Tomoyuki Soma

Omalizumab Attenuates Airway Inflammation and Interleukin-5 Production by Mononuclear Cells in Patients with Severe Allergic Asthma

Background: Omalizumab, an anti-immunoglobulin E monoclonal antibody, has shown an inhibitory effect on airway inflammation, which may be associated with clinical improvement of severe asthma. This study evaluated changes in airway inflammation and cytokine release by the peripheral blood mononuclear cells (PBMCs) of Japanese patients with severe asthma after administration of omalizumab. Methods: Sixteen Japanese patients with severe asthma who were allergic to house-dust mites were enrolled in this study. Eight received omalizumab every 2 or 4 weeks for 16 weeks, and 8 control subjects were treated with conventional drug treatment. Changes in clinical scores for sputum eosinophils and levels of fraction of exhaled nitric oxide (FeNO) were measured at the time of enrollment and at week 16. Cytokines from PBMCs stimulated by house-dust mite (Dermatophagoides farinae) or ionomycin/phorbol myristate acetate (PMA) were measured at baseline and at week 16. Results: In the omalizumab-treated group, decreases in sputum eosinophils and FeNO were observed following treatment. Furthermore, the ex vivo production of interleukin (IL)-5 by PBMCs in response to both mite allergen and ionomycin/PMA decreased significantly. In contrast, interferon (IFN)-γ production was unchanged. There were no changes in any of the parameters observed in the control group. Conclusion: Omalizumab exerts inhibitory effects on airway inflammation in Japanese patients with severe allergic asthma. This treatment attenuates production of IL-5 by PBMCs stimulated with both a specific allergen and a nonspecific activator. Reduction of the Th2 inflammatory cascade likely contributes to clinical benefits; however, further studies are required to clarify these results due to the small sample size in this study.

ATP drives eosinophil effector responses through P2 purinergic receptors.

BACKGROUND Eosinophils recognize various stimuli, such as cytokines, chemokines, immunoglobulins, complement, and external pathogens, resulting in their accumulation in mucosal tissues and the progression of inflammation. Eosinophils are also involved in innate Th2-type immune responses mediated through endogenous danger signals, including IL-33, uric acid (UA), or ATP, in non-sensitized mice exposed to environmental allergens. However, the mechanism involved in eosinophil responses to these danger signals remains insufficiently understood. METHODS We examined migration, adhesion, superoxide production and degranulation of human eosinophils. Isolated eosinophils were incubated with monosodium urate (MSU) crystals and ATPγS, a non-hydrolysable ATP analogue. To determine the involvement of P2 or P2Y2 receptors in eosinophil responses to UA and ATP, eosinophils were preincubated with a pan-P2 receptor inhibitor, oxidized ATP (oATP), or anti-P2Y2 antibody before incubation with MSU crystals or ATPγS. RESULTS MSU crystals induced adhesion of eosinophils to recombinant human (rh)-ICAM-1 and induced production of superoxide. oATP abolished eosinophil responses to MSU crystals, suggesting involvement of endogenous ATP and its receptors. Furthermore, exogenous ATP, as ATPγS, induced migration of eosinophils through a model basement membrane, adhesion to rh-ICAM-1, superoxide generation, and degranulation of eosinophil-derived neurotoxin (EDN). oATP and anti-P2Y2 significantly reduced these eosinophil responses. CONCLUSIONS ATP serves as an essential mediator of functional responses in human eosinophils. Eosinophil responses to ATP may be implicated in airway inflammation in patients with asthma.

