title: Effect of azathioprine on Na(+)/H(+) exchanger activity in dendritic cells.
abstract: Azathioprine is a powerful immunosuppressive drug, which is partially effective by interfering with the maturation and function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs are stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS), paralleled by activation of the Na(+)/H(+) exchanger. The carrier is involved in the regulation of cytosolic pH, cell volume and migration. The present study explored whether azathioprine influences Na(+)/H(+) exchanger activity in DCs. DCs were isolated from murine bone marrow, cytosolic pH (pH(i)) was estimated utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) fluorescence, Na(+)/H(+) exchanger activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNFα release utilizing ELISA, and migration utilizing transwell migration assays. Exposure of DCs to lipopolysaccharide (LPS, 1 μg/ml) led to a transient increase of Na(+)/H(+) exchanger activity, an effect paralleled by ROS formation, increased cell volume, TNFα production and stimulated migration. Azathioprine (10 μM) did not significantly alter the Na(+)/H(+) exchanger activity, cell volume and ROS formation prior to LPS exposure but significantly blunted the LPS-induced stimulation of Na(+)/H(+) exchanger activity, ROS formation, cell swelling, TNFα production and cell migration. In conclusion, azathioprine interferes with the activation of dendritic cell Na(+)/H(+) exchanger by bacterial lipopolysaccharides, an effect likely participating in the anti-inflammatory action of the drug.
authors.affiliation: Department of Physiology, University of Tübingen, Tübingen, Germany.
authors.forename: Madhuri
authors.initials: M
authors.lastname: Bhandaru
authors.forename: Venkanna
authors.initials: V
authors.lastname: Pasham
authors.forename: Wenting
authors.initials: W
authors.lastname: Yang
authors.forename: Diwakar
authors.initials: D
authors.lastname: Bobbala
authors.forename: Anand
authors.initials: A
authors.lastname: Rotte
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D000818:Animals; D001379:Azathioprine / Q000494:pharmacology*; D002465:Cell Movement; D048429:Cell Size; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D003713:Dendritic Cells / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D017382:Reactive Oxygen Species / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D005452:Fluoresceins; D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; D017923:Sodium-Hydrogen Exchangers; D014409:Tumor Necrosis Factor-alpha; C029569:diacetyldichlorofluorescein; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D001379:Azathioprine