Chronic obstructive pulmonary disease (COPD) is a lethal progressive lung disease culminating in permanent airway obstruction and alveolar enlargement. Previous studies suggest CTL involvement in COPD progression; however, their precise role remains unknown. Here, we investigated whether the CTL activation receptor NK cell group 2D (NKG2D) contributes to the development of COPD. Using primary murine lung epithelium isolated from mice chronically exposed to cigarette smoke and cultured epithelial cells exposed to cigarette smoke extract in vitro, we demonstrated induced expression of the NKG2D ligand retinoic acid early transcript 1 (RAET1) as well as NKG2D-mediated cytotoxicity. Furthermore, a genetic model of inducible RAET1 expression on mouse pulmonary epithelial cells yielded a severe emphysematous phenotype characterized by epithelial apoptosis and increased CTL activation, which was reversed by blocking NKG2D activation. We also assessed whether NKG2D ligand expression corresponded with pulmonary disease in human patients by staining airway and peripheral lung tissues from never smokers, smokers with normal lung function, and current and former smokers with COPD. NKG2D ligand expression was independent of NKG2D receptor expression in COPD patients, demonstrating that ligand expression is the limiting factor in CTL activation. These results demonstrate that aberrant, persistent NKG2D ligand expression in the pulmonary epithelium contributes to the development of COPD pathologies.

Receptor-mediated airway smooth muscle (ASM) contraction via G αq , and relaxation via G αs , underlie the bronchospastic features of asthma and its treatment. Asthma models show increased ASM G αi expression, considered the basis for the proasthmatic phenotypes of enhanced bronchial hyperreactivity to contraction mediated by M 3 -muscarinic receptors and diminished relaxation mediated by β 2 -adrenergic receptors (β 2 ARs). A causal effect between G i expression and phenotype has not been established, nor have mechanisms whereby G i modulates G q /G s signaling. To delineate isolated effects of altered G i , transgenic mice were generated overexpressing G αi2 or a G αi2 peptide inhibitor in ASM. Unexpectedly, G αi2 overexpression decreased contractility to methacholine, while G αi2 inhibition enhanced contraction. These opposite phenotypes resulted from different crosstalk loci within the G q signaling network: decreased phospholipase C and increased PKCα, respectively. G αi2 overexpression decreased β 2 AR-mediated airway relaxation, while G αi2 inhibition increased this response, consistent with physiologically relevant coupling of this receptor to both G s and G i . IL-13 transgenic mice (a model of asthma), which developed increased ASM G αi , displayed marked increases in airway hyperresponsiveness when G αi function was inhibited. Increased G αi in asthma is therefore a double-edged sword: a compensatory event mitigating against bronchial hyperreactivity, but a mechanism that evokes β-agonist resistance. By selective intervention within these multipronged signaling modules, advantageous G s /G q activities could provide new asthma therapies. IntroductionAirway smooth muscle (ASM) contraction and relaxation are primarily regulated by G protein-coupled receptors, the former mediated by receptors signaling to G q and the latter by those that couple to G s (1, 2). Many inflammatory cascades in asthma evoke bronchoconstriction by promoting local increases of G q receptor agonists such as acetylcholine, cysteinyl leukotrienes, prostaglandins, and histamine, which activate their cognate receptors expressed on ASM. There appear to be fewer G s -coupled receptors that act via endogenous agonists to counteract bronchoconstriction, but the β 2 -adrenergic receptor (β 2 AR) of ASM is the target of pharmacologically administered β-agonists and is typically highly effective in relaxing constricted airways. The molecular events and critical transduction elements for these 2 classes of receptors are well recognized. Agonist binding to receptors such as the M 3 -muscarinic receptor promote disassociation of heterotrimeric G q into G α and G βγ subunits, with the α subunit activating phospholipase C (PLC; which promotes inositol-3 phosphate and diacylglycerol production) and the latter activating PKC. Receptors such as the β 2 AR act via G αs to stimulate the effector adenylyl cyclase, resulting in cAMP production and activation of PKA. Substantial interest has

Beta(2)-Adrenergic receptors (beta(2)AR) are expressed on airway smooth muscle cells and act to relax the airway on activation by beta-agonists. These agents are utilized for treating asthma but are associated with adverse outcomes. To ascertain the effects of persistent beta(2)AR activation on gene expression, cultured airway smooth muscle cells derived from wild-type (WT) and transgenic mice overexpressing beta(2)AR were subjected to DNA microarray analysis; 319 genes were increased and 164 were decreased. Differential expression was observed in genes from 22 Gene Ontology Slim categories, including those associated with ion transport and calcium ion binding. A 60% decrease (P = 0.008) in phospholamban (PLN), an intracellular Ca(2+) concentration ([Ca(2+)]i)-handling protein that is at a signaling nodal point in cardiomyocytes, was observed in beta(2)AR-overexpressing cells and confirmed at the protein level. To isolate the physiological effect of decreased PLN in airway smooth muscle, airway contraction and relaxation responses were studied in WT and PLN(-/-) mice. PLN(-/-) mice had a markedly reduced constrictive response to methacholine. In contrast, the bronchodilatory effect of beta-agonist was not different between WT and PLN(-/-) mice. These results revealed an unanticipated therapeutic effect of beta-agonists, PLN downregulation, which acts to decrease airway hyperreactivity. Thus agents that inhibit PLN may act synergistically with the bronchodilating action of beta-agonists. A number of other genes related to [Ca(2+)]i are also differentially regulated by beta(2)AR activity, some of which may act to oppose, or augment, the efficacy of chronic beta-agonists. These genes or pathways may also represent additional targets in the treatment of asthma and related obstructive lung diseases.