Rationale: Patients with chronic obstructive pulmonary disease (COPD) show a poor response to corticosteroids. This has been linked to a reduction of histone deacetylase-2 as a result of oxidative stress and is reversed by theophylline. Objectives: To determine the role of phosphoinositide-3-kinase-delta (PI3K-d) on the development of corticosteroid insensitivity in COPD and under oxidative stress, and as a target for theophylline. Methods: Corticosteroid sensitivity was determined as the 50% inhibitory concentration of dexamethasone on tumor necrosis factor-a-induced interleukin-8 release in peripheral blood mononuclear cells from patients with COPD (n 5 17) and compared with that of nonsmoking (n 5 8) and smoking (n 5 7) control subjects. The effect of theophylline and a selective PI3K-d inhibitor (IC87114) on restoration of corticosteroid sensitivity was confirmed in cigarette smoke-exposed mice. Measurements and Main Results: Peripheral blood mononuclear cells of COPD (50% inhibitory concentration of dexamethasone: 156.8 6 32.6 nM) were less corticosteroid sensitive than those of nonsmoking (41.2 6 10.5 nM; P 5 0.018) and smoking control subjects (47.5 6 19.6 nM; P 5 0.031). Corticosteroid insensitivity and reduced histone deacetylase-2 activity after oxidative stress were reversed by a non-selective PI3K inhibitor (LY294002) and low concentrations of theophylline. Theophylline was a potent selective inhibitor of oxidant-activated PI3K-d, which was up-regulated in peripheral lung tissue of patients with COPD. Furthermore, cells with knock-down of PI3K-d failed to develop corticosteroid insensitivity with oxidative stress. Both theophylline and IC87114, combined with dexamethasone, inhibited corticosteroid-insensitive lung inflammation in cigarette-smoke-exposed mice in vivo. Conclusions: Inhibition of oxidative stress dependent PI3K-d activation by a selective inhibitor or theophylline provides a novel approach to reversing corticosteroid insensitivity in COPD.

Corticosteroid insensitivity (CI) is a major barrier to treating severe asthma. Despite intensive research, the molecular mechanism of CI remains uncertain. The aim of this study was to determine abnormality in corticosteroid action in severe asthma and to identify the molecular mechanism of the longacting ␤ 2 -adrenergic agonists (LABAs) formoterol and salmeterol on restoration of corticosteroid sensitivity in severe asthma in vitro. Peripheral blood mononuclear cells (PBMCs) were obtained from 16 subjects with severe corticosteroid-insensitive asthma, 6 subjects with mild corticosteroid-sensitive asthma, and 11 healthy volunteers. Corticosteroid (dexamethasone) sensitivity was determined on tumor necrosis factor-␣ (TNF-␣)-induced interleukin (IL)-8 production. Glucocorticoid receptor (GR) phosphorylation and kinase phosphorylation were evaluated by immunoprecipitation-Western blotting analysis and kinase phosphorylation array in IL-2/IL-4-treated corticosteroid insensitive model in PBMCs. In vitro corticosteroid sensitivity on TNF-␣-induced IL-8 production was significantly lower in patients with severe asthma than in healthy volunteers and patients with mild asthma. This CI seen in severe asthma was associated with reduced GR nuclear translocation and with hyperphosphorylation of GR, which were reversed by LABAs. In IL-2/IL-4-treated PBMCs, LABAs inhibited phosphorylation of Jun-NH 2 -terminal kinase and p38 mitogen-activated protein kinase-␥ (p38MAPK-␥) as well as GR. In addition, cells with p38MAPK-␥ knockdown by RNA interference did not develop CI in the presence of IL-2/IL-4. Furthermore, p38MAPK-␥ protein expression was up-regulated in PBMCs from some patients with severe asthma. In conclusion, p38 MAPK-␥ activation impairs corticosteroid action and p38 MAPK-␥ inhibition by LABAs has potential for the treatment of severe asthma.

Corticosteroid insensitivity represents a major barrier to the treatment of chronic obstructive pulmonary disease (COPD) and severe asthma. It is caused by oxidative stress, leading to reduced histone deacetylase-2 (HDAC2) function through activation of phosphoinositide-3-kinase-␦ (PI3K␦). The tricyclic antidepressant nortriptyline has been identified in high-throughput screens as an agent that increases corticosteroid responsiveness. The aim of this study was to identify the molecular mechanism whereby nortriptyline increases corticosteroid sensitivity. Phosphorylation of Akt, a footprint of PI3K activation, and HDAC activity were evaluated by Western blotting and fluorescent activity assay in U937 monocytic cells. Corticosteroid sensitivity was evaluated by the inhibition of tumor necrosis factor ␣ (TNF␣)-induced interleukin 8 (IL-8) production by budesonide. Hydrogen peroxide (H 2 O 2 ) or cigarette smoke extract (CSE) increased the level of phosphorylated Akt (pAkt) and reduced HDAC activity. Pretreatment with nortriptyline inhibited pAkt induced by CSE and H 2 O 2 as well as restored HDAC activity that had been decreased by H 2 O 2 and CSE. In addition, nortriptyline inhibited PI3K␦ activity, but had no effect on the PI3K␣ and PI3K␥ isoforms. Although CSE reduced the effects of budesonide on TNF␣-induced IL-8 production in U937 cells, nortriptyline reversed CSE-induced corticosteroid insensitivity. Nortriptyline restores corticosteroid sensitivity induced by oxidative stress via direct inhibition of PI3K␦ and is a potential treatment for corticosteroid-insensitive diseases such as COPD and severe asthma.

Objective Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) occurs during the chronic progressive course of idiopathic pulmonary fibrosis. Mortality is estimated to be >70%, because no effective treatment has been established. We evaluated the effectiveness of combination therapy of tacrolimus and methylprednisolone for AE-IPF. Methods Patients of AE-IPF treated with methylprednisolone pulse therapy with or without tacrolimus (targeting 20 ng/mL) during the period between January 2001 and April 2010 were retrospectively reviewed. The primary endpoints were survival rate and duration. We also observed lactate dehydrogenase levels, partial pressure of arterial oxygen/fraction of inspired oxygen ratio (P/F ratio), KL-6, occurrence of re-exacerbation, and computed tomography score. Results Fifteen Japanese patients [tacrolimus group aged 74.2±6.0 years old (n=5), non-tacrolimus group aged 75.1±12.8 years old (n=10)] were identified. Pre-treatment clinical parameters were not significantly different between the two groups. Four of 5 tacrolimus group patients and 1 of 10 non-tacrolimus group patients survived (p<0.05). The median survival durations were >92 days (tacrolimus group) and 38 days (nontacrolimus group) (p<0.05). Lactate dehydrogenase levels and the P/F ratio were also significantly favorable in the tacrolimus group. KL-6 and CT score were not significantly different in both groups. Four re-acute exacerbations were observed only in the non-tacrolimus group. Conclusion Combined tacrolimus and methylprednisolone pulse therapy mitigates AE-IPF, prevents re-acute exacerbation, and contributes to a better prognosis.

SUMMARY:In experimental models of bronchial asthma with mice, airway inflammation and increase in airway hyperreactivity (AHR) are induced by a combination of systemic sensitization and airway challenge with allergens. In this report, we present another possibility: that systemic antigen-specific sensitization alone can induce AHR before the development of inflammation in the airway. Male BALB/c mice were sensitized with ovalbumin (OVA) by a combination of intraperitoneal injection and aerosol inhalation, and various parameters for airway inflammation and hyperreactivity were sequentially analyzed. Bronchial response measured by a noninvasive method (enhanced pause) and the eosinophil count and interleukin (IL)-5 concentration in bronchoalveolar lavage fluid (BALF) gradually increased following the sensitization, and significant increase was achieved after repeated OVA aerosol inhalation along with development of histologic changes of the airway. In contrast, AHR was already significantly increased by systemic sensitization alone, although airway inflammation hardly developed at that time point. BALF IL-4 concentration and the expression of IL-4 mRNA in the lung reached maximal values after the systemic sensitization, then subsequently decreased. Treatment of mice with anti-IL-4 neutralizing antibody during systemic sensitization significantly suppressed this early increase in AHR. In addition, IL-4 gene-targeted mice did not reveal this early increase in AHR by systemic sensitization. These results suggest that an immune response in the lung in an early stage of sensitization can induce airway hyperreactivity before development of an eosinophilic airway inflammation in BALB/c mice and that IL-4 plays an essential role in this process. If this early increase in AHR does occur in sensitized human infants, it could be another therapeutic target for early prevention of the future onset of asthma. (Lab Invest 2001, 81:1385-1396.A fter exposure to allergen(s), atopic bronchial asthma is characterized by airway inflammation and increased serum immunoglobulin (Ig) E levels together with increased airway hyperreactivity (AHR) in response to specific allergens and nonspecific stimuli (Burrows et al, 1989;Sears et al, 1991). In the process of airway inflammation, many kinds of inflammatory cells, such as mast cells (Ying et al, 1997), eosinophils (Lamkhioued et al, 1997), and T lymphocytes (Crimi et al, 1997;De Sanctis et al, 1997), are involved. In addition, various cytokines and growth factors produced by these cells, such as interleukin (IL)-4 (Leonard et al, 1997;Shi et al, 1998a), IL-5 (Shi et al, 1998b;Till et al, 1998), IL-10 (Borish et al, 1996;Robinson et al, 1996), IL-12 (Van Der Pouw Kraan et al, 1997), and IL-13 , may play important roles in the disease process. IL-5 and eosinophils have been recently specifically implicated as key contributors to the development of allergic airway inflammation.The predominance of Th2 phenotype of lymphocyte over Th1 phenotype from a very early stage of life has recently bee...