Chemokine receptor and ligand upregulation in the diaphragm during endotoxemia and Pseudomonas lung infection.

Sepsis-induced diaphragmatic inflammation has been associated with respiratory failure, but the role of chemokines in this process has not been evaluated. Here we sought to study the local expression and molecular regulation of the chemokines, regulated upon activation normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein (MIP)-1alpha, in the murine diaphragm during sepsis. Constitutive expression levels of RANTES and MIP-1alpha, as well as their receptors, CCR1 and CCR5, were significantly higher in diaphragm than limb muscle. Sepsis was induced by acute lipopolysaccharide (LPS) delivery or subacutely by intratracheal administration of live Pseudomonas aeruginosa bacteria. Both sepsis models triggered a marked upregulation of RANTES and MIP-1alpha in the diaphragm. In vitro, stimulation of diaphragmatic muscle cells with LPS also led to RANTES upregulation. Inhibition of the NF-kB pathway using pharmacologic or dominant negative genetic approaches blocked the LPS-induced RANTES upregulation, while free radical scavengers had no effect. We conclude that sepsis leads to greatly increased expression of RANTES, MIP-1alpha and their cognate receptors in the diaphragm. Manipulation of the NF-kB pathway and other regulators of chemokine expression in the diaphragm could represent a novel method for mitigating the skeletal muscle inflammatory response associated with sepsis-induced diaphragmatic dysfunction.

CC family chemokines directly regulate myoblast responses to skeletal muscle injury.

Chemokines have been implicated in the promotion of leucocyte trafficking to diseased muscle. The purpose of this study was to determine whether a subset of inflammatory chemokines are able to directly drive myoblast proliferation, an essential early component of muscle regeneration, in a manner which is entirely independent of leucocytes. Cultured myoblasts (C2C12) were exposed to monocyte chemoattractant protein-1 (MCP-1; CCL2), macrophage inflammatory protein-1alpha (MIP-1alpha; CCL3) or MIP-1beta (CCL4). All chemokines induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) and greatly increased myoblast proliferative responses. Chemokine-induced myoblast proliferation was abolished by pertussis toxin and the MEK1/2 inhibitor U0126, implicating both Galphai-coupled receptors and ERK1/2-dependent signalling. Myoblasts expressed receptors for all of the chemokines tested, and mitogenic responses were specifically inhibited by antibodies directed against CC family chemokine receptors 2 and 5 (CCR2 and CCR5). Within an in vitro myogenic wound healing assay devoid of leucocytes, all chemokines significantly accelerated the time course of myoblast wound closure after mechanical injury. Injections of MCP-1 into cardiotoxin-injured skeletal muscles in vivo also suppressed expression of the differentiation marker myogenin, consistent with a mitogenic effect. Taken together, our results indicate that CC chemokines have potent and direct effects on myoblast behaviour, thus indicating a novel role in muscle repair beyond leucocyte chemoattraction. Therefore, interventions aimed at modulating the balance between myoblast and leucocyte effects of CC chemokines in injured muscle could represent a novel strategy for the treatment of destructive muscle pathologies.

Impact of IL-10 on diaphragmatic cytokine expression and contractility during Pseudomonas Infection.

Severe weakness of the respiratory muscles, with attendant respiratory failure and death, has been documented in sepsis. In this study, we show that during murine pulmonary infection with Pseudomonas aeruginosa, multiple proinflammatory genes are up-regulated not only within the lungs, but also within the diaphragm. Significant induction of TNF-alpha, IL-1alpha, IL-1beta, IL-6, and IL-18 gene expression occurred within the diaphragm in a bacterial dose-dependent manner. We determined whether the anti-inflammatory cytokine IL-10 could blunt proinflammatory gene expression within the diaphragm under these conditions. The IL-10 receptor was found to be expressed by the diaphragm in vivo as well as in primary diaphragmatic muscle cell cultures. Transduction of myoblasts with an adenoviral vector (Ad-IL-10) induced strong IL-10 expression, and intramuscular injection of the same vector in vivo produced significant increases in IL-10 serum levels. Ad-IL-10 treatment of mice infected with P. aeruginosa significantly inhibited the induction of proinflammatory cytokines within the diaphragm, but not in the infected lungs. Ad-IL-10 treatment also led to greatly improved diaphragmatic force production in infected mice. These results suggest that pulmonary infection triggers proinflammatory gene expression by the diaphragm along with diaphragmatic weakness. Shifting the balance between pro- and anti-inflammatory mediators in favor of the latter by IL-10 gene delivery was able to restore normal diaphragmatic force-generating capacity under these conditions, suggesting a possible avenue for therapeutic intervention.

