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Summary Background Tiotropium bromide, a long acting muscarinic receptor inhibitor, is a potent agent for patients with bronchial asthma as well as chronic obstructive pulmonary disease. Objective The aim of this study was to evaluate whether tiotropium bromide can inhibit allergen-induced acute and chronic airway inflammation, T helper (Th)2 cytokine production, and airway remodelling in a murine model of asthma. Methods Balb/c mice were sensitized and challenged acutely or chronically to ovalbumin (OVA). The impact of tiotropium bromide was assessed using these mice models by histologic, morphometric, and molecular techniques. Moreover, the effect of tiotropium bromide on Th2 cytokine production from purified human peripheral blood mononuclear cells (PBMCs) was assessed. Results Treatment with tiotropium bromide significantly reduced airway inflammation and the Th2 cytokine production in bronchoalveolar lavage fluid (BALF) in both acute and chronic models of asthma. The levels of TGF-β1 were also reduced by tiotropium bromide in BALF in a chronic model. The goblet cell metaplasia, thickness of airway smooth muscle, and airway fibrosis were all significantly decreased in tiotropium bromide-treated mice. Moreover, airway hyperresponsiveness (AHR) to serotonin was significantly abrogated by tiotropium bromide in a chronic model. Th2 cytokine production from spleen cells isolated from OVA-sensitized mice was also significantly inhibited by tiotropium bromide and 4-diphenylacetoxy-N-methylpiperidine methiodide, which is a selective antagonist to the M3 receptor. Finally, treatment with tiotropium bromide inhibited the Th2 cytokine production from PBMCs. Conclusion These results indicate that tiotropium bromide can inhibit Th2 cytokine production and airway inflammation, and thus may reduce airway remodelling and AHR in a murine model of asthma. Cite this as: S. Ohta, N. Oda, T. Yokoe, A. Tanaka, Y. Yamamoto, Y. Watanabe, K. Minoguchi, T. Ohnishi, T. Hirose, H. Nagase, K. Ohta and M. Adachi, Clinical & Experimental Allergy, 2010 (40) 1266–1275.

Statins are lipid-lowering agents with pleiotropic effects. We investigated the apoptotic effects of fluvastatin on peripheral CD4+ T cells from healthy subjects. Fluvastatin induced apoptosis in resting CD4+ T cells but not in CD4+ T cells strongly activated with a high concentration of PMA plus ionomycin (PMA/I) analyzed with annexin V and propidium iodide staining. However, CD4+ T cells activated with a low concentration of PMA/I or with anti-CD3 antibodies were apoptotic after treatment with fluvastatin. Activities of caspases-8, -9, and -3 were increased in resting CD4+ T cells treated with fluvastatin (10 microM). In strongly activated CD4+ T cells, fluvastatin inhibited the activation of caspase-8 induced by PMA/I and increased caspase-9 activity. The caspase-3 activity did not differ between untreated and fluvastatin-treated strongly activated CD4+ T cells. Treatment with fluvastatin (10 microM) enhanced cytochrome c release and increased the Bax/Bcl-2 ratio in both resting and strongly activated CD4+ T cells. Although the in vitro concentration of fluvastatin used in this study is higher than in vivo, other factors may sensitize apoptotic cell death of CD4+ T cells in vivo. In conclusion, fluvastatin induces apoptosis in resting T cells but not in strongly activated T cells, a difference that might be due to the interaction between caspase-8 and caspase-9.

Interleukin (IL)-17F is a recently described human cytokine belonging to the IL-17 gene family, but its in vivo function remains to be determined. To this end, a full-length mouse IL-17F cDNA sequence with a 483-bp coding region sequence was first identified. Pulmonary gene transfer of an IL-17F expression construct (pcDNAmIL-17F) in mice was used to investigate its regulatory role. The results showed first that a significant increase in the number of neutrophils was seen in the bronchoalveolar lavage fluids of IL-17F-transduced mice, concomitant with increased expression of genes encoding C-X-C chemokines and inflammatory cytokines when compared with mock and phosphate-buffered saline control animals. Mucosal transfer of the IL-17F gene in ovalbumin (OVA)-sensitized mice before antigen (Ag) challenge enhanced the levels of Ag-induced pulmonary neutrophilia, but not eosinophilia, goblet cell hyperplasia, and mucin gene expression. However, no significant change in the levels of Th2 cytokine expression was noted. A significant enhancement of ventilatory timing in response to inhaled methacholine was also seen in IL-17F-transduced, Ag-sensitized mice, whereas a small but significant increase was found in IL-17F-transduced, naive mice. These results suggest a role for IL-17F in the induction of neutrophilia in the lungs and in the exacerbation of Ag-induced pulmonary inflammation.

