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Introduction: Pulmonary fibrosis includes a spectrum of diseases and is incurable. There is a variation in disease course, but it is often progressive leading to increased breathlessness, impaired quality of life, and decreased life expectancy. Detection of pulmonary fibrosis is challenging, which contributes to considerable delays in diagnosis and treatment. More knowledge about the diagnostic journey from patients' perspective is needed to improve the diagnostic pathway. The aims of this study were to evaluate the time to diagnosis of pulmonary fibrosis, identify potential reasons for delays, and document patients emotions.Methods: Members of European patient organisations, with a self-reported diagnosis of pulmonary fibrosis, were invited to participate in an online survey. The survey assessed the diagnostic pathway retrospectively, focusing on four stages: (1) time from initial symptoms to first appointment in primary care; (2) time to hospital referral; (3) time to first hospital appointment; (4) time to final diagnosis. It comprised open-ended and closed questions focusing on time to diagnosis, factors contributing to delays, diagnostic tests, patient emotions, and information provision.Results: Two hundred and seventy three participants (214 idiopathic pulmonary fibrosis, 28 sarcoidosis, 31 other) from 13 countries responded. Forty percent of individuals took ≥1 year to receive a final diagnosis. Greatest delays were reported in stage 1, with only 50.2% making an appointment within 3 months. For stage 2, 73.3% reported a hospital referral within three primary care visits. However, 9.9% reported six or more visits. After referral, 76.9% of patients were assessed by a specialist within 3 months (stage 3) and 62.6% received a final diagnosis within 3 months of their first hospital visit (stage 4). Emotions during the journey were overall negative. A major need for more information and support during and after the diagnostic process was identified.Conclusion: The time to diagnose pulmonary fibrosis varies widely across Europe. Delays occur at each stage of the diagnostic pathway. Raising awareness about pulmonary fibrosis amongst the general population and healthcare workers is essential to shorten the time to diagnosis. Furthermore, there remains a need to provide patients with sufficient information and support at all stages of their diagnostic journey.

The interplay of type-2 inflammation and antiviral immunity underpins asthma exacerbation pathogenesis. Virus infection induces type-2 inflammation–promoting chemokines CCL17 and CCL22 in asthma; however, mechanisms regulating induction are poorly understood. By using a human rhinovirus (RV) challenge model in human airway epithelial cells in vitro and mice in vivo, we assessed mechanisms regulating CCL17 and CCL22 expression. Subjects with mild to moderate asthma and healthy volunteers were experimentally infected with RV and airway CCL17 and CCL22 protein quantified. In vitro airway epithelial cell- and mouse-RV infection models were then used to define STAT6- and NF-κB–mediated regulation of CCL17 and CCL22 expression. Following RV infection, CCL17 and CCL22 expression was higher in asthma, which differentially correlated with clinical and immunological parameters. Air–liquid interface–differentiated primary epithelial cells from donors with asthma also expressed higher levels of RV-induced CCL22. RV infection boosted type-2 cytokine–induced STAT6 activation. In epithelial cells, type-2 cytokines and STAT6 activation had differential effects on chemokine expression, increasing CCL17 and suppressing CCL22, whereas NF-κB promoted expression of both chemokines. In mice, RV infection activated pulmonary STAT6, which was required for CCL17 but not CCL22 expression. STAT6-knockout mice infected with RV expressed increased levels of NF-κB–regulated chemokines, which was associated with rapid viral clearance. Therefore, RV-induced upregulation of CCL17 and CCL22 was mediated by NF-κB activation, whereas expression was differentially regulated by STAT6. Together, these findings suggest that therapeutic targeting of type-2 STAT6 activation alone will not block all inflammatory pathways during RV infection in asthma.

