Sputum matrix metalloproteinase-12 in patients with chronic obstructive pulmonary disease and asthma: relationship to disease severity.

BACKGROUND: Matrix metalloproteinase (MMP)-12 has been implicated in the pathogenesis of both chronic obstructive pulmonary disease (COPD) and asthma. The influence of disease severity on sputum MMP-12 concentrations and activity is not known. OBJECTIVES: We sought to examine the relationship between disease severity assessed by means of lung function and computed tomography (CT) and induced sputum MMP-12 concentrations and activity in patients with asthma and COPD. METHODS: In 208 subjects (109 asthmatic patients, smokers and never smokers, mild, moderate, and severe; 53 patients with COPD, smokers and exsmokers, mild, moderate, and severe; and 46 healthy control subjects, smokers and never smokers), we measured induced sputum MMP-12 concentrations (ELISA) and enzyme activity (fluorescence resonance energy transfer), sputum cell MMP12 mRNA expression (quantitative PCR [qPCR]), diffusing capacity for carbon monoxide (Dlco), and CT assessment of emphysema (percentage of low-attenuation areas at less -950 Hounsfield units). RESULTS: Sputum MMP-12 concentrations are greater in patients with COPD and smokers with asthma than in healthy nonsmokers (P = .003 and P = .035, respectively) but similar to those seen in healthy smokers. In patients with COPD, disease severity, when measured by means of CT-assessed emphysema, but not by means of spirometry or Dlco values, is directly associated with sputum MMP-12 concentrations and activity. In the asthma groups there is no significant association between disease severity and sputum MMP-12 concentrations or activity. CONCLUSIONS: Sputum MMP-12 concentrations and activity in patients with COPD are directly associated with the extent of emphysema measured by means of CT. This finding supports a role for MMP-12 in the pathogenesis of COPD and might suggest that blocking MMP-12 activity in patients with COPD could prevent the further development of emphysema.

Mast cells reside in myometrium and cervix, but are dispensable in mice for successful pregnancy and labor.

Parturition is associated with myometrial and cervical inflammation. The causes and consequences of this inflammatory response are not clear. Mast cells (MCs) are important inducers of allergic and non-allergic inflammation, and their secreted products can induce myometrial contractions. Thus, mast cell activation has been hypothesized to have a role in initiating labor and/or driving labor-associated inflammation. We report that small numbers of MCs expressing chymase and tryptase are present in the myometrium and cervix of pregnant women. Labor did not lead to any change in mast cell abundance in these tissues, but was associated with reduced expression of the mast-cell regulator FcεR1A, indicative of a change in mast cell properties. This coincided with contraction-dependent myocyte production of interleukin-10 (IL-10), a known suppressor of FcεR1A expression. MCs were also found in the uterine horn and cervical region of pregnant C57BL/6 mice, increasing in number in the cervix, but not the myometrium, with labor. As expected, these cells were absent from mast-cell-deficient Kit(W-sh) mice. Nonetheless, pregnant Kit(W-sh) mice showed no defects in the timing of labor induction or in the upregulation of leukocyte markers during labor. Thus, MCs are present in the uterus and cervix of humans and mice, and our mouse studies suggest that they do not have a vital role in the induction of labor, or in the promotion of labor-associated inflammation.

Corticosteroid Responsiveness Following Mepolizumab in Severe Eosinophilic Asthma-A Randomized, Placebo-Controlled Crossover Trial (MAPLE).

BACKGROUND: Mepolizumab inhibits IL-5 activity and reduces exacerbation frequency and maintenance oral corticosteroid (OCS) dosage in patients with severe eosinophilic asthma (SEA). Some patients remain dependent on OCS despite anti-IL-5 treatment, suggesting residual corticosteroid-responsive mechanisms. OBJECTIVE: To determine the clinical and anti-inflammatory effects of OCS in patients with SEA on mepolizumab. METHODS: We conducted a randomized, triple-blind, placebo-controlled crossover trial of prednisolone (0.5 mg/kg/d, maximum 40 mg/d, for 14 ± 2 days) in adults with SEA after 12 or more weeks of mepolizumab. We compared change in asthma symptoms, quality of life, lung function measured by spirometry and airwave oscillometry, fractional exhaled nitric oxide, and blood and sputum eosinophil cell count after prednisolone and placebo treatment. RESULTS: A total of 27 patients completed the study. Prednisolone did not improve 5-item Asthma Control Questionnaire (mean difference in change for prednisolone vs placebo, -0.23; 95% CI, -0.58 to 0.11), mini-Asthma Quality of Life Questionnaire (0.03; 95% CI, -0.26 to 0.42), St. George's Respiratory Questionnaire (0.24; 95% CI, -3.20 to 3.69), or Visual Analogue Scale scores for overall asthma symptoms (0.11; 95% CI, -0.58 to 0.80). The mean difference for FEV1 in favor of prednisolone was 105 mL (95% CI, -4 to 213 mL); forced expiratory flow at 25% and 75% 484 mL/s (95% CI, 151 to 816 mL/s); fractional exhaled nitric oxide reduction 41% (95% CI, 25% to 54%); blood eosinophil count reduction 49% (95% CI, 31% to 62%); and percentage of sputum eosinophil reduction 71% (95% CI, 26% to 89%). CONCLUSIONS: OCS improved small-airway obstruction and reduced biomarkers of type 2 inflammation but had no significant effect on symptoms or quality of life in patients with SEA receiving treatment with mepolizumab.