Interleukin (IL)-12 production and IL-12 receptor (IL-12R) beta2 chain expression were investigated in patients with allergic asthma successfully treated with rush immunotherapy (RIT) and control patients with mild allergic asthma. Peripheral blood mononuclear cells (PBMCs) were stimulated with Dermatophagoides farinae (Der f), and production of cytokines was measured. Furthermore, the effects of cytokines on IL-12R beta2 chain expression on CD4(+) T cells were investigated. Production by PBMCs of IL-12 and IFN-gamma was significantly higher and production of IL-4 was significantly lower after stimulation with Der f allergen in RIT-treated patients than in control patients. Significant increases in the expression of IL-12R beta2 chain before and after stimulation of CD4(+) T cells with IL-12 or IFN-gamma were observed in RIT-treated patients compared with that in control patients. Allergen RIT increases IL-12 production and IL-12R beta2 chain expression and thus may convert cytokine production from Th2 to Th1 or Th0 type in allergic asthma.

Summary Background Suplatast tosilate is an anti-allergic agent that suppresses cytokine production by human Th2 cells. Objective We investigated the effects of suplatast tosilate on the production of thymus- and activation-regulated chemokine (TARC) by T cells from allergic patients with asthma. Methods Purified protein derivative (PPD)-specific Th1 cell lines and Dermatophagoides farinae (Der f)-specific Th2 cell lines were established from nine patients with house dust mite-allergic asthma. The effects of suplatast tosilate on mRNA expression of TARC and protein production of TARC from antigen-specific Th1 or Th2 cell lines were investigated after stimulation with relevant antigens or phytohemagglutinin (PHA). In addition, the effects of IL-4, IL-10, and IFN-γ on TARC production by Der f-specific Th2 cell lines in the presence or absence of suplatast tosilate were studied. Results Although PPD-specific Th1 cell lines did not produce TARC after stimulation with PPD antigen or PHA, stimulation of Der f-specific Th2 cell lines with Der f antigen or PHA increased production of TARC. Suplatast tosilate significantly and dose-dependently inhibited production of TARC by Der f-specific Th2 cell lines stimulated with either Der f antigen (76.5% inhibition at 100 µg/mL, P

Background: Nebulized lidocaine enables dosages of oral corticosteroids to be tapered in the treatment of severe asthma. Objective: We sought to investigate the effect of lidocaine on T cells from patients with allergic asthma. Methods: PBMCs and CD4 + T cells were isolated from 6 patients with asthma and house dust mite allergy. PBMCs were cultured with lidocaine for up to 7 days and then stained with propidium iodide to evaluate the involvement of apoptosis. In addition, the viability of CD4 + T cells when cultured with lidocaine was investigated. Effects of lidocaine on proliferative response, mRNA expression, and protein production of IL-5 and IFN-γ by PBMCs were investigated after stimulation with Dermatophagoides farinae , purified protein derivative, and phorbol 12-myristate 13-acetate plus calcium ionophore. The effects of lidocaine on the proliferative response of steroid-insensitive PBMCs from 6 nonallergic donors induced by preculture with IL-2 and IL-4 were also investigated. Results: No significant increase in the staining of PBMCs with propidium iodide was observed in the presence of 100 μmol/L lidocaine. The viability of CD4 + T cells was not significantly affected by culture with lidocaine at this concentration. However, lidocaine inhibited, in a dose-dependent manner, the proliferative response and mRNA expression and protein production of IL-5 and IFN-γ of PBMCs stimulated with D farinae , purified protein derivatives, or phorbol 12-myristate 13-acetate plus calcium ionophore. Preincubation of PBMCs with IL-2 and IL-4 significantly decreased the inhibitory effects of both corticosteroids and lidocaine compared with that after preincubation with medium alone. Conclusion: Lidocaine has immunoregulatory effects on T cells. Therefore, lidocaine might be studied as an anti-inflammatory agent in the treatment of severe steroid-dependent asthma. (J Allergy Clin Immunol 2002;109:485-90.)