Effect of Formoterol on Eosinophil Trans-Basement Membrane Migration Induced by Interleukin-8-Stimulated Neutrophils

Background: Neutrophils are often increased in the airways of either chronic severe asthma or acute exacerbations. Neutrophils that have migrated in response to interleukin-8 (IL-8) may lead eosinophils to accumulate in the airways of patients with asthma and possibly aggravate the disease. In this study, we investigated whether formoterol modified the trans-basement membrane migration (TBM) of eosinophils stimulated with neutrophils and IL-8. Methods: Neutrophils and eosinophils were isolated from peripheral blood obtained from healthy donors. Eosinophil TBM was examined using a modified Boydens chamber technique. Neutrophils were preincubated with or without formoterol (0.1 μM) at 37°C for 30 min. Eosinophils were added to the upper compartment of a chamber with a Matrigel-coated transwell insert. Medium containing preincubated neutrophils and IL-8 was added to the lower compartment of the chamber. After a 90-minute incubation, the eosinophils that had migrated into the lower chamber were calculated using eosinophil peroxidase assays. Results: A combination of neutrophils and IL-8 significantly induced the eosinophil TBM; formoterol alone had no effect. However, formoterol modestly but significantly attenuated the TBM of eosinophils stimulated with neutrophils and IL-8. Conclusion: These results suggest that formoterol may act as a therapeutic agent on enhanced eosinophilic inflammation in acute exacerbation or persistent, severe asthma. The effect of formoterol likely involves the inhibition of neutrophil activation.

Effect of beta2-adrenergic agonists on eosinophil adhesion, superoxide anion generation, and degranulation

BACKGROUND Eosinophils play important roles in the development of asthma exacerbation. Viral infection is a major cause of asthma exacerbation, and the expression of IFN-γ-inducible protein of 10 kDa (IP-10) and cysteinyl leukotrienes (cysLTs) is up-regulated in virus-induced asthma. As β2-adrenergic agonists, such as formoterol or salbutamol, are used to treat asthma exacerbation, we examined whether formoterol or salbutamol could modify eosinophil functions such as adhesiveness, particularly those activated by cysLTs or IP-10. METHODS Eosinophils were isolated from the blood of healthy subjects and were pre-incubated with either formoterol or salbutamol, and subsequently stimulated with IL-5, LTD4, or IP-10. Adhesion of eosinophils to intercellular cell adhesion molecule (ICAM)-1 was measured using eosinophil peroxidase assays. The generation of eosinophil superoxide anion (O2(-)) was examined based on the superoxide dismutase-inhibitable reduction of cytochrome C. Eosinophil-derived neurotoxin (EDN) release was evaluated by ELISA as a marker of degranulation. RESULTS Neither formoterol nor salbutamol suppressed the spontaneous adhesion of eosinophils to ICAM-1. However, when eosinophils were activated by IL-5, LTD4, or IP-10, formoterol, but not salbutamol, suppressed the adhesion to ICAM-1. Formoterol also suppressed IL-5, LTD4, or IP-10 induced eosinophil O2(-) generation or EDN release. CONCLUSIONS These findings suggest that formoterol, but not salbutamol, suppresses eosinophil functions enhanced by IL-5, LTD4, or IP-10. As these factors are involved in the development of asthma exacerbation, our results strongly support the hypothesis that administration of formoterol is a novel strategy for treating asthma exacerbation.

Salbutamol Modulates the Balance of Th1 and Th2 Cytokines by Mononuclear Cells from Allergic Asthmatics

Background: There is evidence that excessive use of inhalational β2-agonists induces the deterioration of asthma. Although the exact mechanism of this remains to be elucidated, overuse of β2-agonists may impair the Th1/Th2 balance in asthmatic airways. The aim of the present study was to evaluate whether salbutamol, a representative inhalational β2-agonist, modifies the production of Th1- and Th2-type cytokines by mononuclear cells separated from patients with asthma and healthy volunteers. Methods: Peripheral blood mononuclear cells (PBMCs) obtained from 8 healthy volunteers and 10 patients with mild persistent asthma allergic to house dust mites were treated with either salbutamol or medium alone. PBMCs were then stimulated with either medium alone, house dust mite (Dermatophagoides farina, Df) allergen or a combination of ionomycin plus phorbol 12-myristate 13-acetate ester (PMA). Concentrations of IFN-γ, IL-13, TNF-α and RANTES in the cell supernatants were measured using ELISA. Results: In PBMCs from healthy volunteers, salbutamol did not modify IFN-γ production, but increased the spontaneous production of IL-13. In contrast, salbutamol significantly inhibited the spontaneous and ionomycin- plus PMA-stimulated production of IFN-γ by PBMCs from asthmatics. Salbutamol significantly enhanced both spontaneous and Df-induced production of IL-13 by PBMCs from asthmatics. Salbutamol did not modify the production of TNF-α. Finally, salbutamol enhanced the production of RANTES induced by Df allergen in asthmatics. Conclusions: Salbutamol inhibits IFN-γ and enhances IL-13 production by PBMCs from asthmatics. These effects would promote a Th1/Th2 imbalance in the airways and may therefore contribute to the deterioration of asthma.