The importance of NF-κB activation and deficient anti-viral interferon induction in the pathogenesis of rhinovirus-induced asthma exacerbations is poorly understood. We provide the first in vivo evidence in man and mouse that rhinovirus infection enhanced bronchial epithelial cell NF-κB p65 nuclear expression, NF-κB p65 DNA binding in lung tissue and NF-κB-regulated airway inflammation. In vitro inhibition of NF-κB reduced rhinovirus-induced pro-inflammatory cytokines but did not affect type I/III interferon induction. Rhinovirus-infected p65-deficient mice exhibited reduced neutrophilic inflammation, yet interferon induction, antiviral responses and virus loads were unaffected, indicating that NF-κB p65 is required for pro-inflammatory responses, but redundant in interferon induction by rhinoviruses in vivo. Conversely, IFNAR1(-/-) mice exhibited enhanced neutrophilic inflammation with impaired antiviral immunity and increased rhinovirus replication, demonstrating that interferon signalling was critical to antiviral immunity. We thus provide new mechanistic insights into rhinovirus infection and demonstrate the therapeutic potential of targeting NF-κB p65 (to suppress inflammation but preserve anti-viral immunity) and type I IFN signalling (to enhance deficient anti-viral immunity) to treat rhinovirus-induced exacerbations of airway diseases.

Compelling evidence identifies airway smooth muscle (ASM) not only as a target but also a cellular source for a diverse range of mediators underlying the processes of airway narrowing and airway hyperresponsiveness in diseases such as asthma. These include the growing family of plasma membrane phospholipid-derived polyunsaturated fatty acids broadly characterised by the prostaglandins, leukotrienes, lipoxins, isoprostanes and lysophospholipids. In this review, we describe the enzymatic and non-enzymatic biosynthetic pathways of these lipid mediators and how these are influenced by drug treatment, oxidative stress and airways disease. Additionally, we outline their cognate receptors, many of which are expressed by ASM. We describe potential deleterious and protective roles for these lipid mediators in airway inflammatory and remodelling processes by describing their effects on diverse functions of ASM in asthma that have the potential to contribute to asthma pathogenesis and symptoms. These functions include contractile tone development, cytokine and extracellular matrix production, and cellular proliferation and migration.

Background and purpose: Endothelin-1 (ET-1) is implicated in airway inflammation in asthma, but the mechanisms of its effects are poorly understood. We studied the effect of ET-1 on expression of the chemokine, monocyte chemotactic protein-1 (MCP-1), in primary cultures of human airway smooth muscle cells. Experimental approach: MCP-1 release was measured by elisa. Pharmacological antagonists/inhibitors, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to study ET receptors and kinase cascades. Transcriptional regulation was studied by real-time RT-PCR, transient transfection studies and chromatin immunoprecipitation assay. Major findings were confirmed in cells from three donors and mechanistic studies in cells from one donor. Key results: ET-1 increased MCP-1 release through an ETA and ETB receptor-dependent mechanism. ET-1 increased MCP-1 mRNA levels but not mRNA stability suggesting it was acting transcriptionally. ET-1 increased the activity of an MCP-1 promoter–reporter construct. Serial deletions of the MCP-1 promoter mapped ET-1 effects to a region between −213 and −128 base pairs upstream of the translation start codon, containing consensus sequences for activator protein-1 (AP-1) and nuclear factor-κB (NF-κB). ET-1 promoted binding of AP-1 c-Jun subunit and NF-κB p65 subunit to the MCP-1 promoter. Blocking the inhibitor of κB kinase-2 with 2-[(aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecarboxamide (TPCA-1) decreased ET-1-stimulated MCP-1 production. p38 and p44/p42 mitogen-activated protein kinases were involved in upstream signalling. Conclusions and implications: ET-1 regulated MCP-1 transcriptionally, via NF-κB and AP-1. The upstream signalling involved ETA, ETB receptors, p38 and p44/p42 mitogen-activated protein kinases. These may be targets for novel asthma therapies.

The liver X receptors (LXRα/β) are orphan nuclear receptors that are expressed in a large number of cell types and have been shown to have anti-inflammatory properties. Nuclear receptors have previously proved to be amenable targets for small molecular mass pharmacological agents in asthma, and so the effect of an LXR ligand was assessed in models of allergic airway inflammation. LXR agonist, GW 3965, was profiled in rat and mouse models of allergic asthma. In the Brown Norway rats, GW 3965 (3–30 mg/kg) was unable to reduce the bronchoalveolar lavage eosinophilia associated with this model and had no impact on inflammatory biomarkers (eotaxin and IL-1β). The compound did significantly stimulate ABCA-1 (ATP-binding cassette A1) mRNA expression, indicating that there was adequate exposure/LXR activation. In the mouse model, the LXR ligand surprisingly increased airway reactivity, an effect that was apparent in both the Ag and nonchallenged groups. This increase was not associated with a change in lung tissue inflammation or number of mucus-containing cells. There was, however, a marked increase in airway smooth muscle thickness in both treated groups. We demonstrated an increase in contractile response to exogenous methacholine in isolated airways taken from LXR agonist-treated animals compared with the relevant control tissue. We corroborated these findings in a human system by demonstrating increased proliferation of cultured airway smooth muscle. This phenomenon, if evidenced in man, would indicate that LXR ligands may directly increase airway reactivity, which could be detrimental, especially in patients with existing respiratory disease and with already compromised lung function.