Asthma is characterized by chronic airway inflammation and the recruitment of inflammatory cells, typically eosinophils and lymphocytes, into the airway. Although several chemical mediators are released during the inflammatory process of asthma, evidence strongly suggests that the cysteinyl leukotrienes (LT), LTC4, LTD4, and LTE4, play key roles in asthma. The short-term clinical efficacy of an LT receptor antagonist, pranlukast hydrate, in symptomatic patients with asthma who had already been treated with moderate to high doses of inhaled corticosteroids was therefore investigated. Treatment with pranlukast hydrate for 4 weeks significantly improved respiratory function and decreased asthma symptoms, the rescue use of inhaled β2-agonists, the number of peripheral blood eosinophils and serum levels of eosinophil cationic protein. Furthermore, airway inflammation, as evaluated by the percentage of eosinophils in induced sputum and airway responsiveness to histamine, decreased significantly after treatment. There were no significant changes in these parameters in control patients with asthma whose treatment was not changed over 4 weeks. These preliminary results suggest that pranlukast hydrate, an LT receptor antagonist, is an effective agent in the management of asthma in combination with moderate to high doses of inhaled corticosteroids.

Background: Interleukin (IL)–10 induces a long–term antigen–specific anergy in human CD4+ T cells. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from house dust mite (Dermatophagoides farinae, Der f)–sensitized asthmatic patients. PBMCs were stimulated with Der f antigen for 72 h immediately after purification or after 48 h of resting culture with medium, and IL–10 and IL–5 in the culture supernatant were measured. PBMCs were also stimulated with Der f antigen for 7 days either immediately after purification or after 48 h of resting culture, after which cells were collected. Secondary proliferative responses of these cells to stimulation for 3 days with Der f antigen and mitomycin C–treated PBMCs as antigen–presenting cells or with phorbol myristate acetate (PMA) plus calcium ionophore were investigated. Results: Stimulation of PBMCs with Der f antigen immediately after purification significantly increased the proliferative response and IL–5 production. Stimulation of PBMCs with Der f antigen after resting culture with medium alone for 48 h significantly decreased IL–5 production and markedly increased IL–10 production. Although activation of cells with Der f antigen immediately after purification significantly increased secondary proliferative responses, stimulation after 48 h of resting culture failed to increase secondary proliferative responses. However, proliferation recovered when cells were activated with PMA plus calcium ionophore. Conclusion: These results suggest that antigen–specific Th2 cells are anergized by IL–10 and that Th2 cell tolerance may suppress eosinophilic inflammation in allergic asthma.

