Inhibition of NF-kappaB activity prevents downregulation of alpha1-adrenergic receptors and circulatory failure during CLP-induced sepsis

Abstract

The reduced pressure response to norepinephrine during sepsis has directed our interest to the regulation of alpha1-adrenergic receptors. Because nuclear factor (NF)-kappaB occupies a prominent role in the inflammatory cascade, we hypothesized that NF-kappaB downregulates alpha1-receptors by liberation of proinflammatory cytokines and thereby contributes to septic circulatory failure. Sepsis was induced by cecal ligation and puncture (CLP) in wild-type mice and mice with deficiencies for proinflammatory cytokines, and mice were injected with TNF-alpha, IL-1beta, IFN-gamma, or IL-6. Animals were treated with glucocorticoids or small interfering RNA (siRNA) targeting multiple cytokines and NF-kappaB. Vascular smooth muscle cells were incubated with cytokines and calcium mobilization, mRNA stability assays, and promoter studies with alpha1-promoter-luciferase constructs were performed. Cecal ligation and puncture treatment resulted in a hyperdynamic circulatory failure, diminished calcium response to norepinephrine, and a significant downregulation of alpha1-receptors. Proinflammatory cytokines also downregulated alpha1-receptors by suppressing promoter activity at the level of gene transcription. However, suppression of single proinflammatory cytokines in cytokine knockout mice did not diminish CLP-induced downregulation of alpha1-receptors. In contrast, blocking multiple cytokines via siRNA pretreatment or glucocorticoid administration attenuated CLP-induced cardiovascular failure and downregulation of alpha1-receptors. Furthermore, inhibiting NF-kappaB activity by siRNA reduced the production of cytokines, prevented circulatory failure and downregulation of alpha1-receptors, and improved survival of septic mice. Our findings indicate that NF-kappaB has a central role in augmenting proinflammatory cytokine production during sepsis, which in turn downregulates alpha1-receptor expression. Our data further define a critical role for NF-kappaB in the pathogenesis of septic shock, indicating that targeting NF-kappaB is a desired therapeutic strategy to treat septic vasoplegia.