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. ? ? ?