We have previously shown that interleukin (IL)-1β, transforming growth factor (TGF)-β1, or bradykinin (BK) impair cAMP generation in response to prostacyclin analogs in human pulmonary artery smooth muscle (PASM), suggesting that inflammation can impair the effects of prostacyclin analogs on PASM in pulmonary hypertension. Here we explored the biochemical mechanisms involved. We found that IL-1β, BK, and TGF-β1reduced adenylyl cyclase isoform 1, 2, and 4 mRNA, increased Gαiprotein levels, and reduced prostacyclin receptor (IP receptor) mRNA expression. In contrast, Gαsprotein levels were unchanged. Protein kinase A (PKA) (H-89, KT-2750, PKIm) and p38 mitogen-activated protein (MAP) kinase (SB-202190) inhibitors attenuated these effects, but protein kinase C (bisindolylmaleide) or phosphoinositol 3-kinase (LY-294002) inhibitors did not. Fluorescent kemptide assay and Western blotting confirmed that PKA and p38 MAP kinase were activated by IL-1β, BK, and TGF-β1. These studies suggest that IL-1β, BK, and TGF-β1impair IP receptor-mediated cAMP accumulation by multiple effects on different components of the signaling pathway and that these effects are PKA and p38 MAP kinase dependent.

The prostanoid receptors on human airway smooth muscle cells (HASMC) that augment the release by IL-1β of granulocyte colony-stimulating factor (G-CSF) have been characterized and the signaling pathway elucidated. PCR of HASM cDNA identified products corresponding to EP2, EP3, and EP4receptor subtypes. These findings were corroborated at the protein level by immunocytochemistry. IL-1β promoted the elaboration of G-CSF, which was augmented by PGE2. Cicaprost (IP receptor agonist) was approximately equiactive with PGE2, whereas PGD2, PGF2α, and U-46619 (TP receptor agonist) were over 10-fold less potent. Neither SQ 29,548 nor BW A868C (TP and DP1receptor antagonists, respectively) attenuated the enhancement of G-CSF release evoking any of the prostanoids studied. With respect to PGE2, the EP receptor agonists 16,16-dimethyl PGE2(nonselective), misoprostol (EP2/EP3selective), 17-phenyl-ω-trinor PGE2(EP1selective), ONO-AE1-259, and butaprost (both EP2selective) were full agonists at enhancing G-CSF release. AH 6809 (10 μM) and L-161,982 (2 μM), which can be used in HASMC as selective EP2and EP4receptor antagonists, respectively, failed to displace to the right the PGE2concentration-response curve that described the augmented G-CSF release. In contrast, AH 6809 and L-161,982 in combination competitively antagonized PGE2-induced G-CSF release. Augmentation of G-CSF release by PGE2was mimicked by 8-BrcAMP and abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA). These data demonstrate that PGE2facilitates G-CSF secretion from HASMC through a PKA-dependent mechanism by acting through EP2and EP4prostanoid receptors and that effective antagonism is realized only when both subtypes are blocked concurrently.

