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.

Steroid resistance in severe asthma: current mechanisms and future treatment.

The disproportionate cost of treating asthmatic patients who do not respond to conventional anti-inflammatory therapies makes delineation of the mechanism for glucocorticoid resistance an important field of asthma research. Unbiased cluster analysis indicates that asthma is a syndrome with a number of distinct phenotypes and 5-10% of asthmatics fall into this category of relative glucocorticoid insensitivity. This sub-population is itself divided into smaller subsets which have different underlying mechanisms for this relative glucocorticoid resistance ranging from an inherited genetic basis to specific kinase signalling pathways triggered by exposure to environmental stressors such as cigarette smoking or infection. Whilst the underlying mechanisms are becoming better understood there remains a lack of effective novel therapies. However it is clear that relative glucocorticoid insensitive patients who are smokers should be encouraged to quit, thereby reducing their oxidant load. Novel treatments will consist of either developing new anti-inflammatory treatments targeting pathways aberrantly activated in these patients or of suppressing signalling pathways that attenuate glucocorticoid receptor function and thereby restoring glucocorticoid sensitivity. It will be important to uncover non-invasive biomarkers for aberrant pathway activation and for discerning which components of glucocorticoid receptor activation are abnormal if future treatments are to be tailored to address these specific issues. Conventional combination therapies will continue to be used in the near future but additional add-on treatments using drugs directed against aberrantly expressed inflammatory pathways or mediators along with an inhaled glucocorticoid are likely to prove the most effective new therapies in the future.

Vitamin D inhibits growth of human airway smooth muscle cells through growth factor-induced phosphorylation of retinoblastoma protein and checkpoint kinase 1.

BACKGROUND AND PURPOSE: Airway remodelling in asthma is manifested, in part, as increased airway smooth muscle (ASM) mass, reflecting myocyte proliferation. We hypothesized that calcitriol, a secosteroidal vitamin D receptor (VDR) modulator, would inhibit growth factor-induced myocyte proliferation. EXPERIMENTAL APPROACH: Human ASM cell cultures were derived from bronchial samples taken during surgery. ASM cells were treated with platelet-derived growth factor (PDGF) (10 ng.mL(-1)) for 24 h in the presence of calcitriol, dexamethasone or a checkpoint kinase 1 (Chk1) inhibitor (SB218078). The effects of calcitriol on PDGF-mediated cell proliferation were assessed by thymidine incorporation assay, propidium iodide-based cell cycle analysis, caspase-3 assay and immunoblotting for specific cell cycle modulators. KEY RESULTS: Calcitriol, but not dexamethasone, inhibited PDGF-induced ASM DNA synthesis concentration dependently (IC(50)= 520 +/- 52 nM). These effects were associated with VDR-mediated expression of cytochrome CYP24A1 with no effects on ASM apoptosis. Calcitriol substantially inhibited (P < 0.01) PDGF-stimulated cell growth in ASM derived from both normal (59 +/- 8%) and asthmatic subjects (57 +/- 9%). Calcitriol inhibited PDGF-induced phosphorylation of retinoblastoma protein (Rb) and Chk1, with no effects on PDGF-mediated activation of extracellular signal-regulated kinases 1/2, PI3-kinase and S6 kinase, or expression of p21(Waf/Cip-1), p27(Kip1), cyclin D and E2F-1. Consistent with these observations, SB218078 also inhibited (IC(50)= 450 +/- 100 pM) PDGF-induced cell cycle progression. CONCLUSIONS AND IMPLICATIONS: Calcitriol decreased PDGF-induced ASM cell growth by inhibiting Rb and Chk1 phosphorylation.

Vitamin D and glucocorticoids differentially modulate chemokine expression in human airway smooth muscle cells.

