CFTR Expression and Function in Human Neutrophils

描述(申请人提供)：中性粒细胞(PMN或中性粒细胞)，负责清除病原体和细胞碎片的主要吞噬细胞，合成并维持丰富的髓过氧化物酶(MPO)。这种特殊的氧化酶将过氧化氢和氯离子转化为次氯酸(HOCI)，次氯酸是最有效的杀菌氧化剂之一。尽管如果MPO被分泌或泄漏，这种反应可以发生在细胞外，但它主要包含在PMN吞噬酶体中。我们的初步数据和文献表明，囊性纤维化跨膜传导调节因子(CFTR)是一种cAMP调节的氯离子通道，在中性粒细胞及其吞噬酶体内表达。重要的是，囊性纤维化(CF)患者中性粒细胞中的基因缺陷会损害吞噬溶酶体中Hoci的产生。这种缺陷会影响对吞噬细菌的氯化和杀灭。基于这些发现，我们假设CFTR在调节吞噬溶酶体中的氯水平方面起着关键作用。因此，CFTR的功能障碍可能会影响该细胞器对氯的利用率，从而影响PMN的杀菌能力。这项建议的长期目标是确认CFTR在PMN中的表达及其在吞噬溶酶体的氯离子分泌中的功能。我们提出了四个具体的目标：1)鉴定CFTR在正常人和CF患者静止期中性粒细胞中的亚细胞定位；2)鉴定正常人和CF中性粒细胞分离的吞噬溶酶体中CFTR的功能；3)检测CFTR在调节活的中性粒细胞吞噬溶酶体氯浓度中的作用；以及4)比较正常、CF和MPO缺乏的中性粒细胞的杀菌能力。通过完成这四个特定的目标，我们将了解cftr在氯离子分泌到吞噬溶酶体中的重要性。这些研究将扩展我们目前对CFTR在非上皮细胞中所起作用的理解，并建立起功能失调的氯通道功能与中性粒细胞杀菌能力之间的联系。显然，所获得的知识与寻找治疗CF的有效疗法高度相关，CF是高加索人最致命的遗传病。

DESCRIPTION (provided by applicant): Neutrophilic polymorphonuclear leukocytes (PMNs or neutrophils), the main phagocytes responsible for elimination of pathogens and cell debris, synthesize and maintain abundant myeloperoxidase (MPO). This special oxidase converts hydrogen peroxide and chloride anion to hypochlorous acid (HOCI), one of the most potent oxidants for bacterial killing. Even though this reaction can occur extracellularly if MPO is secreted or leaked out, it is largely contained within PMN phagolysosomes. Our preliminary data and publication have demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP- regulated chloride channel, is expressed in neutrophils and their phagolysosomes. Importantly, the gene defects in PMNs from cystic fibrosis (CF) patients have an impaired HOCI production in phagolysosomes. Such a deficiency affects the chlorination and killing of phagocytosed bacteria. Based on these findings, we hypothesize that CFTR plays a pivotal role in regulating chloride levels in phagolysosomes. Therefore, a dysfunctional CFTR in CF may affect the chloride availability to this organelle, thus compromising the bacterial killing capacity of the PMNs. The long-term objective of this proposal is to confirm CFTR expression in PMNs and its function in chloride secretion to phagolysosomes. We put forward four specific aims: 1) to identify the subcellular localization of CFTR in resting neutrophils from normal subjects and CF patients; 2) to characterize CFTR function in isolated phagolysosomes from normal and CF neutrophils; 3) to examine the role of CFTR in regulation of phagolysosomal chloride concentration in live neutrophils; and 4) to compare the microbicidal abilities of normal, CF and MPO-deficient neutrophils. By completion of the four specific aims, we will understand the importance of CFTR in secretion of chloride anion to phagolysosomes. Such studies will extend our current understanding of the role played by CFTR in non-epithelial cells and establish the link of a dysfunctional chloride channel function with the neutrophil bacterial killing capacity. Apparently, the obtained knowledge is highly relevant to searching effective therapies for the treatment of CF, the most fatal genetic disease in Caucasians.