8-Isoprostanes are bioactive lipid mediators formed via the nonenzymatic peroxidation of arachidonic acid by free radicals and reactive oxygen species. However, their cognate receptors, biological actions, and signaling pathways are poorly studied. Here, we report the effect of a variety of E- and Falpha-ring 8-isoprostanes on the release of granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) from human airway smooth muscle (HASM) cells stimulated with interleukin-1beta (IL-1beta). The elaboration of GM-CSF and G-CSF by IL-1beta was inhibited and augmented, respectively, in a concentration-dependent manner by 8-iso-prostaglandin (PG) E(1) and 8-iso-PGE(2), but not by 8-iso-PGF(1alpha), 8-iso-PGF(2alpha), and 8-iso-PGF(3)alpha. AH 6809 (6-isopropoxy-9-oxoxanthine-2-carboxylic acid), an EP(1)-/EP(2)-/DP-receptor blocking drug, antagonized the inhibitory effect of 8-iso-PGE(1) and 8-iso-PGE(2) on GM-CSF output with an affinity consistent with an interaction at prostanoid receptors of the EP(2)-subtype. In contrast, the facilitation by 8-iso-PGE(1) and 8-iso-PGE(2) of G-CSF release was unaffected by AH 6809 and the selective EP(4)-receptor antagonist L-161,982 [4'-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide]. However, when used in combination, AH 6809 and L-161,982 displaced 5-fold to the right the 8-iso-PGE and 8-iso-PGE concentration-response curves. The opposing (1)effect of E-ring (2)8-isoprostanes on GM-CSF and G-CSF release was mimicked by 8-bromo-cAMP and abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA). Together, these data demonstrate that E-ring 8-isoprostanes regulate the secretion of GM-CSF and G-CSF from HASM cells by a cAMP- and PKA-dependent mechanism. Moreover, antagonist studies revealed that 8-iso-PGE(1) and 8-iso-PGE(2) act solely via EP(2) -receptors to inhibit GM-CSF release, whereas both EP(2)- and EP(4)-receptor subtypes positively regulate G-CSF output.

1. The prostanoid receptor(s) on human airways smooth muscle (HASM) cells that mediates the inhibitory effect of PGE(2) on interleukin (IL)-1 beta-induced granulocyte/macrophage colony-stimulating factor (GM-CSF) release has been classified. 2. IL-1 beta evoked the release of GM-CSF from HASM cells, which was suppressed by PGE(2), 16,16-dimethyl PGE(2) (nonselective), misoprostol (EP(2)/EP(3)-selective), ONO-AE1-259 and butaprost (both EP(2)-selective) with pIC(50) values of 8.61, 7.13, 5.64, 8.79 and 5.43, respectively. EP-receptor agonists that have selectivity for the EP(1)-(17-phenyl-omega-trinor PGE(2)) and EP(3)-receptor (sulprostone) subtypes as well as cicaprost (IP-selective), PGD(2), PGF(2 alpha) and U-46619 (TP-selective) were poorly active or inactive at concentrations up to 10 microM. 3. AH 6809, a drug that can be used to selectively block EP(2)-receptors in HASM cells, antagonised the inhibitory effect of PGE(2), 16,16-dimethyl PGE(2) and ONO-AE1-259 with apparent pA(2) values of 5.85, 6.09 and 6.1 respectively. In contrast, the EP(4)-receptor antagonists, AH 23848B and L-161,982, failed to displace to the right the concentration-response curves that described the inhibition of GM-CSF release evoked by PGE(2) and ONO-AE1-259. 4. Inhibition of GM-CSF release by PGE(2) and 8-Br-cAMP was abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA) but not by H-89, a purported small molecule inhibitor of PKA. 5. We conclude that prostanoid receptors of the EP(2)-subtype mediate the inhibitory effect of PGE(2) on GM-CSF release from HASM cells by recruiting a PKA-dependent pathway. In addition, the data illustrate that caution should be exercised when using H-89 in studies designed to assess the role of PKA in biological processes.