Toll-like receptors differentially regulate CC and CXC chemokines in skeletal muscle via NF-kappaB and calcineurin.

Immunologically active molecules such as cytokines and chemokines have been implicated in skeletal muscle weakness during sepsis as well as recovery from muscle injury. In sepsis, Toll-like receptors (TLRs) act as key sentinel molecules of the innate immune system. Here we determined skeletal muscle cell responses of two prototypical CC and CXC chemokine genes (monocyte chemoattractant protein 1 [MCP-1] and KC, respectively), to stimulation with specific TLR ligands. In addition, we examined whether NF-kappaB and calcineurin signaling are involved in these responses. Differentiated myotubes and intact whole muscles expressed TLR2, TLR4, TLR5, and TLR9. Stimulation with ligands for TLR2 (peptidoglycan) or TLR4 (LPS) elicited robust and equivalent levels of MCP-1 and KC mRNA expression, whereas stimulation of TLR5 (by flagellin) required gamma interferon priming to induce similar effects. Although both TLR2 and TLR4 ligands activated the NF-kappaB pathway, NF-kappaB reporter activity was approximately 20-fold greater after TLR4 stimulation than after TLR2 stimulation. Inhibitory effects of NF-kappaB blockade on TLR-mediated chemokine gene expression, by either pharmacological (pyrrolidine dithiocarbamate) or molecular (IKKbeta dominant-negative transfection) methods, were also more pronounced during TLR4 stimulation. In contrast, inhibitory effects on TLR-mediated chemokine expression of calcineurin blockade (by FK506) were greater for TLR2 than for TLR4 stimulation. MCP-1 and KC mRNA levels also demonstrated differential responses to NF-kappaB and calcineurin blockade during stimulation with specific TLR ligands. We conclude that skeletal muscle cells differentially utilize the NF-kappaB and calcineurin pathways in a TLR-specific manner to enable complex regulation of CC and CXC chemokine gene expression.

Endotoxin triggers nuclear factor-kappaB-dependent up-regulation of multiple proinflammatory genes in the diaphragm.

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)-kappaB 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]-alpha, TNF-alpha receptor p55, interleukin [IL]-1alpha, IL-1beta, IL-6, macrophage inflammatory peptide-2, intercellular adhesion molecule-1, Fas ligand, and inducible nitric oxide synthase) and ELISAs (TNF-alpha, 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-kappaB 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-kappaB pathway and dominant-negative molecular inhibition of IKB kinase-beta 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-kappaB pathway. We suggest the diaphragm may be relatively predisposed to proinflammatory responses.

Endothelin-1 regulates proliferative responses, both alone and synergistically with PDGF, in rat tracheal smooth muscle cells.

The peptide, endothelin-1 (ET-1) regulates proliferative responses in numerous cell types. Recently, a dual ET receptor antagonist was shown to prevent the increase in airway smooth muscle cell (SMC) proliferation that accompanies airway smooth muscle remodeling in a rat model of experimental asthma. Thus, we used [(3)H]-thymidine incorporation assays and western immunoblotting to identify signaling pathways that regulate proliferative responses in cultured rat tracheal SMC. Our data indicate that ET-1 activation of the ET A receptor subtype induced [(3)H]-thymidine incorporation and activation of ERK 1/2 in primary rat tracheal SMC. ET-1-induced [(3)H]-thymidine incorporation and activation of ERK 1/2 were inhibited by pretreatment of SMC with pertussis toxin or down regulation of phorbol ester responsive isoforms of PKC. While ET- 1-induced ERK 1/2 activation was unaffected following inhibition of Rho kinase, ET-1-induced [(3)H]-thymidine incorporation was abrogated. ET-1 also potentiated [(3)H]-thymidine incorporation as well as cell proliferation of SMC stimulated with PDGF-BB and this response did not appear to be regulated by ERK1/ 2. These data demonstrate that ET-1 induces activation of multiple G proteins that regulate rat tracheal SMC proliferative responses, likely through signaling pathways downstream of ERK1/2 and Rho kinase.