Rush immunotherapy (RIT) has been shown to be effective in allergic asthma.We investigated the mechanisms of RIT on the basis of cytokine production by T-cell lines and airway inflammation and responsiveness.Subjects were 8 patients with house dust mite-allergic asthma treated with dust mite extract RIT for 6 months and 6 RIT-untreated control patients. IL-5 production by Dermatophagoides farinae -specific T-cell lines, eosinophil percentages, and eosinophil cationic protein (ECP) in induced sputum and airway responsiveness to allergen and histamine were evaluated before and after treatment. Changes in eosinophil percentages and ECP in induced sputum and responsiveness to histamine 24 hours after allergen inhalation were also studied.After 6 months of RIT, percentages of total eosinophils (43. 0% +/- 6.90% to 16.8% +/- 2.48%; P.01), percentages of EG2(+ ) eosinophils (32.6% +/- 6.39% to 19.7% +/- 4.68%; P.01) and ECP (362.7 +/- 125.3 ng/mL to 26.2 +/- 5.15 ng/mL; P.05) decreased in induced sputum, and IL-5 production by T-cell lines decreased (617 +/- 93.2 pg/mL to 200.0 +/- 34.1 pg/mL; P.01). RIT decreased both early- and late-phase bronchoconstriction (early phase: 33.2% +/- 3. 46% to 25.4% +/- 1.42%; P.03; late phase: 16.2% +/- 3.52% to 6.2% +/- 1.96%; P.03) and suppressed increases in the percentages of total (61.8% +/- 4.89% to 42.0% +/- 4.67%; P.01) and EG2-positive eosinophils (55.54% +/- 7.21% to 36.5% +/- 6.43%; P.01) and ECP (685.6 +/- 217.0 ng/mL to 85.4 +/- 23.4 ng/mL; P.05) in induced sputum after allergen inhalation. RIT also decreased airway responsiveness to dust mite (1:303.7 +/- 123.7 wt/vol to 1:65.0 +/- 13.2 wt/vol; P.03) and to histamine before (397.1 +/- 206.9 microgra/mL to 1391.3 +/- 283.3 microgram/mL; P.03) and after allergen inhalation (139.2 +/- 36.5 microgram/mL to 629.1 +/- 196.3 microgram/mL; P.03).RIT decreases airway inflammation and airway hyperresponsiveness before and after bronchial provocation with allergen, possibly by inhibiting both allergen-specific T-cell- and mast cell-dependent pathways. RIT is an effective antiinflammatory treatment in allergic asthma.

A 24-year-old female patient with chronic dry cough, without wheezing and other symptoms, was diagnosed as atopic cough variant asthma (CVA) sensitized with house dust (HD) mite. To investigate the effect of a leukotriene (LT) receptor antagonist (pranlukast hydrate, ONON 450 mg/day), cough score, respiratory function, cough receptor sensitivity to capsaicin, airway inflammation evaluated by hypertonic saline inhalation and airway reactivity to histamine were studied before and after treatment for 4 weeks. Furthermore, the effect of a LT receptor antagonist on HD allergen-induced bronchoconstrictive and cough responses was investigated. Treatment with a LT receptor antagonist resulted in disappearance of cough, improvement of respiratory function, decrease in eosinophil percentage in induced sputum and increase in capsaicin and histamine threshold. Although bronchoprovocation with HD extract induced an immediate bronchoconstriction followed by cough responses before treatment, inhibition of both bronchoconstrictive and cough responses was observed after treatment. These results suggest that LT is involved in the mechanism of cough in this patient with atopic CVA.

A new family of cytokines, IL-17, has recently been defined that reveals a distinct ligand-receptor signaling system. Functional studies have provided evidence for its importance in the regulation of immune responses. Notably, 3 members, IL-17A, IL-17E (IL-25), and IL-17F, have been best characterized both in vitro and in vivo , and have been shown to be proinflammatory in nature. This proinflammatory activity is exemplified by their involvement in pulmonary inflammatory responses, in which both IL-17A and IL-17F are involved in the recruitment of neutrophils, and IL-17E is able to induce T H 2 cytokine production and eosinophilia. Although the elucidation of a detailed mechanism of action continues to be an active area of research, the potent inflammatory activity and its association with various human disease states suggest this new cytokine family as an important contributor to the pathophysiology of human disease conditions, in particular the pulmonary diseases.

Suplatast tosilate (IPD-1151T) is an antiallergic agent that suppresses airway eosinophil infiltration in asthma. We investigated the effects of IPD-1151T on proliferative response and cytokine production by human antigen-specific T cell lines. Purified protein derivatives (PPD)-specific T helper 1 (Th1) cell lines and Dermatophagoides farinae (Der f)-specific T helper 2 (Th2) cell lines were established from patients with asthma sensitized with house dust mite. Stimulation of PPD-specific and Der f-specific T cell lines with relevant antigens resulted in production of mostly interferon (IFN)-gamma and of interleukin (IL)-4 and IL-5, respectively. IPD-1151T did not inhibit the proliferative responses of either the Th1 or Th2 cell line to antigens. Although IPD-1151T did not inhibit IFN-gamma production by PPD-specific Th1 cell lines, it did inhibit IL-4 and IL-5 production by antigen-stimulated Der f-specific Th2 cell lines in a dose-dependent manner. IPD-1151T directly inhibited cytokine production by Der f-specific Th2 cell lines stimulated with immobilized anti-CD3 antibodies. Although IPD-1151T did not inhibit the clonal expansion of memory T cells among PBMCs into PPD-specific Th1 and Th2 cell lines, it did inhibit IL-4 and IL-5 production by Der f-specific Th2 cell lines but not IFN-gamma production by PPD-specific Th1 cell lines. These results suggest that IPD-1151T selectively inhibits Th2-type cytokine production.

