Inhibition of the NF-k B Inflammatory Signalling Pathway by Carbon Dioxide Involves Reduced Kinetics of Multiple Kinases

Introduction Inflammation in the lung epithelium can arise from a number of sources, including recognition of pathogens by Toll-Like Receptors (TLRs), ventilator induced lung injury (VILI) and endogenous cytokines (TNF, IL-1) [1]. These lead to activation of a cytoplasmic signalling cascade, ultimately resulting in translocation of NF-B to the nucleus, binding to specific promoter regions and subsequent transcription of inflammation associated genes. Recent studies have demonstrated the ability of elevated carbon dioxide in ventilation strategies, coupled with the associated acidosis of fluids and tissues, to modulate disease course in the lung. Degradation of IB, which releases NF-B to allow translocation, has been shown to be inhibited [2], and elements of the homologous Drosophila inflammatory signalling system have been shown to have inhibited function under hypercapnic acidosis (HCA) [3]. Here we have sought to more closely examine several key elements of the mammalian signalling cascade, particularly protein phosphorylation events, under conditions of HCA. This will lead to a more complete understanding of the mechanism by which elevated carbon dioxide concentrations can alleviate forms of acute lung injury. Materials and Methods The A549 human immortal pulmonary alveolar cell line was used due to ease of transfection. Immunoblotting with phospho-specific antibodies was used to detect active kinases. Immunoprecipitation of transfected protein, in vitro kinase assays and subsequent detection of phospho-proteins by ELISA or Western blot was used to assess kinase activity. Results Overexpression of Neutrophil Induced Kinase (NIK) or Inhibitor of Kappa-B Kinase (IKK) led to increased translation of a κB-luciferase reporter construct (Figure) and secretion of the chemokine IL-8. This induction could be partially but significantly reversed by HCA (pCO2 15% v pCO2 5%). The kinase active phosphorylated state of IKK was also reduced by exposure of the cells to increased CO2. This phenomenon was observed both with and without further induction of phosphorylation by treatment with TNFα. The actual kinase activity of IKK was shown to be inhibited, not just its phosphorylation state, by HCA, as judged by a decreased ability to phosphorylate IκBα, which targets it for degradation. Finally, an equal amount of isolated, cell-free, activated IKK preparation was shown to have inhibited kinase activity under HCA.