Rationale: Benralizumab is a humanized, afucosylated anti-IL-5Rα monoclonal antibody shown to reduce sputum and blood eosinophils and improve FEV1 in a Phase 2a study in adults with GOLD II-IV COPD1. An exploratory analysis investigated its effect on blood basophils and potential serum pharmacodynamic markers. Methods: The randomized, double-blind, placebo-controlled study was conducted in subjects on standard of care(n=101) with >1 COPD exacerbation and >3.0% sputum eosinophils in previous year1. Subjects received subcutaneous benralizumab (100 mg) or placebo every 28-days (3 doses) then every 56-days (5-doses). Blood and serum were collected at Day 1 (baseline) and Days 29, 113, and 225. Blood basophils were evaluated using a hematology analyzer; serum was analyzed for biomarkers using multi-analyte protein profiling. Results: In comparison with placebo, benralizumab significantly suppressed (p<0.05) mean absolute blood–basophil count at Days 29 (23 vs 50 cells/μL), 113 (24 vs 54 cells/μL), and 225 (35 vs 56 cells/μL), with no significant effect on neutrophil counts, and was associated with significant (p<0.05) increases in CCL11/eotaxin-1 and CCL24/eotaxin-2 at Day 225 (105% and 45% respective increases relative to baseline). Conclusion: Eosinophils, blood basophils and the eosinophil chemokines were modulated with benralizumab administration and could serve as serum markers of treatment. Increased levels of the chemokines are not unexpected and may result from accumulation following removal of eosinophils. Other mechanisms may be involved. 1Brightling CE et al. Lancet Resp Med. 2014. doi: 10.1016/S2213-2600(14)70187-0.

BACKGROUND AND PURPOSE Previous studies have linked a reduction in pH in airway, caused by either environmental factors, microaspiration of gastric acid or inflammation, with airway smooth muscle (ASM) contraction and increased airway resistance. Neural mechanisms have been shown to mediate airway contraction in response to reductions in airway pH to < 6.5; whether reduced extracellular pH (pHo) has direct effects on ASM is unknown. EXPERIMENTAL APPROACH Intracellular signalling events stimulated by reduced pHo in human cultured ASM cells were examined by immunoblotting, phosphoinositide hydrolysis and calcium mobilization assays. ASM cell contractile state was examined using magnetic twisting cytometry. The expression of putative proton-sensing GPCRs in ASM was assessed by real-time PCR. The role of ovarian cancer G protein-coupled receptor 1 (OGR1 or GPR68) in acid-induced ASM signalling and contraction was assessed in cultures subjected to siRNA-mediated OGR1 knockdown. KEY RESULTS ASM cells responded to incremental reductions in pHo (from pH 8.0 to pH 6.8) by activating multiple signalling pathways, involving p42/p44, PKB, PKA and calcium mobilization. Coincidently, ASM cells contracted in response to decreased pHo with similar dose-dependence. Real-time PCR suggested OGR1 was the only proton-sensing GPCR expressed in ASM cells. Both acid-induced signalling (with the exception of PKB activation) and contraction were significantly attenuated by knockdown of OGR1. CONCLUSIONS AND IMPLICATIONS These studies reveal OGR1 to be a physiologically relevant GPCR in ASM cells, capable of pleiotropic signalling and mediating contraction in response to small reductions in extracellular pH. Accordingly, ASM OGR1 may contribute to asthma pathology and represent a therapeutic target in obstructive lung diseases.