Background: It has been reported that protein tyrosine phosphorylation is essential for cytokine production in mouse Th1 cells but not in Th2 cells. However, little is known about the difference of signal transduction between human antigen–specific Th1 and Th2 cell clones. Methods: Purified protein derivative–specific Th1 and Dermatophagoides farinae–specific Th2 cell clones were established. T cell clones were stimulated with anti–CD3 Abs and further treated with goat antimouse immunoglobulin Abs for cross–linking of TCR/CD3 complex. Results: In contrast to murine Th2 clones, tyrosine phosphorylation including both ZAP–70 and phospholipase–C–Ál was necessary for cell activation in both types of human T cell clones. This was further supported by the observation that genistein (protein tyrosine kinase inhibitor) inhibits IFN–Á production for Th1 and IL–4 for Th2 in a dose–dependent manner. Conclusion: Protein tyrosine phosphorylation is thus an essential component in transducing the activation signal from TCR–CD3 complex both in human Th1 and Th2 cells.

Rush immunotherapy (RI), a modified allergen-specific immunotherapeutic procedure, is an effective treatment for extrinsic (atopic) asthma, although the precise mechanism of its action is unclear. We have thus investigated the effect of RI on T cell response in seven mite-allergen-sensitive asthmatic patients who were successfully treated with RI. The proliferative response to mite allergen profoundly decreased after 3 months of therapy compared to the response before therapy; the response, however, recovered 18 months after RI. Regarding cytokine production patterns of mite-specific T cells, RI brought about a shift in cytokine profiles from Th2 to Th0 or Th1 in mite-specific T cell clones. The data indicate that the efficacy of RI is due to modification of T cell responses to mite antigens. Allergen RI results in the conversion of Th2 to Th1 and Th0 cells and/or selection of Th1 and Th0 cells over Th2 cells and thus may improve both clinical symptoms and airway inflammation in asthmatics.

Rationale We have previously reported on the discovery and in vitro function of human ML-1 (IL-17F) gene. However, in vivo function of IL-17F remains to be determined. To this end, mouse IL-17F gene was identified and its in vivo function was investigated using a mucosal gene transfer approach. Methods The full-length mouse IL-17F (mIL-17F) cDNA sequence was identified using both 5'- and 3'-RACE. An expression construct (pcDNAmIL17F) of the mIL-17F coding region sequence was generated. The resulting expression construct (pcDNAmIL17F) was propagated and purified. Groups of mice were administered PBS, a control expression vector, or pcDNAmIL17F by the intratracheal route. 72h after instillation, bronchial alveolar lavage fluid (BALF) samples were analyzed for differential cell counts. Gene expression of pulmonary inflammatory cytokines and C-X-C chemokines was performed by RT-PCR. Airwayhyperreactivity (AHR) to methacholine (Mch) was also measured using whole body plethysmography. Results A full-length cDNA with 1178 bp was identified, including a 486-bp coding region sequence. Mucosal transfer of IL-17F gene in Balb/c mice demonstrated a significant increase in the number of BALF neutrophils in mice treated with pcDNAmIL17F. While no significant difference in AHR to Mch was seen in different treatment groups, a significant increase in AHR to Mch in ovalbumin (OVA)-sensitized and challenged mice was noted in mice treated with pcDNAmIL17F when compared to those seen in PBS and mock-treated, OVA-sensitized and challenged mice. Conclusions IL-17F induced significant pulmonary neutrophilia in mice, suggesting a role for this new cytokine in neutrophil-associated pulmonary inflammatory responses.