Abstract
NADPH oxidase activity, phagocytosis, and cell migration are essential functions of polymorphonuclear leukocytes (PMNs) in host defense. The cytoskeletal reorganization necessary to perform these functions has been extensively studied, but the role of cell volume regulation, which is likely dependent upon anion channels, has not been defined. Mice lacking the anion channel ClC-3 (Clcn3(-/-)) died from presumed sepsis following intravascular catheter placement, whereas Clcn3(+/+) littermates survived. We hypothesized that ClC-3 has a critical role in host defense and reasoned that PMN function would be compromised in these mice. Clcn3(-/-) PMNs displayed markedly reduced NADPH oxidase activity in response to opsonized zymosan and modestly reduced activity after phorbol 12-myristate 13-acetate. Human PMNs treated with the anion channel inhibitors niflumic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid had a very similar defect. ClC-3 protein was detected in the secretory vesicles and secondary granules of resting PMNs and was up-regulated to the phagosomal membrane. Clcn3(-/-) PMNs and human PMNs lacking normal anion channel function both exhibited reduced uptake of opsonized zymosan at 1, 5, and 10 min in a synchronized phagocytosis assay. Niflumic acid-treated PMNs also had impaired transendothelial migration in vitro, whereas migration in vivo was not altered in Clcn3(-/-) PMNs. Selective inhibition of the swelling-activated chloride channel with tamoxifen profoundly reduced PMN migration but had no effect on NADPH oxidase activity. In summary, PMNs lacking normal anion channel function exhibited reduced NADPH oxidase activity, diminished phagocytosis, and impaired migration. ClC-3 was specifically involved in the respiratory burst and phagocytosis.
Highlights
The current investigation suggests that anion channels in general, and ClC-3 in particular, are critical regulators of several PMN responses
Functional alterations in human PMNs treated with each of the three anion channel inhibitors are compared with control PMNs
We acknowledge that the data presented do not offer a definitive explanation for the abnormal immune phenotype of the Clcn3(Ϫ/Ϫ) mice, we have defined several elements of the cell biology of neutrophil function that require anion channels
Summary
Hanks’ Balanced Salt Solution (HBSS), Hepes-RPMI, L-glutamine, and Dulbecco’s phosphate-buffered saline (DPBS) were obtained from BioWhittaker (Walkersville, MD). Hanks’ buffers made with alternative anions for studies of anion selectivity were made in sterile water as follows (concentrations in mM): NaCl, NaBr, or NaI (138 mM); KCl 5 mM, CaCl2 (1 mM), MgSO41⁄7H2O (0.4 mM), Na2HPO41⁄72H2O (0.4 mM), K2HPO41⁄72H2O (0.4 mM) MgCl1⁄76H2O (0.5 mM), and NaHCO3 (4.2 mM). 1% human serum albumin and 0.1% dextrose were added, and pH was adjusted to 7.4 with HCl. Hanks’ buffers made with alternative anions for studies of anion selectivity were made in sterile water as follows (concentrations in mM): NaCl, NaBr, or NaI (138 mM); KCl 5 mM, CaCl2 (1 mM), MgSO41⁄7H2O (0.4 mM), Na2HPO41⁄72H2O (0.4 mM), K2HPO41⁄72H2O (0.4 mM) MgCl1⁄76H2O (0.5 mM), and NaHCO3 (4.2 mM). Murine PMNs were separated from the collected peritoneal leukocyte population by Hypaque-Ficoll density centrifugation as described below
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