Landscape of electrophilic and inflammatory stress-mediated gene regulation in human lymphoblastoid cell lines.

Human lymphoblastoid cell lines (LCLs) are valuable for the functional analyses of diseases. We have established more than 4200 LCLs as one of the resources of an integrated biobank. While oxidative and inflammatory stresses play critical roles in the onset and progression of various diseases, the responsiveness of LCLs, especially that of biobank-made LCLs, to these stresses has not been established. To address how LCLs respond to these stresses, in this study, we performed RNA sequencing of eleven human LCLs that were treated with an electrophile, diethyl maleate (DEM) and/or an inflammatory mediator, lipopolysaccharide (LPS). We found that over two thousand genes, including those regulated by a master regulator of the electrophilic/oxidative stress response, NRF2, were upregulated in LCLs treated with DEM, while approximately three hundred genes, including inflammation-related genes, were upregulated in LPS-treated LCLs. Of the LPS-induced genes, a subset of proinflammatory genes was repressed by DEM, supporting the notion that DEM suppresses the expression of proinflammatory genes through NRF2 activation. Conversely, a part of DEM-induced gene was repressed by LPS, suggesting reciprocal interference between electrophilic and inflammatory stress-mediated pathways. These data clearly demonstrate that LCLs maintain, by and large, responsive pathways against oxidative and inflammatory stresses and further endorse the usefulness of the LCL supply from the biobank.

Essential role of B7-H1 in double-stranded RNA-induced augmentation of an asthma phenotype in mice.

Clinical and epidemiological studies have shown the contribution of viral infection to the development of allergic asthma. Many RNA viruses, pathogenic for the respiratory tract, generate double-stranded (ds)RNA during their replication. Typical innate immune responses triggered by dsRNA involve the endosomal and cytoplasmic pathways. The former is mediated by Toll/IL-1R domain-containing adaptor inducing IFN-ß (TRIF), and the latter by IFN-ß promoter stimulator 1 (IPS-1). We explored the effect of polyinocinic polycytidilic acid, a synthetic dsRNA, on the development of an asthma phenotype in mice. Administration of dsRNA during ovalbumin sensitization augmented airway eosinophilia and airway hyperresponsiveness after an antigen challenge, which was associated with enhanced induction of IL-13-producing CD8(+) T cells. The augmentation was induced in IPS-1-deficient mice but not in TRIF-deficient mice. The interactions between dendritic cells (DCs) and T cells are regulated by B7-family costimulatory molecules, including B7-H1 (also known as PD-L1), a putative ligand for programmed death-1 (PD-1). Treatment of bone marrow-derived DCs with dsRNA enhanced B7-H1 expression in a TRIF-dependent manner. Additionally, dsRNA increased B7-H1 expression on DCs in the draining lymph nodes of ovalbumin-sensitized mice. The augmentation of the asthma phenotype was prevented by the treatment of mice with anti-B7-H1 mAb but not with anti-PD-1 mAb. The augmentation was not induced in B7-H1-deficient mice. These results suggest that dsRNA-triggered activation of the innate immune system in sensitization leads to augmentation of the asthma phenotype via IL-13 mainly from CD8(+) T cells. B7-H1 plays a crucial role in the process without requiring interaction with PD-1.

Demethylallosamidin, a chitinase inhibitor, suppresses airway inflammation and hyperresponsiveness.

Acidic mammalian chitinase is upregulated in response to allergen exposure in the lung. We investigated the effects of chitinase inhibitors, allosamidin (Allo) and demethylallosamidin (Dma), on asthmatic responses. Mice were subjected to IL-13 instillation into the airways or to ovalbumin sensitization plus exposure with or without treatment of Allo or Dma. Airway hyperresponsiveness (AHR) and inflammation were evaluated. Allo and Dma attenuated airway eosinophilia and the upregulation of eotaxin after IL-13 instillation, while Dma, but not Allo, suppressed AHR in IL-13-induced asthma. Allo or Dma suppressed the elevated chitinase activity in BAL fluids after IL-13 to similar levels. The bronchoprotective PGE(2) levels in BAL fluids were elevated after IL-13 instillation. Allo, but not Dma, suppressed the overproduction of PGE(2) and the expression of COX-2 and PGE synthase-1 induced by IL-13. In ovalbumin-induced asthma, Dma suppressed AHR more strongly than Allo. These findings suggest that Dma attenuates asthmatic responses induced by IL-13 without affecting PGE(2) synthesis. Dma may have potential as therapeutic agents for asthma.

