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Endotoxin Triggers Nuclear Factor-B–dependent Up-regulation of Multiple Proinflammatory Genes in the Diaphragm

Meakins-Christie Laboratories, McGill University; and Respiratory Division, McGill University Health Center, Montreal, Quebec, Canada

Correspondence and requests for reprints should be addressed to Basil J. Petrof, M.D., Respiratory Division, Room L411, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, PQ, Canada H3A 1A1. E-mail: basil.petrof{at}mcgill.ca

Background: Sepsis-induced diaphragmatic force loss and failure are associated with an increased exposure of the muscle to proinflammatory mediators.

Objectives: Our objectives were to test the hypothesis that force-inhibiting mediators may arise in large part from the diaphragm itself and to evaluate the roles of mechanical stress, free radicals, and the nuclear factor (NF)-B transcription factor pathway in endotoxin (LPS)-induced proinflammatory responses of the diaphragm.

Methods: Murine diaphragm and limb muscle cells were exposed to LPS in vitro and in vivo. Proinflammatory gene expression was measured using RNase protection assays (tumor necrosis factor [TNF]-, TNF- receptor p55, interleukin [IL]-1, IL-1, IL-6, macrophage inflammatory peptide-2, intercellular adhesion molecule-1, Fas ligand, and inducible nitric oxide synthase) and ELISAs (TNF-, IL-6, and macrophage inflammatory peptide-2). Cyclical muscle cell stretch and free-radical scavengers (N-acetylcysteine and catalase) were used to alter mechanical and oxidative stress levels, respectively. Pharmacologic (pyrrolidinedithiocarbamate) and dominant-negative transfection strategies were used to inhibit the NF-B pathway.

Results: In primary diaphragm muscle cell cultures, modulation of mechanical stress levels or free-radical exposure did not alter responses to LPS stimulation. However, pharmacologic blockade of the NF-B pathway and dominant-negative molecular inhibition of IKB kinase- strongly suppressed LPS-induced proinflammatory gene expression. In vivo, acute endotoxemia induced significantly greater mRNA and protein levels for proinflammatory mediators in the diaphragm as compared with limb muscle. Basal expression levels of proinflammatory genes were significantly higher in the diaphragm.

Conclusions: Constitutive and LPS-induced proinflammatory gene expression are exaggerated in the diaphragm compared with limb muscles and are critically dependent on the NF-B pathway. We suggest the diaphragm may be relatively predisposed to proinflammatory responses.

Key Words: cytokines • diaphragm • inflammation • LPS • nuclear factor-B

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