Validation of antibodies for the specific detection of human TRPA1.

The transient receptor potential cation channel family member ankyrin 1 (TRPA1) is a potential target for several diseases, but detection of human TRPA1 (hTRPA1) protein in cells and tissues is problematic as rigorous antibody validation is lacking. We expressed hTRPA1 in a TRPA1-negative cell line to evaluate 5 commercially available antibodies by western blotting, immunofluorescence, immunocytochemistry and flow cytometry. The three most cited anti-TRPA1 antibodies lacked sensitivity and/or specificity, but two mouse monoclonal anti-TRPA1 antibodies detected hTRPA1 specifically in the above assays. This enabled the development of a flow cytometry assay, which demonstrated strong expression of TRPA1 in human lung myofibroblasts, human airway smooth muscle cells but not lung mast cells. The most cited anti-TRPA1 antibodies lack sensitivity and/or specificity for hTRPA1. We have identified two anti-TRPA1 antibodies which detect hTRPA1 specifically. Previously published data regarding human TRPA1 protein expression may need revisiting.

Increased ß2-adrenoceptor phosphorylation in airway smooth muscle in severe asthma: possible role of mast cell-derived growth factors.

The purpose of this study was to investigate whether growth factors produced by activated human lung mast cells (HLMCs) impair ß2 -adrenoceptor (ß2 -AR) function in human airway smooth muscle (ASM) cells. Protein array analysis confirmed the presence of various growth factors, including transforming growth factor (TGF)-ß1, in the supernatants of high-affinity IgE receptor (FcεRI)-activated HLMCs which, when applied to ASM cells, impaired albuterol-induced cyclic adenosine monophosphate (cAMP) production, an effect that was prevented following neutralization of TGF-ß1. This blunted ß2 -AR response was reproduced by treating ASM cells with TGF-ß1 or fibroblast growth factor (FGF)-2, which induced ß2 -AR phosphorylation at tyrosine residues Tyr141 and Tyr350 , and significantly reduced the maximal bronchorelaxant responses to isoproterenol in human precision cut lung slices (PCLS). Finally, ASM cells isolated from severe asthmatics displayed constitutive elevated ß2 -AR phosphorylation at both Tyr141 and Tyr350 and a reduced relaxant response to albuterol. This study shows for the first time that abnormal ß2 -AR phosphorylation/function in ASM cells that is induced rapidly by HLMC-derived growth factors, is present constitutively in cells from severe asthmatics.

Protein phosphatase 5 mediates corticosteroid insensitivity in airway smooth muscle in patients with severe asthma.

BACKGROUND: The mechanisms driving glucocorticoid (GC) insensitivity in patients with severe asthma are still unknown. Recent evidence suggests the existence of GC-insensitive pathways in airway smooth muscle (ASM) caused by a defect in GC receptor (GRα) function. We examined whether other mechanisms could potentially explain the reduced sensitivity of ASM cells to GC in severe asthmatics. METHODS: Airway smooth muscle cells from healthy and severe asthmatic subjects were treated with TNF-α and responses to corticosteroids in both cohorts were compared by ELISA, immunoblot, immunohistochemistry and real-time PCR. Immunohistochemistry and flow cytometry assays were used to assess the expression of the protein phosphatase PP5 in endobronchial biopsies and ASM cells. RESULTS: The production of CCL11 and CCL5 by TNF-α was insensitive to both fluticasone and dexamethasone in ASM cells from severe asthmatic compared to that in healthy subjects. Fluticasone-induced GRα nuclear translocation, phosphorylation at serine 211 and expression of GC-induced leucine zipper (GILZ) were significantly reduced in ASM cells from severe asthmatics compared to responses in healthy subjects. Levels of PP5 were increased in ASM cells from severe asthmatics and PP5 knockdown using siRNA restored fluticasone repressive action on chemokine production and its ability to induce GRα nuclear translocation and GRE-dependent GILZ expression. In vivo PP5 expression was also increased in the ASM bundles in endobronchial biopsies in severe asthmatics. CONCLUSIONS: PP5-dependent impairment of GRα function represents a novel mechanism driving GC insensitivity in ASM in severe asthma.

Abnormal corticosteroid signalling in airway smooth muscle: mechanisms and perspectives for the treatment of severe asthma.

Growing in vivo evidence supports the concept that airway smooth muscle produces various immunomodulatory factors that could contribute to asthma pathogenesis via the regulation of airway inflammation, airway narrowing and remodelling. Targeting ASM using bronchial thermoplasty has provided undeniable clinical benefits for patients with uncontrolled severe asthma who are refractory to glucocorticoid therapy. The present review will explain why the failure of glucocorticoids to adequately manage patients with severe asthma could derive from their inability to affect the immunomodulatory potential of ASM. We will support the view that ASM sensitivity to glucocorticoid therapy can be blunted in severe asthma and will describe some of the factors and mechanisms that could be responsible for glucocorticoid insensitivity.

Mechanisms of glucocorticoid action and insensitivity in airways disease.

Glucocorticoids are the mainstay for the treatment of chronic inflammatory diseases including asthma and chronic obstructive pulmonary disease (COPD). However, it has been recognized that glucocorticoids do not work well in certain patient populations suggesting reduced sensitivity. The ultimate biologic responses to glucocorticoids are determined by not only the concentration of glucocorticoids but also the differences between individuals in glucocorticoid sensitivity, which is influenced by multiple factors. Studies are emerging to understand these mechanisms in detail, which would help in increasing glucocorticoid sensitivity in patients with chronic airways disease. This review aims to highlight both classical and emerging concepts of the anti-inflammatory mechanisms of glucocorticoids and also review some novel strategies to overcome steroid insensitivity in airways disease.