Functional expression of IgG-Fc receptors in human airway smooth muscle cells.

IgE-Fc receptors and IgG-Fc receptors are expressed on hematopoietic cells, but some evidence suggests that these receptors are also found on nonhematopoietic cells, including human airway smooth muscle (hASM) cells. Our study characterizes the expression of IgE-Fc receptors (FcεRI/CD23) and IgG-Fc receptors (FcγRs-I, -II, and -III) in cultured hASM cells by flow cytometry and Western blotting, and the functional activity of receptors was determined through quantification of cell proliferation and released cytokines. Expression of Fc receptor-linked intracellular signaling proteins and phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1/2 and p38(MAPK) in hASM cells was examined by Western blotting. Expression of FcεRI and CD23 was not detectable in hASM cells. However, FcγRI and FcγRII were shown to be expressed on these cells. Specific antibodies, validated using transfected cell lines, revealed that the inhibitory IgG receptor, FcγRIIb, was the most abundant Fc receptor subtype expressed. Although cross-linking FcγR with heat-aggregated γ globulin (HAGG) did not induce detectable cell stimulation, pretreating hASM cells with HAGG significantly inhibited IL-1α-induced increases in cytokine levels and basic fibroblast growth factor-induced cell proliferation. This inhibitory effect of HAGG was abrogated by preincubation of cells with an anti-FcγRIIb antigen-binding fragment (Fab). Expression of proteins involved in the canonical FcγRIIb inhibitory signaling pathway was established in hASM cells. Pretreatment of hASM cells with HAGG significantly inhibited IL-1α- and basic fibroblast growth factor-induced extracellular signal-regulated kinase 1/2 and p38(MAPK) phosphorylation. This study identifies functional expression of FcγRIIb in hASM cells, with the potential to suppress their remodeling and immunomodulatory roles.

IL-33 amplifies the polarization of alternatively activated macrophages that contribute to airway inflammation.

Alternatively activated macrophages (AAM) play a crucial role in type 2 immunity. Mice deficient in ST2, a receptor for the latest member of the IL-1 family, IL-33, have impaired type 2 immune responses. We therefore reasoned that IL-33/ST2 signaling may be involved in the differentiation and activation of AAM during airway inflammation. We report here that IL-33 changed the quiescent phenotype of alveolar macrophages toward an AAM phenotype that expressed mannose receptor, IL-4Ralpha, and produced high levels of CCL24 and CCL17 in an IL-13-dependent manner during IL-33-induced airway inflammation. Neutralization of AAM-derived CCL24 led to an amelioration of IL-33-induced eosinophilia in the lungs. Moreover, depletion of alveolar macrophages reduced IL-33-induced airway inflammation. Additionally, the attenuated OVA-induced airway inflammation in ST2(-/-) mice was associated with a decrease in AAM differentiation. In vitro, IL-33 amplified IL-13-induced polarization of alveolar- and bone marrow-derived macrophage toward an AAM phenotype by increasing the expression of arginase I, Ym1, as well as the production of CCL24 and CCL17. IL-13/IL-4Ralpha signaling was crucial for IL-33-driven AAM amplification by inducing the expression of ST2L. Finally, we showed that IL-33 was more abundantly expressed in the lung epithelial cells of asthma patients than those from healthy controls, suggesting that IL-33 may be involved in lung macrophage activation in clinical asthma. Taken together, we demonstrate here that IL-33/ST2 plays a significant role in the amplification of AAM polarization and chemokine production which contribute to innate and Ag-induced airway inflammation.

Ion channel regulation of intracellular calcium and airway smooth muscle function.

