Briggs R T, Karnovsky M L, Karnovsky M J
J Clin Invest. 1977 Jun;59(6):1088-98. doi: 10.1172/JCI108732.
The ability of polymorphonuclear leukocytes (PMN) to produce H(2)O(2) in response to phagocytic stimulation was examined cytochemically using leukocytes from normal individuals and patients with chronic granulomatous disease (CGD). Normal PMN oxidized diaminobenzidine within the phagocytic vacuole by a reaction dependent upon endogenous H(2)O(2) and myeloperoxidase. CGD PMN failed to oxidize diaminobenzidine, which is consistent with the biochemical data showing a lack of H(2)O(2)-generating capacity. A plasma membrane enzyme (oxidase) activated by phagocytosis is capable of H(2)O(2) production in PMN. The localization of this oxidase activity was explored in CGD PMN using a cytochemical technique specific for H(2)O(2). The enzyme activity is stimulated by exogenous NADH, but not NADPH. Reaction product formation, indicative of activity of the oxidase, is dependent upon precipitation of cerium ions by the enzymatically generated H(2)O(2). The advantage of this approach is that enzyme activity of individual cells can be assessed, allowing determination of numbers of reactive cells in the population and their relative degrees of reactivity. NADH oxidase was found to be active both on the plasma membrane and within the phagocytic vacuoles of control PMN, whereas those cells from three CGD patients showed greatly reduced activity in both these sites. Assessment of the reactivity of individual cells showed the number of cells with oxidase activity in CGD to be significantly reduced when compared to control values. Additionally, of those cells that do react, a higher percentage of them are only weakly reactive. Omission of NADH from the incubation medium reduced the percentage of control cells showing enzyme activity but had no effect on CGD PMN, implying that the enzyme is not saturated with substrate in control cells, but in CGD the diminished enzyme is fully saturated. The defect may lie in the fact that in CGD patients there are fewer cells capable of peroxide generation, and a majority of these reactive cells produce only reduced amounts of this bactericidal agent.
利用正常个体和慢性肉芽肿病（CGD）患者的白细胞，通过细胞化学方法检测了多形核白细胞（PMN）在吞噬刺激下产生H₂O₂的能力。正常PMN通过依赖内源性H₂O₂和髓过氧化物酶的反应，使吞噬泡内的二氨基联苯胺氧化。CGD患者的PMN不能氧化二氨基联苯胺，这与生化数据显示缺乏H₂O₂生成能力一致。一种由吞噬作用激活的质膜酶（氧化酶）能够在PMN中产生H₂O₂。利用一种对H₂O₂特异的细胞化学技术，在CGD患者的PMN中探索了这种氧化酶活性的定位。该酶活性受外源性NADH刺激，但不受NADPH刺激。氧化酶活性的反应产物形成依赖于酶促产生的H₂O₂使铈离子沉淀。这种方法的优点是可以评估单个细胞的酶活性，从而确定群体中反应性细胞的数量及其相对反应程度。发现NADH氧化酶在对照PMN的质膜和吞噬泡内均有活性，而三名CGD患者的细胞在这两个部位的活性均大大降低。对单个细胞反应性的评估显示，与对照值相比，CGD中具有氧化酶活性的细胞数量显著减少。此外，在那些有反应的细胞中，较高比例的细胞仅具有弱反应性。在孵育培养基中省略NADH可降低显示酶活性的对照细胞百分比，但对CGD患者的PMN无影响，这意味着对照细胞中的酶未被底物饱和，而在CGD中，减少的酶已完全饱和。缺陷可能在于，CGD患者中能够产生过氧化物的细胞较少，并且这些反应性细胞中的大多数仅产生少量这种杀菌剂。
