至少抑制65%。此外，针对铜蓝蛋白mRNA不同区域的多个反义寡脱氧核苷酸可使LDL氧化的抑制率高达95%。反义寡核苷酸的特异性作用已通过抑制铜蓝蛋白合成以及外源性铜蓝蛋白克服抑制作用的能力得到验证。总之，这些结果与一种机制相符，即细胞来源的铜蓝蛋白参与U937单核细胞对LDL的氧化。数据还表明，除铜蓝蛋白外的细胞因子，可能是活性氧和/或脂氧合酶，对于LDL氧化是必不可少的，并且与铜蓝蛋白协同作用来氧化LDL。