Airway hyper-responsiveness associated with asthma is mediated by airway smooth muscle cells (SMCs) and has a complicated etiology involving increases in cell contraction and proliferation and the secretion of inflammatory mediators. Although these pathological changes are diverse, a common feature associated with their regulation is a change in intracellular Ca(2+) concentration ([Ca(2+)](i)). Because the [Ca(2+)](i) itself is a function of the activity and expression of a variety of ion channels, in both the plasma membrane and sarcoplasmic reticulum of the SMC, the modification of this ion channel activity may predispose airway SMCs to hyper-responsiveness. Our objective is to review how ion channels determine the [Ca(2+)](i) and influence the function of airway SMCs and emphasize the potential of ion channels as sites for therapeutic approaches to asthma.

Insights into frequent asthma exacerbations from a primary care perspective and the implications of UK National Review of Asthma Deaths recommendations.

The United Kingdom National Review of Asthma Deaths (NRAD) recommends that patients who require ≥3 courses of oral corticosteroids (OCS) for exacerbations in the past year or those on British Thoracic Society (BTS) Step 4/5 treatment must be referred to a specialist asthma service. The aim of the study was to identify the proportion of asthma patients in primary care that fulfil NRAD criteria for specialist referral and factors associated with frequent exacerbations. A total of 2639 adult asthma patients from 10 primary care practices in Glasgow, UK were retrospectively studied between 2014 and 2015. Frequent exacerbators and short-acting ß2-agonist (SABA) over-users were identified if they received ≥2 confirmed OCS courses for asthma and ≥13 SABA inhalers in the past year, respectively. Community dispensing data were used to assess treatment adherence defined as taking ≥75% of prescribed inhaled corticosteroid (ICS) dose. The study population included 185 (7%) frequent exacerbators, 137 (5%) SABA over-users, and 319 (12%) patients on BTS Step 4/5 treatment. Among frequent exacerbators, 41% required BTS Step 4/5 treatment, 46% had suboptimal ICS adherence, 42% had not attended an asthma review in the past year and 42% had no previous input from a specialist asthma service. Older age, female gender, BTS Step 4/5, SABA over-use and co-existing COPD diagnosis increased the risk of frequent exacerbations independently. Fourteen per 100 asthma patients would fulfil the NRAD criteria for specialist referral. Better collaboration between primary and secondary care asthma services is needed to improve chronic asthma care.

KCa3.1 Ca2+ activated K+ channels regulate human airway smooth muscle proliferation.

Airway smooth muscle cell hyperplasia contributes to airway remodeling and hyperreactivity characteristic of asthma. Changes to potassium channel activity in proliferating human airway smooth muscle (HASM) cells have been described, but no regulatory role in proliferation has been attributed to them. We sought to investigate the expression of the intermediate conductance calcium-activated potassium channel K(Ca)3.1 in HASM cells and investigate its role in proliferation. Smooth muscle cells derived from human airways were grown in vitro and K(Ca)3.1 channel expression was measured using Western blot, RT-PCR, and patch clamp electrophysiology. Pharmacologic inhibitors of the channel were used in assays of cellular proliferation, and flow cytometry was used to identify cell cycle regulation. HASM cells expressed K(Ca)3.1 channel mRNA, protein, and activity with up-regulation evident after transforming growth factor-beta stimulation. Pharmacologic inhibition of K(Ca)3.1 led to growth arrest in cells stimulated to proliferate with mitogens. These inhibitors did not cause cellular toxicity or induce apoptosis. We have demonstrated, for the first time, the expression of K(Ca)3.1 channels in HASM cells. In addition, we have shown that K(Ca)3.1 channels are important in HASM cell proliferation, making these channels a potential therapeutic target in airway remodeling.

Corticosteroid insensitivity in smokers with asthma : clinical evidence, mechanisms, and management.

Corticosteroids are the most effective treatment for asthma, but the therapeutic response varies considerably between individuals. Several clinical studies have found that smokers with asthma are insensitive to the beneficial effects of short- to medium-term inhaled corticosteroid treatment compared with non-smokers with asthma. It is estimated that 25% of adults in most industrialized countries smoke cigarettes, and similar surveys amongst asthmatic individuals suggest that the prevalence of smoking in this grouping mirrors that found in the general population. Therefore, cigarette smoking is probably the most common cause of corticosteroid insensitivity in asthma. Cigarette smoking and asthma are also associated with poor symptom control and an accelerated rate of decline in lung function. The mechanism of corticosteroid insensitivity in smokers with asthma is currently unexplained but could be due to alterations in airway inflammatory cell phenotypes, changes in glucocorticoid receptor alpha/beta ratio, and/or reduced histone deacetylase activity. Smoking cessation should be encouraged in all smokers with asthma. Short-term benefits include improvements in lung function and asthma control. However, the numbers of sustained quitters is disappointingly small. Additional or alternative drugs need to be identified to treat those individuals who are unable to stop smoking or who have persistent symptoms following smoking cessation.