Eosinophils Do Not Enhance the Trans-Basement Membrane Migration of Neutrophils

Background: There is increasing evidence that both neutrophilic and eosinophilic inflammation persist in the airways of patients with severe asthma. We have reported a positive relationship between the concentrations of eosinophils and neutrophils in sputum from severe asthmatics, suggesting a possible role of eosinophils in regulating neutrophilic inflammation. The aim of this study was to investigate whether activated eosinophils modify the trans-basement membrane migration (TBM) of neutrophils. Methods: Eosinophils and neutrophils were isolated from peripheral blood drawn from healthy donors. The TBM of neutrophils in response to a variety of chemoattractants was evaluated in the presence or absence of eosinophils by using the chambers with a Matrigel®-coated Transwell® insert. Results: As expected, eotaxin (10 nM) and RANTES (10 nM), but not IL-8 (10 nM), induced the TBM of eosinophils. On the contrary, only IL-8 induced the TBM of neutrophils. When eosinophils were coincubated with neutrophils and stimulated with IL-8, the TBM of eosinophils was significantly augmented. On the other hand, when neutrophils were coincubated with eosinophils and stimulated with eotaxin or RANTES, the TBM of neutrophils was not modified. Conclusions: Neutrophils migrated by IL-8 may lead eosinophils to accumulate in the airways of patients with severe asthma. On the other hand, it is unlikely that eosinophils migrated by chemoattractants such as CC chemokines regulate neutrophilic inflammation.

Inhibitory Effect of Budesonide Alone and in Combination with Formoterol on IL-5 and RANTES Production from Mononuclear Cells

Background:The use of a budesonide (BUD)/formoterol (FOR) combination (Symbicort®) for both maintenance and reliever therapy reduces the exacerbation of asthma better than the traditional fixed-dose regimens. In the early stage of exacerbation, the combination therapy could intervene with the development of subsequent airway inflammation. The objective of the study was to evaluate whether in the early stage of cell activation the use of BUD/FOR combination would modify the adhesion of eosinophils or production of cytokines from mononuclear cells. Methods:Human peripheral blood eosinophils, pretreated with BUD (0.1 µM), FOR (0.1 µM) or BUD/FOR combination for 30 min at 37°C, were stimulated with IL-5, leukotriene D4 or phorbol myristate acetate, and eosinophil adhesion was evaluated using an eosinophil peroxidase assay. BUD (0.1 µM), FOR (0.1 µM) or BUD/FOR combination was added to the peripheral blood mononuclear cells stimulated with ionomycin plus phorbol myristate acetate. Concentrations of IL-5 and RANTES in the cell supernatant were measured using ELISA. Results:BUD, FOR or BUD/FOR combination did not modify the eosinophil adhesion induced by any stimulators. In contrast, BUD or BUD/FOR combination significantly reduced the productions of IL-5 and RANTES in peripheral blood mononuclear cells (BUD: p < 0.0001, p = 0.027 vs. control; BUD/FOR: p < 0.0001, p = 0.031 vs. control) when the drugs were added 15 min, but not 4 h, following cell stimulation. Conclusion: In the early stage of exacerbation of asthma, inhaled BUD may suppress the progression of the allergic inflammatory cascade by inhibiting the mononuclear cells. These results also underline the importance of using BUD in rescue inhaler.

