Amiloride blockades lipopolysaccharide-induced proinflammatory cytokine biosynthesis in an IkappaB-alpha/NF-kappaB-dependent mechanism. Evidence for the amplification of an antiinflammatory pathway in the alveolar epithelium.
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 kappaB (IkappaB-alpha)/nuclear factor kappaB (NF- kappaB)-sensitive pathway. Amiloride and HMA suppressed the phosphorylation of IkappaB-alpha mediated by LPS, thereby allowing its cytosolic accumulation. Furthermore, both inhibitors interfered with the nuclear translocation of selective NF-kappaB subunits, an effect associated with blockading the DNA-binding activity of NF-kappaB. Recombinant IL-10 blockaded LPS-induced biosynthesis of IL-1beta and TNF-alpha and reduced NF-kappaB activation. Immunoneutralization of endogenous IL-10 reversed the inhibitory effect of amiloride on proinflammatory cytokines and restored the DNA-binding activity of NF-kappaB. 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 IkappaB-alpha/NF-kappaB signaling transduction pathway.
Haddad
John J
JJ
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. haddadj@anesthesia.ucsf.edu
Land
Stephen C
SC
D021581:Active Transport, Cell Nucleus; D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D004232:Diuretics / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000276:immunology / Q000378:metabolism*; D005786:Gene Expression Regulation; D020672:I-kappa B Proteins; D007375:Interleukin-1 / Q000096:biosynthesis; D016753:Interleukin-10 / Q000096:biosynthesis; D015850:Interleukin-6 / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008168:Lung / Q000166:cytology / Q000196:embryology; D008954:Models, Biological; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000096:biosynthesis*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D015854:Up-Regulation
D000970:Antineoplastic Agents; D016207:Cytokines; D004268:DNA-Binding Proteins; D004232:Diuretics; D020672:I-kappa B Proteins; D007375:Interleukin-1; D015850:Interleukin-6; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606206:Nfkbia protein, rat; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D000072000:NF-KappaB Inhibitor alpha; C057360:5-(N,N-hexamethylene)amiloride; D000584:Amiloride