In the present study, we examined the effect of E-ring 8-isoprostanes on cholinergic neurotransmission in guinea-pig trachea and identified the receptor(s) involved. As isoprostanes are isomeric with prostaglandins, PGE2 and sulprostone (a selective EP3-receptor agonist) were examined in parallel. 8-Iso-PGE1, 8-iso-PGE2 (0.1 nM–1 μM), sulprostone (1 nM–1 μM) and PGE2 (1 μM) suppressed EFS-evoked [3H]ACh release from guinea-pig trachea in a concentration-dependent manner, producing 39.5, 53.9, 61.2 and 59.9% inhibition, respectively, at 1 μM. It should be noted that an established maximum effective concentration was not determined. Neither SQ 29,548 (1 μM; a TP-receptor antagonist) nor AH 6809 (10 μM; an EP1-/EP2-/DP-receptor antagonist) reversed the inhibitory effect of these compounds. L-798,106, a novel and highly selective EP3-receptor antagonist, produced a parallel shift to the right of the concentration–response curves that described the inhibitory action of sulprostone on EFS-evoked contractile responses in guinea-pig vas deferens (an established EP3-receptor-expressing tissue), from which a mean pA2 of 7.48 was derived. On guinea-pig trachea, L-798,106 also antagonised sulprostone-induced inhibition of EFS-induced twitch responses, with similar potency (mean pA2=7.82). The inhibitory effects of 8-iso-PGE1, 8-iso-PGE2, sulprostone and PGE2 on EFS-induced [3H]ACh release was blocked by L-798,106 at a concentration (10 μM) that binds only weakly to human recombinant EP1-, EP2- and EP4-receptor subtypes expressed in HEK 293 cells. These data suggest that E-ring 8-isoprostanes, PGE2 and sulprostone inhibit EFS-evoked [3H]ACh release from cholinergic nerves innervating guinea-pig trachea, by interacting with prejunctional prostanoid receptors of the EP3-subtype. British Journal of Pharmacology (2004) 141, 600–609. doi:10.1038/sj.bjp.0705648

Chronic respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD) represent a significant health burden worldwide and are a major unmet medical need. Asthma affects over 300 million people and leads to 250,000 deaths per year, with an increasing prevalence particularly in developing countries. Although a large proportion of asthmatics are maintained on beta agonists and corticosteroids, there still remains a group of patients where these medicines fail to modulate symptoms and who may therefore benefit from monoclonal antibody based drugs that are aimed at controlling the disease. COPD is a cigarette smoke-driven chronic inflammatory airway disease with an increasing global prevalence. Given that current therapies fail to prevent disease progression or mortality, this patient population is also a focus for the development of monoclonal antibody therapies. At present anti-IgE (omalizumab, Xolair®) is the only monoclonal antibody based drug approved in the respiratory space for the treatment of asthma. However, an increasing number of antibodies targeting key mediators/pathways of disease are in clinical development for both asthma and COPD, including targeting the Th2 pathway for asthma (anti-IL-4/5/13) and the pro-inflammatory cytokine IL-1 for COPD. This review will examine the antibody engineering approaches used to develop the next generation of antibodies, with a focus on respiratory disease.

Background Asthma and chronic obstructive pulmonary disease are airway inflammatory diseases characterised by airflow obstruction. Currently approved bronchodilators such as long-acting β 2 adrenoceptor agonists are the mainstay treatments but often fail to relieve symptoms of chronic obstructive pulmonary disease and severe asthma and safety concerns have been raised over long-term use. The aim of the study was to identify the receptor involved in prostaglandin E 2 (PGE 2 )-induced relaxation in guinea pig, murine, monkey, rat and human airways in vitro . Methods Using an extensive range of pharmacological tools, the relaxant potential of PGE 2 and selective agonists for the EP 1–4 receptors in the presence and absence of selective antagonists in guinea pig, murine, monkey, rat and human isolated airways was investigated. Results In agreement with previous studies, it was found that the EP 2 receptor mediates PGE 2 -induced relaxation of guinea pig, murine and monkey trachea and that the EP 4 receptor mediates PGE 2 -induced relaxation of the rat trachea. These data have been confirmed in murine airways from EP 2 receptor-deficient mice ( Ptger2 ). In contrast to previous publications, a role for the EP 4 receptor in relaxant responses in human airways in vitro was found. Relaxant activity of AH13205 (EP 2 agonist) was also demonstrated in guinea pig but not human airway tissue, which may explain its failure in clinical studies. Conclusion Identification of the receptor mediating PGE 2 -induced relaxation represents a key step in developing a novel bronchodilator therapy. These data explain the lack of bronchodilator activity observed with selective EP 2 receptor agonists in clinical studies.

Due to recent safety concerns with beta 2 adrenoceptor agonists (e.g. salbutamol), there has been an ongoing attempt to identify novel bronchodilator agents for use in asthma and COPD. Due to its beneficial action on airway calibre, prostaglandin E2 (PGE2) was previously investigated as a potential therapy, however in addition to bronchodilator activity, in clinical studies PGE2 also produced tussive effects and bronchial irritancy. As PGE2 mediates its actions via a number of different prostanoid receptors, it was hoped that by selectively targeting individual receptors, the beneficial actions could be selected. In a previous study (Nials et al 1993), the EP2 receptor was identified as mediating PGE2-induced relaxation however when tested clinically, an EP2 agonist (AH13205) produced disappointing results.

The relative roles of the endosomal TLR3/7/8 versus the intracellular RNA helicases RIG-I and MDA5 in viral infection is much debated. We investigated the roles of each pattern recognition receptor in rhinovirus infection using primary bronchial epithelial cells. TLR3 was constitutively expressed; however, RIG-I and MDA5 were inducible by 8–12 h following rhinovirus infection. Bronchial epithelial tissue from normal volunteers challenged with rhinovirus in vivo exhibited low levels of RIG-I and MDA5 that were increased at day 4 post infection. Inhibition of TLR3, RIG-I and MDA5 by siRNA reduced innate cytokine mRNA, and increased rhinovirus replication. Inhibition of TLR3 and TRIF using siRNA reduced rhinovirus induced RNA helicases. Furthermore, IFNAR1 deficient mice exhibited RIG-I and MDA5 induction early during RV1B infection in an interferon independent manner. Hence anti-viral defense within bronchial epithelium requires co-ordinated recognition of rhinovirus infection, initially via TLR3/TRIF and later via inducible RNA helicases., Author Summary Host-pathogen interactions are mediated by pattern recognition receptors that identify conserved structures of micro-organisms that are distinct from self. During a viral infection, important pattern recognition receptors include the endosomal Toll-like receptors (TLRs), and a second set of cytoplasmic pattern recognition receptors known as the RNA helicases. Many studies have highlighted the importance of TLR3, TLR7/8 and the RNA helicases in providing robust anti-viral immunity via interferon induction and inflammation. Both endosomal TLR and cytoplasmic RNA helicase mediated pathways are believed to exist as separate yet non-redundant entities; however, little thought is given to why both systems exist, and few studies also consider how both pathways together contribute to anti-viral immunity. Using models of rhinovirus infection in primary bronchial epithelial cell culture in vitro and experimental infection in mouse and human models in vivo, we show that the RNA helicases are preferentially induced early in the infection cycle via TLR3 mediated signaling events, and work in a co-ordinated, systematic manner. The results help understand the complex events that determine effective innate immunity to rhinovirus infection and how these processes contribute to virus induced exacerbations of asthma and chronic obstructive pulmonary disease.

The transcription factor NF-κB plays a pivotal role in regulating inflammatory gene expression. Its effects are optimized by various coactivators, including histone acetyltransferases (HATs) such as CREB-binding protein/p300 and p300/CBP-associated factor (p/CAF). The molecular mechanisms regulating cofactor recruitment are poorly understood. In this study, we describe a novel role for protein kinase C (PKC) βΙΙ in augmenting NF-κB-mediated TNF-α-induced transcription of the target gene CCL11 in human airway smooth muscle cells by phosphorylating the HAT p/CAF. Studies using reporters, overexpression strategies, kinase-dead and HAT-defective mutants, and chromatin immunoprecipitation showed that PKCβII activation was not involved in NF-κB translocation, but facilitated NF-κB-mediated CCL11 transcription by colocalizing with and phosphorylating p/CAF, and thereby acetylating histone H4 and promoting p65 association with the CCL11 promoter. The effect was dependent on p/CAF’s HAT activity. Furthermore, mouse embryonic fibroblasts from PKCβ knockout mice showed markedly reduced TNF-α-induced CCL11 expression and NF-κB reporter activity that was restored on PKCβII overexpression, suggesting a critical role for this pathway. These data suggest a novel important biological role for PKCβΙΙ in NF-κB-mediated CCL11 transcription by p/CAF activation and histone H4 acetylation.

The immuno-modulatory properties of airway smooth muscle have become of increasing importance in our understanding of the mechanisms underlying chronic inflammation and structural remodeling of the airway wall in asthma and chronic obstructive pulmonary disease (COPD). ASM cells respond to many cytokines, growth factors and lipid mediators to produce a wide array of immuno-modulatory molecules which may in turn orchestrate and perpetuate the disease process in asthma and COPD. Despite numerous studies of the cellular effects of cytokines on cultured ASM, few have identified intracellular signaling pathways by which cytokines modulate or induce these cellular responses. In this review we provide an overview of the transcriptional mechanisms as well as intracellular signaling pathways regulating cytokine functions in ASM cells. The recent discovery of toll-like receptors in ASM cells represents a significant development in our understanding of the immuno-modulatory capabilities of ASM cells. Thus, we also review emerging evidence of the inflammatory response to toll-like receptor activation in ASM cells. © 2009 Elsevier Ltd. All rights reserved.

Mast cells are central in the development of several allergic diseases and contain a number of pre-formed mediators. beta-tryptase, the most abundant mast cell product, is increasingly recognized as a key inflammatory mediator, as it causes the release of cytokines, particularly the chemokine IL-8, from both inflammatory and structural cells. The molecular mechanisms, however, remain largely unknown. In this study we sought to investigate whether beta-tryptase could induce IL-8 expression in human airway smooth muscle (ASM) cells and to explore the molecular mechanisms involved. We found that purified human beta-tryptase stimulated IL-8 production in a time- and concentration-dependent manner, which was inhibited by protease inhibitors and mimicked by recombinant human beta-tryptase, but not by the protease-activated receptor-2 (PAR-2) agonist SLIGKV-NH(2), consistent with the low-level expression of PAR-2 protein in these cells. beta-tryptase also up-regulated IL-8 mRNA expression, as analyzed by RT-PCR and real-time PCR, which was abolished by the transcription inhibitor actinomycin D. Reporter gene assay showed that beta-tryptase-induced IL-8 transcription was mediated by the transcription factors activator protein-1, CCAAT/enhancer binding protein, and NF-kappaB, and chromatin immunoprecipitation assay demonstrated that beta-tryptase induced in vivo binding of these transcription factors to the IL-8 gene promoter. Furthermore, beta-tryptase stabilized IL-8 mRNA, suggesting additional post-transcriptional regulation. Collectively these findings show that beta-tryptase up-regulates IL-8 expression in ASM cells through a PAR-2-independent proteolytic mechanism and coordinated transcriptional and post-transcriptional regulation, which may be of particular importance in understanding the role and the mechanisms of action of beta-tryptase in regulating chemokine expression in mast cell-related disorders.

1. The prostanoid receptor(s) on human airways smooth muscle (HASM) cells that mediates the inhibitory effect of PGE(2) on interleukin (IL)-1 beta-induced granulocyte/macrophage colony-stimulating factor (GM-CSF) release has been classified. 2. IL-1 beta evoked the release of GM-CSF from HASM cells, which was suppressed by PGE(2), 16,16-dimethyl PGE(2) (nonselective), misoprostol (EP(2)/EP(3)-selective), ONO-AE1-259 and butaprost (both EP(2)-selective) with pIC(50) values of 8.61, 7.13, 5.64, 8.79 and 5.43, respectively. EP-receptor agonists that have selectivity for the EP(1)-(17-phenyl-omega-trinor PGE(2)) and EP(3)-receptor (sulprostone) subtypes as well as cicaprost (IP-selective), PGD(2), PGF(2 alpha) and U-46619 (TP-selective) were poorly active or inactive at concentrations up to 10 microM. 3. AH 6809, a drug that can be used to selectively block EP(2)-receptors in HASM cells, antagonised the inhibitory effect of PGE(2), 16,16-dimethyl PGE(2) and ONO-AE1-259 with apparent pA(2) values of 5.85, 6.09 and 6.1 respectively. In contrast, the EP(4)-receptor antagonists, AH 23848B and L-161,982, failed to displace to the right the concentration-response curves that described the inhibition of GM-CSF release evoked by PGE(2) and ONO-AE1-259. 4. Inhibition of GM-CSF release by PGE(2) and 8-Br-cAMP was abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA) but not by H-89, a purported small molecule inhibitor of PKA. 5. We conclude that prostanoid receptors of the EP(2)-subtype mediate the inhibitory effect of PGE(2) on GM-CSF release from HASM cells by recruiting a PKA-dependent pathway. In addition, the data illustrate that caution should be exercised when using H-89 in studies designed to assess the role of PKA in biological processes.