Morgan Deri, Capasso Melania, Musset Boris, Cherny Vladimir V, Ríos Eduardo, Dyer Martin J S, DeCoursey Thomas E
Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL 60612, USA.
Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):18022-7. doi: 10.1073/pnas.0905565106. Epub 2009 Oct 5.
Phagocytosis of microbial invaders represents a fundamental defense mechanism of the innate immune system. The subsequent killing of microbes is initiated by the respiratory burst, in which nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates vast amounts of superoxide anion, precursor to bactericidal reactive oxygen species. Cytoplasmic pH regulation is crucial because NADPH oxidase functions optimally at neutral pH, yet produces enormous quantities of protons. We monitored pH(i) in individual human neutrophils during phagocytosis of opsonized zymosan, using confocal imaging of the pH sensing dye SNARF-1, enhanced by shifted excitation and emission ratioing, or SEER. Despite long-standing dogma that Na(+)/H(+) antiport regulates pH during the phagocyte respiratory burst, we show here that voltage-gated proton channels are the first transporter to respond. During the initial phagocytotic event, pH(i) decreased sharply, and recovery required both Na(+)/H(+) antiport and proton current. Inhibiting myeloperoxidase attenuated the acidification, suggesting that diffusion of HOCl into the cytosol comprises a substantial acid load. Inhibiting proton channels with Zn(2+) resulted in profound acidification to levels that inhibit NADPH oxidase. The pH changes accompanying phagocytosis in bone marrow phagocytes from HVCN1-deficient mice mirrored those in control mouse cells treated with Zn(2+). Both the rate and extent of acidification in HVCN1-deficient cells were twice larger than in control cells. In summary, acid extrusion by proton channels is essential to the production of reactive oxygen species during phagocytosis.
吞噬微生物入侵者是先天免疫系统的一种基本防御机制。随后对微生物的杀伤由呼吸爆发启动，在呼吸爆发过程中，烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶产生大量超氧阴离子，这是杀菌性活性氧的前体。细胞质pH调节至关重要，因为NADPH氧化酶在中性pH下功能最佳，但会产生大量质子。我们使用pH传感染料SNARF-1的共聚焦成像监测了调理酵母聚糖吞噬过程中单个人类中性粒细胞的细胞内pH（pH(i)），通过移动激发和发射比率增强成像，即SEER成像。尽管长期以来的观点认为，在吞噬细胞呼吸爆发期间，Na(+)/H(+)反向转运体调节pH，但我们在此表明电压门控质子通道是首先做出反应的转运体。在最初的吞噬事件中，pH(i)急剧下降，恢复需要Na(+)/H(+)反向转运体和质子电流。抑制髓过氧化物酶可减弱酸化作用，这表明次氯酸扩散到细胞质中构成了大量的酸负荷。用Zn(2+)抑制质子通道会导致深度酸化，达到抑制NADPH氧化酶的水平。来自HVCN1缺陷小鼠的骨髓吞噬细胞在吞噬过程中伴随的pH变化与用Zn(2+)处理的对照小鼠细胞中的变化相似。HVCN1缺陷细胞中的酸化速率和程度均是对照细胞的两倍。总之，质子通道介导的酸外排对于吞噬过程中活性氧的产生至关重要。