Effects of older age and age of asthma onset on clinical and inflammatory variables in severe refractory asthma.

BACKGROUND: Asthma in the elderly as well as asthma of adult-onset has been associated with increased morbidity, but little is known specifically about the effects of age on clinical and inflammatory outcomes in severe refractory asthma. The aims of the study were to examine the effects of age [<65 versus ≥65 years] and age of onset of asthma [childhood-onset, <18 versus adult-onset, ≥18 years] on clinical and inflammatory variables in patients with severe asthma. METHODS: In 1042 subjects with refractory asthma recruited to the British Thoracic Society Severe Asthma Registry, we compared patient demographics, disease characteristics and biomarkers of inflammation in patients aged <65 years (n = 896) versus ≥65 years (n = 146) and onset at age <18 years (n = 430) versus ≥18 years (n = 526). RESULTS: Severe asthma patients aged ≥65 years had improved symptom control, better asthma quality of life and in the last year, less emergency visits and rescue oral steroid courses [3 (1-6) versus 5 (2-7), p < 0.001] than severe asthmatics aged <65 years. Blood eosinophils were lower in the elderly group. Patients with severe adult-onset asthma had similar symptom control, lung function and health-care utilization compared to severe childhood-onset asthma. Adult-onset asthmatics had higher blood eosinophils and were less atopic. CONCLUSIONS: Patients with severe refractory asthma aged ≥65 years exhibit better clinical and health care outcomes and have lower blood eosinophils compared to those aged <65 years. Severe refractory adult-onset asthma is associated with similar levels of asthma control, higher blood eosinophils and less atopy than severe refractory childhood-onset asthma.

Effectiveness of bronchial thermoplasty in severe asthma in 'real life' patients compared with those recruited to clinical trials in the same centre.

Published information on the effectiveness of bronchial thermoplasty (BT) for severe asthma in 'real life' patients is limited. We compared safety and efficacy outcomes 12 months post procedure in 10 clinic patients and 15 patients recruited to clinical trials of BT at the same centre. Baseline asthma severity was greater in the clinic group. Adverse events were similar. Clinical improvements occurred in 50% of the clinic patients compared with 73% of the research patients.

Remodelling of the PDE4 cAMP phosphodiesterase isoform profile upon monocyte-macrophage differentiation of human U937 cells.

Monocytes and macrophages provide key targets for the action of novel anti-inflammatory therapeutics targeted at inhibition of PDE4 cAMP-specific phosphodiesterases. PDE4 enzymes provide the dominant cAMP phosphodiesterase activity in U937 human monocytic cells. Differentiation of U937 monocytic cells to a macrophage-like phenotype causes a marked reduction in total cellular PDE4 activity. Monocytic U937 cells express the long PDE4A4, PDE4D5 and PDE4D3 isoforms plus the short PDE4B2 isoform. Differentiation of U937 cells to a macrophage-like phenotype causes a marked downregulation of PDE4D3 and PDE4D5, elicits a marked upregulation of PDE4B2 and induces the novel PDE4A10 long isoform. Comparable patterns are found in human peripheral blood monocytes and macrophages differentiated from them. Immunopurification of PDE4 subfamilies identifies long PDE4D isoforms as providing the major PDE4 activity in U937 monocytic cells. In U937 macrophage-like cells, the activity of the short PDE4B2 isoform predominates. No indication of either the expression or induction of PDE4C was evident. Activation of ERK exerts an inhibitory effect on total PDE4 activity in monocytic U937 cells, where the activity of long PDE4 isoforms predominates. The effect of ERK activation is switched to one of overall stimulation of total PDE4 activity in macrophage U937 cells, where the activity of the short PDE4B2 isoform predominates.10 The profound differentiation-induced changes in PDE4 isoform profile identified here suggests that the development of inhibitors specific for particular PDE4 isoforms may allow for selective effects on monocytes and macrophages to be achieved.

Differential expression of PDE4 cAMP phosphodiesterase isoforms in inflammatory cells of smokers with COPD, smokers without COPD, and nonsmokers.

