title: Interleukin-10 stabilizes inhibitory kappaB-alpha in human monocytes.
abstract: Interleukin-10 (IL-10) protects animals from lethal endotoxemia. This beneficial effect is mediated, in part, by inhibition of inflammatory cytokine production, including tumor necrosis factor-alpha (TNF-alpha). Evidence suggests that IL-10 may inhibit activation of the transcription factor nuclear factor-kappaB (NF-kappaB) through an unknown mechanism. NF-kappaB activation in response to inflammatory signals is dependent upon degradation of its associated inhibitory peptide, inhibitory kappaB-alpha (IkappaB-alpha). We hypothesized that IL-10 prevents human monocyte NF-kappaB activation and resultant TNF-alpha production by stabilization of IkappaB-alpha. The purpose of this study was to determine the effect of IL-10 on lipopolysaccharide (LPS)-induced human monocyte TNF-alpha production, NF-kappaB activation, and IkappaB-alpha degradation. Monocytes were isolated from human donors. Cells were stimulated with endotoxin (LPS, 100 ng/mL) with and without human IL-10 (10 ng/mL). Following stimulation, TNF-alpha was measured in cell supernatants by ELISA, NF-kappaB activity by electrophoretic mobility shift assay, and IkappaB-alpha levels by Western blot. We observed that after LPS stimulation of human monocytes, TNF-alpha increased to 798+/-67 pg/mL (p < .001 versus control). IL-10 attenuated LPS-stimulated TNF-alpha production (297+/-54; p < .001 versus LPS alone). After LPS stimulation in human monocytes, IkappaB-alpha protein levels decreased, and NF-kappaB DNA binding increased. IL-10 pretreatment prevented LPS-induced decreases in IkappaB-alpha protein levels and attenuated NF-kappaB DNA binding. IL-10 appears to prevent activation of NF-kappaB by preserving IkappaB-alpha protein levels, leading to a reduction in TNF-alpha release.
authors.affiliation: Department of Surgery, University of Colorado Health Sciences Center, Denver 80262, USA.
authors.forename: B D
authors.initials: BD
authors.lastname: Shames
authors.forename: C H
authors.initials: CH
authors.lastname: Selzman
authors.forename: D R
authors.initials: DR
authors.lastname: Meldrum
authors.forename: E J
authors.initials: EJ
authors.lastname: Pulido
authors.forename: H A
authors.initials: HA
authors.lastname: Barton
authors.forename: X
authors.initials: X
authors.lastname: Meng
authors.forename: A H
authors.initials: AH
authors.lastname: Harken
authors.forename: R C
authors.initials: RC
authors.lastname: McIntyre
mesh_terms: D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019446:Endotoxemia / Q000517:prevention & control; D006801:Humans; D020672:I-kappa B Proteins; D066298:In Vitro Techniques; D016753:Interleukin-10 / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000633:toxicity; D009000:Monocytes / Q000187:drug effects* / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA