Functional role of cation-chloride cotransporter in neutrophil activation

• Main Authors: Jun-Hao Liang, 梁峻豪
• Other Authors: Meng-Ru Shen
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/54821624838863078346

碩士 === 國立成功大學 === 藥理學研究所 === 97 === Polymorphonuclear neutrophils (PMNs), also referred to as neutrophils, are the most abundant and important type of white blood cells and form an essential part of the immune system. Several studies indicated that there is a dramatic and irreversible efflux of intracellular chloride during neutrophil phagocytosis. This chloride movement leads to high chloride concentration in phagosome or outermembrane microenvironment in order to interact with hydrogen peroxide (H2O2) to produce hypochlorous acid (HOCl) that is the most critical bacterial killing chemical. Some literatures show that the H+/ 2Cl- antiporter chloride channel 7 (ClC-7) is the primary chloride permeation pathway in lysosome, that can fuse with phagosome. ClC-3, another member of ClC family regulates phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to generate oxidants after endotoxin priming. Other studies proved that the neutrophils isolated from cystic fibrosis (CF) patients have poor bacterial killing activity due to dysfunction of CF transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. On the other hand, the K+ influx of the phagosomal membrane makes the vacuole hypertonic and it is suitable for activating granule enzyme. The identity of this protein which can mediate K+ influx is controversial. All above results suggest that the Cl- and K+ transport systems are important for NADPH oxidase activity or HOCl generation. However, it is not clear about the dominant Cl- and K+ permeation pathway for the Cl- and K+ movements during neutrophil activation. So far, all researches focused on the relationship between chloride channels and neutrophil function. This study aims to investigate the role of cation-chloride cotransporter (CCC) including K-Cl cotransport (KCC) and Na-K-Cl cotransport (NKCC) during neutrophil activation. We at first examined the role of cation-chloride cotransporters in NADPH oxidase activation by utilizing different cation-chloride cotransport inhibitors and lucigenin-enhanced chemiluminescence assay. DIOA, the KCC blocker inhibits phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP) or lipopolysaccharides-induced NADPH activity of human neutrophils. In contrast, bumetanide, the NKCC blocker does not inhibit this activation. This implies KCC, instead of NKCC plays a functional role in PMA, fMLP or lipopolysaccride-induced NADPH activity of human neutrophils. Western blotting and RT-PCR showed that human neutrophils express all KCC isoforms (KCC1-KCC4). In addition, PMA-induced isotopic 36Cl and 86Rb efflux can be abolished by DIOA. This result indicated KCC mediates ion movements of PMA-induced Cl- and K+ efflux in human neutrophils. Besides, we found that KCC expresses and is important for PMA or fMLP-induced NADPH activity in differentiated neutrophil-like HL-60 cells (dHL-60). Consistently, KCC is necessary for PMA or fMLP-induced NADPH activity in murine neutrophils by chemiluminescence assay. Finally, we found that KCC3 knockout PMNs have poor PMA-induced NADPH oxidase activity compare to wildtype PMNs. That indicated KCC3 participates in the activation. This study indicated that KCC can regulate NADPH oxidase activity and provides K+ and Cl- movements which are necessary for bacterial killing activity of PMNs.