Renoprotective effect of the L/N-type calcium channel antagonist cilnidipine on puromycin aminonucleoside-induced nephrosis in rats.

The renoprotective effect of cilnidipine ((+/-)-2-methoxyethyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate, CAS 132203-70-4), a L/N-type calcium channel antagonist, on puromycin aminonucleoside (PAN)-induced nephrosis was investigated in rats. In the Experiment I, rats were given an intravenous injection of PAN (70 mg/kg). Cilnidipine (3 mg/kg/day) and enalapril (CAS 75847-73-3, 5 mg/kg/day) were administered orally from 6 days after treatment with PAN (day 6) to day 26, and urinary analysis was performed on days 9, 15, 20 and 27. In the Experiment II, nephrosis was also induced by intravenous injection of PAN (70 or 100 mg/kg) in rats which were treated with cilnidipine and enalapril from days 6 to 10. Systolic blood pressure was measured on day 7 and urinary analysis was performed on day 10. On day 11, serum was collected and the kidneys were removed for immunofluorescence staining for nephrin and podocin proteins. In PAN-treated rats, the daily urinary protein excretion was dramatically elevated on day 5, reached a peak on day 9 and gradually returned to a normal level from days 15 to 27. Cilnidipine (3 mg/kg/ day) significantly suppressed the increase in proteinuria on day 9 and also improved the decrease in creatinine clearance without evident effect on the blood pressure. Furthermore, the elevations in serum total cholesterol and triglyceride tended to be suppressed by cilnidipine. The expression of nephrin and podocin proteins in PAN-treated rats showed the granular pattern in the glomeruli, while the intensity of staining seemed to be dependent on the urinary protein excretion level in the cilnidipine-treated rats. The results obtained in this study suggest a renoprotective effect of cilnidipine in PAN-induced nephrosis in rats.

Frequency of Foxp3+CD4CD25+ T cells is associated with the phenotypes of allergic asthma.

BACKGROUND AND OBJECTIVE: A forkhead/winged-helix family transcriptional repressor, Foxp3, is highly expressed on CD4(+)CD25(+) T regulatory cells. The role of Foxp3(+)CD4(+)CD25(+) T regulatory cells in asthma remains to be elucidated. Using mouse models and peripheral blood mononuclear cells (PBMC) from subjects with allergic asthma, we aimed to explore whether Foxp3(+)CD4(+)CD25(+) T regulatory cells associate with asthma phenotypes. METHODS: Foxp3(+)CD4(+)CD25(+) T cells were detected by FACS and the correlation between the frequency of Foxp3(+)CD4(+)CD25(+) T cells and asthma phenotypes was assessed. RESULTS: The frequency of Foxp3(+)CD4(+)CD25(+) T cells among total CD4(+)CD25(+) T cells in the lungs showed an inverse correlation with eosinophilic inflammation in BALB/c, A/J and C57BL/6 strains. In addition, the frequency of Foxp3(+)CD4(+)CD25(+) T cells was inversely correlated with BHR and allergen-specific IgE levels in the serum of A/J mice. In BALB/c mice, the frequency of Foxp3(+)CD4(+)CD25(+) T cells correlated with the level of IL-10 in BAL fluid. The inverse correlation between the frequency of Foxp3(+)CD4(+)CD25(+) T cells and eosinophilic inflammation disappeared when mice were treated with anti-IL-10 receptor mAb during allergen challenge. Interestingly, intracellular cytokine staining of lung cells revealed that IL-10 was predominantly produced by Foxp3(-)CD4(+)CD25(+) T cells. The frequency of Foxp3(+)CD4(+)CD25(+) T cells among total CD4(+)CD25(+) T cells in PBMC of asthmatics was significantly lower than that of healthy subjects, although there was no significant correlation between the frequency of Foxp3(+)CD4(+)CD25(+) T cells and asthma severity. CONCLUSIONS: These results suggest a role for lung Foxp3(+)CD4(+)CD25(+) T cells in the regulation of asthma phenotypes, presumably through an IL-10-mediated mechanism.

B7-DC induced by IL-13 works as a feedback regulator in the effector phase of allergic asthma.

B7-DC is a costimulatory molecule belonging to the B7 family. We previously found that treatment with anti-B7-DC mAb during the effector phase enhances asthma phenotypes in mice. We investigated the mechanisms of B7-DC induction and how B7-DC regulates asthma phenotypes. In allergen-challenged IFN-gamma-deficient mice, anti-B7-DC mAb failed to enhance the asthma phenotypes although the induction of B7-DC on dendritic cells of the mice was comparable with that on dendritic cells of wild-type mice. B7-DC on dendritic cells was up-regulated by IL-13 in vitro. The induction of B7-DC on dendritic cells after allergen challenge was attenuated by blockade of IL-13 in vivo. The asthma phenotypes were enhanced in B7-DC-deficient mice, more than in wild-type mice. The enhancement was concurrent with the down-regulation of IFN-gamma and up-regulation of IL-13. These results suggest that B7-DC induced by IL-13 works as a feedback regulator by up-regulating IFN-gamma production during the effector phase of allergic asthma.

Effects of salmeterol in patients with persistent asthma receiving inhaled corticosteroid plus theophylline.

BACKGROUND: Patients with severe asthma require multiple therapies to improve lung function and reduce symptoms. The use of long-acting inhaled beta(2)-agonists plus theophylline in addition to high doses of inhaled corticosteroids (ICSs) for the treatment of severe asthma has not been extensively studied. OBJECTIVE: The purpose of this study was to investigate the efficacy and safety of salmeterol combined with high-dose ICSs plus theophylline in severe asthma. METHODS: We undertook a randomized, placebo-controlled, crossover study to compare the effect of a single dose of inhaled salmeterol (50 microg) or a placebo in patients with severe asthma whose conditions were not being adequately controlled by therapies with high-dose ICSs plus oral theophylline with or without leukotriene receptor antagonists. RESULTS: Twenty patients took part in the trial. Compared with the placebo, the inhalation of salmeterol significantly increased the FEV(1). Even in the 9 patients treated with high-dose ICSs plus theophylline plus a leukotriene receptor antagonist, the FEV(1) increased significantly more after salmeterol than after the placebo. CONCLUSION: Patients with severe asthma receiving high-dose ICSs plus theophylline may benefit from the addition of salmeterol.

Niflumic acid suppresses interleukin-13-induced asthma phenotypes.

RATIONALE: Chloride channels have been implicated in the regulation of mucus production in epithelial cells. Expression of hCLCA1, a calcium-activated chloride channel, has been reported to be increased in the airway epithelium of patients with asthma. Interleukin (IL)-13 induces the cardinal features of bronchial asthma, and glucocorticoids are not sufficient to suppress IL-13-induced airway hyperresponsiveness or goblet cell hyperplasia. OBJECTIVES: We studied the effects of chloride channel inhibitors in IL-13-induced asthma. METHODS: The effects of niflumic acid (NA), a relatively specific blocker of calcium-activated chloride channel (CLCA), on goblet cell hyperplasia, eosinophil accumulation, and airway hyperresponsiveness were evaluated after IL-13 instillation into the airways. Because IL-13-dependent features rely on JAK/STAT6 signaling, the effect of NA on phosphorylation of JAK2 and STAT6 after IL-13 stimulation was examined in airway epithelial cells in vitro. The expression of the mCLCA family in mouse lung after IL-13 local administration in vivo was analyzed using reverse transcription-polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: Treatment with NA inhibited not only IL-13-induced goblet cell hyperplasia but also airway hyperresponsiveness and eosinophilic infiltration. NA suppressed the eotaxin levels in bronchoalveolar lavage fluids and overexpression of the MUC5AC gene, a marker of goblet cell hyperplasia, in the lung after IL-13 instillation. NA suppressed JAK2 activation, STAT6 activation, and eotaxin expression in epithelial cells. The expression of mCLCA3 (mouse homolog hCLCA1), but not that of other CLCA family members, was up-regulated by IL-13. CONCLUSIONS: These findings suggest that a chloride channel inhibitor can control IL-13-mediated airway features at least by suppressing JAK/STAT6 activation.

Exacerbation of experimental allergic asthma by augmented Th2 responses in WSX-1-deficient mice.

WSX-1 (IL-27R) is a class I cytokine receptor with homology to gp130 and IL-12 receptors and is typically expressed on CD4+ T lymphocytes. Although previous reports have clarified that IL-27/WSX-1 signaling plays critical roles in both Th1 differentiation and attenuation of cell activation and proinflammatory cytokine production during some bacterial or protozoan infections, little is known about the importance of WSX-1 in cytokine-mediated diseases of allergic origin. To this aim, we took advantage of WSX-1-deficient (WSX-1(-/-)) mice and induced experimental asthma, in which Th2 cytokines are central modulators of the pathology. OVA-challenged WSX-1(-/-) mice showed marked enhancement of airway responsiveness with goblet cell hyperplasia, pulmonary eosinophil infiltration, and increased serum IgE levels compared with wild-type mice. Production of Th2 cytokines, which are largely responsible for the pathogenesis of asthma, was augmented in the lung or in the culture supernatants of peribronchial lymph node CD4+ T cells from WSX-1(-/-) mice compared with those from wild-type mice. Surprisingly, IFN-gamma production was also enhanced in WSX-1(-/-) mice, albeit at a low concentration. The cytokine overproduction, thus, seems independent from the Th1-promoting property of WSX-1. These results demonstrated that IL-27/WSX-1 also plays an important role in the down-regulation of airway hyper-reactivity and lung inflammation during the development of allergic asthma through its suppressive effect on cytokine production.

Expression of B7-H1 and B7-DC on the airway epithelium is enhanced by double-stranded RNA.

Viral infection in the airway provokes various immune responses, including Th1 and Th2 responses, which are partly initiated by double-stranded RNA (dsRNA), a viral product for its replication. B7-H1 (PD-L1) and B7-DC (PD-L2) are B7-family molecules that bind to programmed death-1 (PD-1) on lymphocytes and are implicated in peripheral tolerance. We investigated the effect of dsRNA on the expression of B7-H1 and B7-DC on airway epithelial cell lines. B7-H1 and B7-DC were constitutively expressed on the cells, and their expression was profoundly upregulated by stimulation with an analog of viral dsRNA, polyinosinic-polycytidylic acid. B7-H1 and B7-DC were also upregulated by stimulation with IFN-gamma, IL-13, and the supernatant from T cell clones. A relatively high concentration of dexamethasone (1 microM) was required to suppress the upregulation of B7-H1 or B7-DC. These results suggest that epithelial B7-H1 and B7-DC play a role in virus-associated immune responses in the airways.

Different roles of interleukin-10 in onset and resolution of asthmatic responses in allergen-challenged mice.

OBJECTIVE: Although interleukin (IL)-10 is an immunoregulatory cytokine produced by various cells including T cells, its precise role in asthma remains uncertain. The aim of this study was to investigate the role of IL-10 in experimental asthma using ovalbumin (OVA)-sensitized mice. METHODOLOGY: Mice were challenged with OVA aerosol, and airway responsiveness and inflammation were measured. OVA-specific IL-10-producing CD4+ T cells were counted from lung cells collected by enzymatic digestion and stimulated ex vivo with OVA. The effects of an anti-IL-10 antibody on airway responsiveness and inflammation were also evaluated. RESULTS: The OVA challenge caused airway hyperresponsiveness and eosinophilic inflammation. A significant increase in IL-10-producing CD4+ T cells was observed, mainly in the CD45RB(low) subset, for several days after the OVA challenge. Anti-IL-10 antibody treatment before the OVA challenge did not affect eosinophilic inflammation but significantly inhibited airway hyperresponsiveness 24 h after the OVA challenge. However, anti-IL-10 antibody treatment just before the last OVA challenge significantly attenuated the resolution of eosinophilic inflammation without affecting airway responsiveness 2 weeks after the OVA challenge. CONCLUSIONS: Intrinsic IL-10 may have a distinct role in the early and late phases of asthmatic responses. In the early phase, IL-10 induces airway hyperresponsiveness, while in the late phase IL-10 contributes to the resolution of eosinophilic inflammation.

Spred-1 negatively regulates allergen-induced airway eosinophilia and hyperresponsiveness.

T helper 2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, play a critical role in allergic asthma. These cytokines transmit signals through the Janus kinase/signal transducer and activator of transcription (STAT) and the Ras-extracellular signal-regulated kinase (ERK) signaling pathways. Although the suppressor of cytokine signaling (SOCS) family proteins have been shown to regulate the STAT pathway, the mechanism regulating the ERK pathway has not been clarified. The Sprouty-related Ena/VASP homology 1-domain-containing protein (Spred)-1 has recently been identified as a negative regulator of growth factor-mediated, Ras-dependent ERK activation. Here, using Spred-1-deficient mice, we demonstrated that Spred-1 negatively regulates allergen-induced airway eosinophilia and hyperresponsiveness, without affecting helper T cell differentiation. Biochemical assays indicate that Spred-1 suppresses IL-5-dependent cell proliferation and ERK activation. These data indicate that Spred-1 negatively controls eosinophil numbers and functions by modulating IL-5 signaling in allergic asthma.

Activation of PI3K-Akt pathway mediates antiapoptotic effects of beta-adrenergic agonist in airway eosinophils.

beta-Adrenoceptor agonists reportedly decrease spontaneous apoptosis of peripheral blood eosinophils; however, its signaling pathway is unknown. Survival signals can be elicited by the activation of phosphatidylinositol 3-kinase (PI3K) and Akt, both of which are known to be potent regulators of apoptosis, and Akt in turn inactivates Forkhead transcription factors, including FKHR (Forkhead in rhabdomyosarcoma). We have investigated the effect of beta-agonists on apoptosis of local eosinophils isolated from the airways and the involvement of PI3K, Akt, and FKHR in its survival signal. Eosinophils obtained from immunized mice by bronchoalveolar lavage after allergen provocation underwent apoptosis in a time-dependent manner. Incubation of eosinophils with isoproterenol or formoterol dose-dependently inhibited both spontaneous eosinophil apoptosis and apoptosis induced by Fas receptor activation. Incubation with cAMP or forskolin also inhibited eosinophil apoptosis. The PI3K inhibitors wortmannin and LY-294002 and an Akt inhibitor, 1-L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, but not a mitogen-activated protein kinase kinase inhibitor PD-98059, blocked isoproterenol-mediated eosinophil survival. Wortmannin also inhibited cAMP-mediated eosinophil survival. Isoproterenol rapidly induced phosphorylation of Akt and FKHR in eosinophils in a PI3K-dependent manner. These findings indicate that the PI3K-Akt-FKHR pathway conveys a critical survival signal induced by beta-agonists in airway eosinophils.

Decrease of interleukin-10-producing T cells in the peripheral blood of severe unstable atopic asthmatics.

BACKGROUND: Although IL-10 is known as an immunoregulatory cytokine produced by various cells including T cells, its basic profile in atopic asthma remains uncertain. OBJECTIVE: The profiles of IL-10 production in circulating CD4+ T cells of atopic asthmatics were investigated with respect to clinical severity. METHODS: Forty atopic asthmatics were divided into three groups: mild, and severe but stable and severe unstable asthmatics. Eosinophils were counted in the peripheral blood and sputum, and exhaled nitric oxide was assessed. PBMCs were stimulated with or without anti-CD3 and anti-CD28 antibodies and then processed for detecting IL-10-producing CD4+ cells using flow cytometry. RESULTS: There was no difference in the eosinophil count in blood or sputum and in nitric oxide level among the three groups. IL-10-producing CD4+ cells were mainly detected in a CD45RO+ memory population. The frequency of IL-10-producing cells after stimulation was significantly lower in the severe unstable group compared to the mild group. In addition, the frequency of IL-10-producing cells in the severe unstable group was significantly lower than that in the severe stable group despite the fact that both groups received similar treatments with high-dose inhaled corticosteroids. The IL-10 production of CD4+CD45RO+ cells in response to dexamethasone did not differ among the three groups. CONCLUSIONS: IL-10-producing CD4+CD45RO+ cells in the peripheral blood are decreased in severe unstable asthmatics, which is not explained by the effect of high-dose inhaled corticosteroid medication.

B7-DC regulates asthmatic response by an IFN-gamma-dependent mechanism.

B7-H1 (PD-L1) and B7-DC (PD-L2) are the ligands for programmed death-1 (PD-1), which is a member of the CD28/CTLA-4 family and has been implicated in peripheral tolerance. We investigated the roles of B7-H1 and B7-DC in a murine OVA-induced allergic asthma model. B7-H1 was constitutively expressed on dendritic cells, macrophages, B cells, and T cells in the lungs of naive mice, and its expression could be dramatically increased after allergen challenge. In contrast, B7-DC expression was scarcely expressed on dendritic cells in naive mice, but was up-regulated after allergen challenge, although the up-regulation of B7-DC expression on macrophages was minimal. Treatment of mice with anti-B7-DC mAb at the time of allergen challenge, but not at the time of sensitization, significantly increased their airway hyper-reactivity and eosinophilia. Such treatment also resulted in the increased production of IL-5 and IL-13, and decreased IFN-gamma production in the lungs and draining lymph node cells. These changes were diminished when mice were depleted of IFN-gamma by anti-IFN-gamma mAb pretreatment. Interestingly, treatment with anti-B7-H1 or anti-PD-1 mAb did not significantly affect the asthmatic response. These results suggest a unique role for B7-DC in the regulation of asthmatic response through an IFN-gamma-dependent, but PD-1-independent, mechanism.