title: Rapamycin sensitive ROS formation and Na(+)/H(+) exchanger activity in dendritic cells.
abstract: Rapamycin, a widely used immunosuppressive drug, has been shown to interfere with the function of dendritic cells (DCs), antigen-presenting cells contributing to the initiation of primary immune responses and the establishment of immunological memory. DC function is governed by the Na(+)/H(+) exchanger (NHE), which is activated by bacterial lipopolysaccharides (LPS) and is required for LPS-induced cell swelling, reactive oxygen species (ROS) production and TNF-α release. The present study explored, whether rapamycin influences NHE activity and/or ROS formation in DCs. Mouse DCs were treated with LPS in the absence and presence of rapamycin (100 nM). ROS production was determined from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, and TNF-α production utilizing ELISA. In the absence of LPS, rapamycin did not significantly modify cytosolic pH, NHE activity or cell volume but significantly decreased ROS formation. LPS stimulated NHE activity, enhanced forward scatter, increased ROS formation, and triggered TNF-α release, effects all blunted in the presence of rapamycin. NADPH oxidase inhibitor Vas-2870 (10 μM) mimicked the effect of rapamycin on LPS induced stimulation of NHE activity and TNF-α release. The effect of rapamycin on TNF-α release was also mimicked by the antioxidant ROS scavenger Tempol (30 μM) and partially reversed by additional application of tert-butylhydroperoxide (10 μM). In conclusion, in DCs rapamycin disrupts LPS induced ROS formation with subsequent inhibition of NHE activity, cell swelling and TNF-α release.
authors.affiliation: Department of Physiology, University of Tübingen, Tübingen, Germany.
authors.forename: Anand
authors.initials: A
authors.lastname: Rotte
authors.forename: Venkanna
authors.initials: V
authors.lastname: Pasham
authors.forename: Madhuri
authors.initials: M
authors.lastname: Bhandaru
authors.forename: Diwakar
authors.initials: D
authors.lastname: Bobbala
authors.forename: Christine
authors.initials: C
authors.lastname: Zelenak
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D000818:Animals; D001583:Benzoxazoles / Q000494:pharmacology; D048429:Cell Size; D002478:Cells, Cultured; D003497:Cyclic N-Oxides / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D003713:Dendritic Cells / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry; 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*; D020123:Sirolimus / Q000494:pharmacology*; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D013113:Spin Labels; D014230:Triazoles / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
chemical_list: C511179:3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo(4,5-d)pyrimidine; D001583:Benzoxazoles; D003497:Cyclic N-Oxides; D005452:Fluoresceins; D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; C480499:Slc9c1 protein, mouse; D017923:Sodium-Hydrogen Exchangers; D013113:Spin Labels; D014230:Triazoles; D014409:Tumor Necrosis Factor-alpha; C029569:diacetyldichlorofluorescein; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D020122:tert-Butylhydroperoxide; C001803:tempol; D020123:Sirolimus