MEASUREMENT OF SUPEROXIDE RELEASE IN THE PHAGOVACUOLES OF IMMUNE COMPLEX-STIMULATED HUMAN NEUTROPHILS
MEASUREMENT OF SUPEROXIDE RELEASE IN THE PHAGOVACUOLES OF IMMUNE COMPLEX-STIMULATED HUMAN NEUTROPHILS
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DOI：
10.1016/0022-1759(90)90052-w
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发表时间：
1990-07-03
影响因子：
2.2
通讯作者：
SIMONS, ER
中科院分区：
文献类型：
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作者：
RYAN, TC;WEIL, GJ;SIMONS, ER
免疫复合物刺激人嗜中性粒细胞，除其他事件外，还导致吞噬空泡的形成，刺激激活的氧化爆发和脱粒产物被释放到吞噬空泡中。为了监测在吞噬空泡的爆发活动，我们已经开发了这种中性粒细胞激动剂的荧光染料偶联衍生物。荧光染料2“，7”-二氯二氢荧光素（DCFH）（2“，7”-二氯荧光素（DCF）的非荧光还原形式）已与牛血清白蛋白（BSA）共价连接，可用于与抗BSA免疫球蛋白形成免疫复合物。与非衍生化对照相比，所得复合物是刺激所有免疫复合物介导的中性粒细胞应答的有效激动剂。在暴露于过氧化氢时，刺激连接的探针被转化为其氧化的、完全荧光的形式，其荧光与探针氧化的程度线性相关。使用流式细胞术，我们已经证明，探针刺激复合物是能够监测的动力学的活性氧的生产由膜结合的NADPH氧化酶，大概是在免疫复合物激活的中性粒细胞的吞噬空泡。我们已经发现，免疫复合物介导的激活的氧化爆发内的吞噬隔室之前的滞后约30秒，然后由一个大的持续释放的超氧化物依赖性过氧化氢。然而，来自慢性肉芽肿病患者的中性粒细胞的探针荧光没有持续增加，这一发现与这些细胞中缺乏氧化爆发活性一致。因此，DCFH-免疫复合物偶联物提供了一种有效的探针，用于监测活化的嗜中性粒细胞形成的吞噬空泡内氧化产物的局部释放动力学，并可用于进一步检查人嗜中性粒细胞响应“生理性"宿主防御激动剂如免疫复合物的活化和活性。
Immune complex stimulation of human neutrophils elicits, among other events, the formation of phagocytic vacuoles into which the products of the stimulus activated oxidative burst and degranulation are released. In order to monitor burst activity in the phagocytic vacuole, we have developed a fluorochrome-coupled derivative of this neutrophil agonist. The fluorochrome 2'',7''-dichlorodihydrofluorescein (DCFH) (the nonfluorescent, reduced form of 2'',7''-dichlorofluorescein (DCF)) has been covalently linked to bovine serum albumin (BSA), which can be used to form an immune complex with anti-BSA immunoglobulin. The resultant complex is an effective agonist for stimulating all immune complex-mediated neutrophil responses, as compared to nonderivatized controls. Upon exposure to hydrogen peroxide the stimulus-linked probe is converted to its oxidized, fully fluorescent form, the fluorescence of which is linearly related to the extent of probe oxidation. Using flow cytometry, we have demonstrated that the probe-stimulus complex is capable of monitoring the kinetics of the production of activated oxygen species by the membrane bound NADPH-oxidase enzyme, presumably within the phagocytic vacuoles of immune complex-activated neutrophils. We have found that the immune complex-mediated activation of the oxidative burst within the phagocytic compartment is preceded by a lag of approximately 30 s followed by a large sustained release of superoxide dependent hydrogen peroxide. Neutrophils from patients with chronic granulomatous disease, however, demonstrated no sustained increase in probe fluorescence, a finding consistent with the lack of oxidative burst actvity in these cells. The DCFH-immune complex conjugate therefore provides an effective probe for monitoring the kinetics of the localized release of oxidative products within the forming phagocytic vacuoles of activated neutrophils, and may be used to further examine both the activation and activity of human neutrophils in response to ''physiologic'' host denfense agonists such as immune complexes.