Evidence suggests inhibition of leukocyte trafficking mitigates, in part, ozone-induced inflammation. In the present study, the authors postulated that inhibition of myristoylated alanine-rich C kinase substrate (MARCKS), an 82-kDa protein with multiple biological roles, could inhibit ozone-induced leukocyte trafficking and cytokine secretions. BALB/c mice (n = 5/cohort) were exposed to ozone (100 ppb) or forced air (FA) for 4 hours. MARCKS-inhibiting peptides, MANS, BIO-11000, BIO-11006, or scrambled control peptide RNS, were intratracheally administered prior to ozone exposure. Ozone selectively enhanced bronchoalveolar lavage (BAL) levels of killer cells (KCs; 6 +/- 0.9-fold), interleukin-6 (IL-6; 12.7 +/- 1.9-fold), and tumor necrosis factor (TNF; 2.1 +/- 0.5-fold) as compared to cohorts exposed to FA. Additionally, ozone increased BAL neutrophils by 21% +/- 2% with no significant (P > .05) changes in other cell types. MANS, BIO-11000, and BIO-11006 significantly reduced ozone-induced KC secretion by 66% +/- 14%, 47% +/- 15%, and 71.1% +/- 14%, and IL-6 secretion by 69% +/- 12%, 40% +/- 7%, and 86.1% +/- 11%, respectively. Ozone-mediated increases in BAL neutrophils were reduced by MANS (86% +/- 7%) and BIO-11006 (84% +/- 2.5%), but not BIO-11000. These studies identify for the first time the novel potential of MARCKS protein inhibitors in abrogating ozone-induced increases in neutrophils, cytokines, and chemokines in BAL fluid. BIO-11006 is being developed as a treatment for chronic obstructive pulmonary disorder (COPD) and is currently being evaluated in a phase 2 clinical study.

Asthma, a chronic disease characterized by airway hyperreactivity, is an extraordinarily common cause of pulmonary impairment. Despite considerable research effort, asthma mortality rates continue to rise and the primary defects that underlie airway hyperreactivity remain unknown although an intrinsic abnormality of airways smooth muscle has been postulated [1–4].

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 (IC50= 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 p21Waf/Cip‐1, p27Kip1, cyclin D and E2F‐1. Consistent with these observations, SB218078 also inhibited (IC50= 450 ± 100 pM) PDGF‐induced cell cycle progression.Conclusions and implications: Calcitriol decreased PDGF‐induced ASM cell growth by inhibiting Rb and Chk1 phosphorylation.This Research Paper is the subject of a Commentary in this issue by Clifford and Knox (pp. 1426–1428). To view this article visit http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009

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 1 alpha, 25-dihydroxy vitamin D-3 (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 (TNF alpha) and/or interferon gamma (IFN gamma) 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 TNF alpha-treated cells, calcitriol inhibited RANTES and IP-10 secretion in a concentration-dependent manner. FKN levels were negligible. In TNF alpha/IFN gamma-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.

Mucus hyper-secretion is a pathological feature associated with airway diseases such as allergic asthma. Multiple lines of evidence suggest that allergen-specific CD4+ Th2 cells, through the release of a variety of cytokines and chemokines play a critical role in initiating and sustaining this abnormality. With this background, we investigated the potential role of IL-4 and IL-9, two pleotropic Th2 cytokines predominant in asthmatic lungs, in modulating the expression of airway mucin gene MUC8. Initially, the presence of IL-4 and IL-9 receptors on NCI-H650 cells was established by immunohistochemical methods. Real time PCR analysis of total RNA from IL-4 and IL-9 treated cultures revealed a time and concentration dependant enhancement, reaching peak expression levels at 2.5 ng/ml and 10 ng/ml respectively, after 8 h of exposure. Nuclear-run on assays revealed transcriptional regulation of MUC8 upon stimulus with either cytokines. Pre-treatments with a JAK-3 selective inhibitor, WHI-P131 suppressed IL-4 and IL-9 modulated MUC8-increase in a concentration dependant fashion. Western blotting analysis of cell lysates from cytokine treated confluent cultures revealed phosporylation of STAT-6 transcription factor within 10 min of exposure. Our findings reveal that Th2 cytokines such as IL-9 and IL-4 increase MUC8 expression at the transcriptional level via a JAK3/STAT-6 selective pathway. This study was supported in part by NIH grant, HL34012.