BACKGROUND AND PURPOSE: Chemokines play a critical role in the pathogenesis of asthma and facilitate the recruitment of inflammatory cells in the airways. Evidence now suggests that airway smooth muscle (ASM) may serve as a source of chemokines in inflamed airways. Although vitamin D has potent anti-inflammatory properties in vitro in some cell types, its effects on ASM cells remain unclear. Here, we investigated whether 1alpha, 25-dihydroxy vitamin D3 (calcitriol) modulated chemokine production in ASM. EXPERIMENTAL APPROACH: Human ASM cell cultures were derived from tracheal samples taken during surgery. ASM cells were treated with tumour necrosis factor alpha (TNFalpha) and/or interferon gamma (IFNgamma) for 24 h in the presence of calcitriol and/or the glucocorticoid fluticasone added 2 h before. RANTES (regulated upon activation, normal T-cell expressed and secreted), interferon-inducible protein 10 (IP-10) and fractalkine (FKN) levels in cell supernatants were measured by ELISA. KEY RESULTS: In TNFalpha-treated cells, calcitriol inhibited RANTES and IP-10 secretion in a concentration-dependent manner. FKN levels were negligible. In TNFalpha/IFNgamma-treated cells, whereas fluticasone or calcitriol alone partially inhibited RANTES secretion (by 38 and 20%, respectively), the combination of both drugs additively inhibited RANTES secretion (by 60%). No effect was observed on IP-10 secretion. Whereas fluticasone enhanced FKN secretion (by 50%), calcitriol significantly decreased FKN levels (by 50%). Interestingly, calcitriol blocked the stimulatory effect of fluticasone on FKN secretion, which was inhibited by 60% with the combination of calcitriol and fluticasone. CONCLUSIONS AND IMPLICATIONS: These findings suggest that vitamin D uniquely modulates human ASM expression of chemokines and may exert some beneficial effects in the treatment of steroid-resistant patients with asthma.

Early hepatic cytokine mRNA expression in experimental rat fasciolosis.

We studied the development of the cellular response, particularly with respect to Th1 and Th2 cytokine mRNA levels, in rat liver during the first 14 days of experimental infection with Fasciola hepatica. We analysed the panel of cytokines involved in initiation of the inflammatory and immune response. The levels of various mRNAs, particularly those primarily associated with the acute inflammatory response, and those commonly associated with T-cell proliferation and differentiation, were assessed by reverse transcription-polymerase chain reaction (RT-PCR) in liver samples. We also investigated the immune and inflammatory mediators balance in the liver, draining lymph node and spleen, by RT-competitive PCR quantification of mRNA levels for IL-4, IL-10 and IFN-gamma. Our data provide the first evidence that, in the early phase of infection, the inflammatory response in the liver of infected animals is transiently depressed or delayed. A Th0 profile was initially observed in the liver and hepatic lymph node, which developed into a Th2 profile 2 weeks after infection in the liver only. In the spleen, cytokine down-regulation was initiated and maintained during this period, suggesting that the parasite acts differently locally and in the periphery.

Local hepatic immune response in rats during primary infection with Fasciola hepatica.

The distribution of lymphocyte subpopulations (TCD4+, TCD8+, TCD43+ and Ig+ cells), macrophages and eosinophils were analysed in the inflammatory infiltrates associated with hepatic lesions and in hepatic lymph nodes (HLN) from rats experimentally infected with F. hepatica and necropsied 1, 2, 3, 4, 6 and 8 week post infection (WPI). We also investigated the fixation of immunoglobulin isotypes on migrating flukes in the liver. As early as 1WPI, portal tract areas surrounding migratory tunnels were infiltrated with immune and inflammatory cells. The dominant cells were eosinophils and to lesser extent, macrophages and lymphocytes (TCD4+, TCD8+ and B). Most of the inflammatory and immune cells reached the posterior part of flukes, whereas in front of the parasites these cells were fewer in number. Except for eosinophils, no immune cells penetrated through granuloma consisting of hepatic necrotic cells. As early as 1WPI, IgM could be detected in the liver, and to a lesser extent IgA, IgG2a and IgG2b. At 2WPI, IgE and IgG1 began being detected. IgG2c was detectable at 3WPI. In HLN, we observed numerous microscopic follicles in the cortical zone with proliferation of germinal centres and medullary cords. The protective role of infiltrating cell populations and immunoglobulin isotypes and possible mechanisms of immune evasion by the parasite are discussed.