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Am. J. Respir. Cell Mol. Biol., Volume 26, Number 1, January, 2002 114-126

Amiloride Blockades Lipopolysaccharide-Induced Proinflammatory Cytokine Biosynthesis in an Ikappa B-alpha /NF-kappa B-Dependent Mechanism
Evidence for the Amplification of an Antiinflammatory Pathway in the Alveolar Epithelium

John J. Haddad and Stephen C. Land

Oxygen Signaling Group, Center for Research into Human Development, Tayside Institute of Child Health, Faculty of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, Scotland, United Kingdom

It has been previously reported that amiloride suppresses inflammatory cytokine biosynthesis. However, the molecular mechanism involved has yet to be ascertained. Therefore, the immunoregulatory potential mediated by amiloride and the underlying signaling transduction pathway was investigated. Exposure of alveolar epithelial cells to amiloride or its analog, 5-(N,N-hexamethylene)-amiloride (HMA), reduced, in a dose-dependent manner, lipopolysaccharide (LPS)-induced secretion of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha . This inhibitory effect was associated with the augmentation of a counter antiinflammatory response, mediated by IL-6 and IL-10. Analysis of the mechanism implicated revealed the involvement of an inhibitory kappa B (Ikappa B-alpha )/nuclear factor kappa B (NF- kappa B)-sensitive pathway. Amiloride and HMA suppressed the phosphorylation of Ikappa B-alpha mediated by LPS, thereby allowing its cytosolic accumulation. Furthermore, both inhibitors interfered with the nuclear translocation of selective NF-kappa B subunits, an effect associated with blockading the DNA-binding activity of NF-kappa B. Recombinant IL-10 blockaded LPS-induced biosynthesis of IL-1beta and TNF-alpha and reduced NF-kappa B activation. Immunoneutralization of endogenous IL-10 reversed the inhibitory effect of amiloride on proinflammatory cytokines and restored the DNA-binding activity of NF-kappa B. These results indicate that amiloride acts as a novel dual immunoregulator in the alveolar epithelium: it downregulates an inflammatory signal and at the same time upregulates an antiinflammatory response. This biphasic effect is IL-10 sensitive and is associated with the selective targeting of the Ikappa B-alpha /NF-kappa B signaling transduction pathway.

Abbreviations: one-way analysis of variance, ANOVA; bovine serum albumin, BSA; Dulbecco's modified Eagle's medium, DMEM; enzyme-linked immunosorbent assay, ELISA; fetal calf serum, FCS; Hanks' balanced salt solution, HBSS; 5-(N,N-hexamethylene)-amiloride, HMA; inhibitory kappa B, Ikappa B; inhibitory kappa B kinase, IKK; interleukin, IL; lipopolysaccharide, LPS; mitogen-activated protein kinase, MAPK; nuclear factor kappa B, NF-kappa B; NF-kappa B inducing kinase, NIK; phosphate-buffered saline, PBS; sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE; superoxide anion, O2-·; tumor necrosis factor-alpha , TNF-alpha .




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Copyright © 2002 American Thoracic Society.