Trans-basement membrane migration of eosinophils induced by LPS-stimulated neutrophils from human peripheral blood in vitro

In the airways of severe asthmatics, an increase of neutrophils and eosinophils is often observed despite high-dose corticosteroid therapy. We previously reported that interleukin-8-stimulated neutrophils induced trans-basement membrane migration (TBM) of eosinophils, suggesting the link between neutrophils and eosinophils. Concentrations of lipopolysaccharide (LPS) in the airway increase in severe asthma. As neutrophils express Toll-like receptor (TLR)4 and can release chemoattractants for eosinophils, we investigated whether LPS-stimulated neutrophils modify eosinophil TBM. Neutrophils and eosinophils were isolated from peripheral blood of healthy volunteers and severe asthmatics. Eosinophil TBM was examined using a modified Boydens chamber technique. Eosinophils were added to the upper compartment, and neutrophils and LPS were added to the lower compartment. Migrated eosinophils were measured by eosinophil peroxidase assays. LPS-stimulated neutrophils induced eosinophil TBM (about 10-fold increase), although LPS or neutrophils alone did not. A leukotriene B4 receptor antagonist, a platelet-activating factor receptor antagonist or an anti-TLR4 antibody decreased eosinophil TBM enhanced by LPS-stimulated neutrophils by almost half. Neutrophils from severe asthmatics induced eosinophil TBM and lower concentrations of LPS augmented neutrophil-induced eosinophil TBM. These results suggest that the combination of neutrophils and LPS leads eosinophils to accumulate in the airways, possibly involved the pathogenesis of severe asthma. LPS-stimulated neutrophils induced eosinophil TBM, which may be involved in the pathogenesis of severe asthma http://ow.ly/UR4jP

Changes in Airway Inflammation and Hyperresponsiveness after Inhaled Corticosteroid Cessation in Allergic Asthma

Background: Most patients with asthma are currently controlled by pharmacotherapeutic means such as inhaled corticosteroid (ICS). However, whether ICS actually induces remission of asthma remains unknown. The present study evaluates changes in airway inflammation and hyperresponsiveness in adult patients with asthma after stopping ICS. Methods: We enrolled 11 patients with allergic asthma (7 males and 4 females; mean age, 52.3 years) who had been asymptomatic and had no exacerbation by low-dose ICS. Airway hyperresponsiveness (AHR) was assessed using methacholine challenge, and induced sputum was evaluated before and every 3 months after ICS cessation during the 1-year follow-up. Results: Among the 11 asthmatics, AHR increased in 10 (90.9%) and asthma clinically relapsed in 4 (36.4%) within 1 year of ICS cessation. AHR increased in all 7 asthmatics that were sensitized to Dermatophagoides farinae and asthma clinically relapsed in 4 (57.1%) of them. Furthermore, eosinophil numbers and IL-4 concentrations in the sputum significantly increased after ICS cessation. Conclusions: Remission with normal airway response to methacholine (no AHR) might be rare in adult patients with allergic asthma, and sensitization to house dust mites appears to play an important role in relapse. Therefore, ICS cessation should be carefully considered in patients sensitive to house dust mites. Serial determination of eosinophil counts or IL-4 concentrations in sputum might be appropriate for monitoring and preventing asthma relapse in adults.

Eosinophil transendothelial migration induced by the bronchoalveolar lavage fluid of acute eosinophilic pneumonia

Acute eosinophilic pneumonia (AEP) is characterized by a massive pulmonary infiltration of eosinophils. Mechanisms regulating the selective accumulation of eosinophils in AEP have not been fully established. The objective of this study was to evaluate the mechanisms of eosinophil accumulation in alveolar spaces through examination of bronchoalveolar lavage fluid (BALF) from AEP patients (AEP‐BALF).