The expression profile of a panel of 15 cAMP phosphodiesterase isoforms was determined for inflammatory cell types of relevance to chronic obstructive pulmonary disease (COPD). In particular, the expression profiles for bronchoalveolar macrophages, peripheral blood monocytes, T lymphocytes, and neutrophils from smokers with and without COPD were compared. The phosphodiesterase expression profile was also analyzed for peripheral blood monocytes, T lymphocytes, and neutrophils from nonsmokers and compared with smokers. Qualitative RT-PCR identified transcripts for PDE4A10, PDE4A7, PDE4B1, PDE4B2, PDE4D1, and PDE4D2 isoforms as well as transcripts for both PDE3B and PDE7A in T cells, monocytes, and macrophages in all subjects. Transcripts for PDE4B3 and PDE4D4 were not observed in any of the cell types investigated. PDE4C was detected in all cells analyzed except for T cells. The long PDE4A4, PDE4D3, and PDE4D5 isoforms exhibited cell type-specific expression patterns. Semiquantitative and real-time quantitative RT-PCR were used to analyze differential expression between disease states and between cell types. PDE4A4 was found significantly upregulated in lung macrophages from smokers with COPD when compared with control smokers. Furthermore, PDE4A4 as well as PDE4B2 transcripts were detected in higher amounts in peripheral blood monocytes of smokers when compared with nonsmokers. Finally, PDE4D5 and PDE4C were differentially regulated in lung macrophages when compared with monocytes of the same subjects, irrespective of the disease state. The data obtained suggest that PDE4A4 may be relevant as a macrophage-specific anti-inflammatory target for COPD.

Cyclic AMP-dependent transcriptional up-regulation of phosphodiesterase 4D5 in human airway smooth muscle cells. Identification and characterization of a novel PDE4D5 promoter.

Phosphodiesterase 4D (PDE4D), part of the complex cAMP-specific PDE4 family, plays a pivotal role in the regulation of airway smooth muscle relaxation by catalyzing the hydolysis of cAMP. Its gene on chromosome 5q12 encodes 5 splice variants, which show tissue-dependent expression and regulation. The genomic arrangement of PDE4D was determined using in silico methods, and a putative promoter of one of the protein kinase A-activated, long isoforms, PDE4D5 was identified. Promoter-luciferase constructs, transiently transfected into a beta(2) adrenoreceptor-expressing CHO-K1 cell line, were used to demonstrate that the PDE4D5 promoter up-regulated reporter gene expression in response to increased cell cAMP. Site-directed mutagenesis of the cAMP-response element (CRE) at position -201 identified this as the principal component of the mechanism underlying this cAMP responsiveness. In the second part of this study, cAMP-dependent induction of PDE4D5 transcript in primary cultured human airway smooth muscle cells (hASMs) was demonstrated using both qualitative reverse-transcriptase PCR and quantitative real-time PCR. Isolated PDE4D5 isoenzyme activity, measured after selective immunoprecipitation from hASMs, confirmed that this increase in expression led to an up-regulation of functional activity. We present evidence for cAMP-driven PDE4D5 up-regulation in hASMs and suggest a CRE-containing, isoform-specific promoter as the primary mechanism.

Molecular cloning and subcellular distribution of the novel PDE4B4 cAMP-specific phosphodiesterase isoform.

We have isolated cDNAs encoding PDE4B4, a new cAMP-specific phosphodiesterase (PDE4) isoform with novel properties. The amino acid sequence of PDE4B4 demonstrates that it is encoded by the PDE4B gene, but that it differs from the previously isolated PDE4B1, PDE4B2 and PDE4B3 isoforms by the presence of a novel N-terminal region of 17 amino acids. PDE4B4 contains both of the upstream conserved region 1 (UCR1) and UCR2 regulatory units that are characteristic of 'long' PDE4 isoforms. RNase protection demonstrated that PDE4B4 mRNA is expressed preferentially in liver, skeletal muscle and various regions of the brain, which differs from the pattern of tissue distribution of the other known PDE4B long forms, PDE4B1 and PDE4B3. Expression of PDE4B4 cDNA in COS7 cells produced a protein of 85 kDa under denaturing conditions. Subcellular fractionation of recombinant, COS7-cell expressed PDE4B4 showed that the protein was localized within the cytosol, which was confirmed by confocal microscopic analysis of living COS7 cells transfected with a green fluorescent protein-PDE4B4 chimaera. PDE4B4 exhibited a K(m) for cAMP of 5.4 microM and a V(max), relative to that of the long PDE4B1 isoform, of 2.1. PDE4B4 was inhibited by the prototypical PDE4 inhibitor rolipram [4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidinone] with an IC(50) of 83 nM. Treatment of COS7 cells with forskolin, to elevate cAMP levels, produced activation of PDE4B4, which was associated with the phosphorylation of PDE4B4 on Ser-56 within UCR1. The unique tissue distribution and intracellular targeting of PDE4B4 suggests that this isoform may have a distinct functional role in regulating cAMP levels in specific cell types.