Oxygen radical-induced erythrocyte hemolysis by neutrophils. Critical role of iron and lactoferrin.
Oxygen radical-induced erythrocyte hemolysis by neutrophils. Critical role of iron and lactoferrin.
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DOI：
10.1172/jci112095
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发表时间：
1985-09
期刊：
影响因子：
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通讯作者：
G. Vercellotti;B S van Asbeck;H. S. Jacob
中科院分区：
文献类型：
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作者：
G. Vercellotti;B S van Asbeck;H. S. Jacob
人中性粒细胞（PMN），当被诸如佛波醇肉豆蔻酸酯（PMA）的趋化因子刺激时，破坏红细胞和其他靶点。细胞毒性取决于中性粒细胞产生的活性氧代谢产物，但确切的有毒物质及其生产模式是一个有争议的问题。使用51铬标记的红细胞作为目标，我们比较了各种反应性O2产生系统的能力，溶解红细胞，以及氧化血红蛋白高铁血红蛋白。将PMA激活的PMN或黄嘌呤氧化酶加乙醛以提供类似水平的超氧化物的量添加到靶红细胞。中性粒细胞裂解68.3 +/- 2.9%（SEM）的靶细胞，而黄嘌呤氧化酶系统几乎无效（2.3 +/- 0.8%）。与此相反，高铁血红蛋白的形成黄嘌呤氧化酶加乙醛显着大于刺激的中性粒细胞（P <0.001）。添加试剂H2 O2或H2 O2生成系统（葡萄糖加葡萄糖氧化酶）也出现了类似的二分法;这两种情况都不会导致51 Cr释放，但会诱导10-70%高铁血红蛋白形成。因此，虽然O2-和H2 O2可以穿过红细胞膜并迅速氧化血红蛋白，但它们显然不会损伤细胞膜。中性粒细胞的颗粒成分是促进靶细胞溶解所必需的，这一事实表明，无颗粒的中性粒细胞胞质（胞质），虽然加入产生等量的O2-作为完整的中性粒细胞，对靶红细胞的溶解显着减少（P <0.01）。铁被证明是直接参与溶解效率的补充研究与2 μ M的柠檬酸铁，这种补充增加PMN的细胞毒性约30%，但红细胞裂解的叶绿体（约3%的增加）的影响要小得多，并没有影响裂解酶的氧自由基生成系统。这些结果表明一个铁liganding部分，是大量存在于中性粒细胞中的一个关键的作用，所以在血小板，而不是在所有纯化的酶系统-一个部分，我们推测催化非常有毒的O2物种的产生在附近的并列红细胞的目标。明显的候选者是乳铁蛋白（LF），事实上，抗乳铁蛋白IgG（而不是非特异性IgG）可将PMN细胞毒性降低85%以上。再加入10（-8）M纯LF到原生质体中，使其促进溶血的能力增加了48.4 +/- 0.9%--接近完整PMN的水平。我们得出结论，O-2和H2 O2是不足以介导靶细胞裂解，但需要铁结合LF，这反过来，可能会产生和集中有毒的O2自由基，如OH，目标膜网站。
Human neutrophils (PMN), when stimulated with such chemotaxins as phorbol myristate acetate (PMA), destroy erythrocytes and other targets. Cytotoxicity depends on PMN-generated reactive oxygen metabolites, yet the exact toxic specie and its mode of production is a matter of some dispute. Using 51Cr-labeled erythrocytes as targets, we compared various reactive-O2 generating systems for their abilities to lyse erythrocytes as well as to oxidize hemoglobin to methemoglobin. PMA-activated PMNs or xanthine oxidase plus acetaldehyde were added to target erythrocytes in amounts that provided similar levels of superoxide. PMNs lysed 68.3 +/- 2.9% (SEM) of targets, whereas the xanthine oxidase system was virtually impotent (2.3 +/- 0.8%). In contrast, methemoglobin formation by xanthine oxidase plus acetaldehyde was significantly greater than that caused by stimulated PMNs (P less than 0.001). A similar dichotomy was noted with added reagent H2O2 or the H2O2-generating system, glucose plus glucose oxidase; neither of these caused 51Cr release, but induced 10-70% methemoglobin formation. Thus, although O2- and H2O2 can cross the erythrocyte membrane and rapidly oxidize hemoglobin, they do so evidently without damaging the cell membrane. That a granule constituent of PMNs is required to promote target cell lysis was suggested by the fact that agranular PMN cytoplasts (neutroplasts), although added to generate equal amounts of O2- as intact PMNs, were significantly less lytic to target erythrocytes (P less than 0.01). Iron was shown to be directly involved in lytic efficiency by supplementation studies with 2 microM iron citrate; such supplementation increased PMN cytotoxicity by approximately 30%, but had much less effect on erythrocyte lysis by neutroplasts (approximately 3% increase), and no effect on lysis in the enzymatic oxygen radical-generating systems. These results suggest a critical role for an iron-liganding moiety that is abundantly present in PMN, marginally so in neutroplasts, and not at all in purified enzymatic systems--a moiety that we presume catalyzes very toxic O2 specie generation in the vicinity of juxtaposed erythrocyte targets. The obvious candidate is lactoferrin (LF), and indeed, antilactoferrin IgG, but not nonspecific IgG, reduced PMN cytotoxicity by greater than 85%. Re-adding 10(-8) M pure LF to neutroplasts increased their ability to promote hemolysis by 48.4 +/- 0.9%--to a level near that of intact PMNs. We conclude that O-2 and H2O2 are not sufficient to mediate target cell lysis, but require iron bound to LF, which, in turn, probably generates and focuses toxic O2 radicals, such as OH, to target membrane sites.