Specific immunotherapy reduces the antigen-dependent production of eosinophil chemotactic activity from mononuclear cells in patients with atopic asthma

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Background: Eosinophil transendothelilal migration across vascular endothelial cells is an initial step of eosinophil accumulation in allergic inflammation. There is increasing evidence that specific immunotherapy (SIT) modulates the production of inflammatory molecules from mononuclear cells. Objective: The present study was undertaken to examine whether SIT modifies eosinophil transendothelial migration induced by the supernatants of antigen-stimulated mononuclear cells from atopic asthmatics. Methods:Dermatophagoides farinae(Df)-sensitive mild persistent asthmatics were divided into a SIT-treated group and a control group. Peripheral blood mononuclear cells (PBMC) were isolated before and after SIT using the rush protocol, and cultured for 96 h at 37°C in the presence or absence of Df antigen. Eosinophils were isolated from the blood of healthy subjects, and put on transwell filters coated with pulmonary microvascular endothelial cell monolayers stimulated with IL-4 plus TNF-α. The supernatants of PBMC were applied to the lower compartment and the transmigration of eosinophils was examined. Results:Df stimulation of PBMC resulted in an augmentation of eosinophil transendothelial migration. This enhancement was abrogated following SIT. In the control group, the antigen-induced effect on eosinophil transmigration did not show an interval change. Conclusion: SIT attenuates eosinophil transendothelial migration induced by antigen-stimulated mononuclear cells.

Section: Introductionsupporting

confidence: 52%

Immunotherapy Attenuates Eosinophil Transendothelial Migration Induced by the Supernatants of Antigen-Stimulated Mononuclear Cells from Atopic Asthmatics

Nagata

Saito²,

Kikuchi³

et al. 2004

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“…PBMCs were isolated by the Ficoll-Hypaque density gradient technique and adjusted to a final concentration of 2 × 10 6 cells/ml in RPMI culture medium (RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 m M L -glutamine). To assess the effects of the drugs, PBMCs were incubated in the presence or absence of 0.1–1 µ M salbutamol for 24 h. Subsequently, PBMCs of both normal volunteers and asthma patients were cultured for72 h at 37°C in 5% CO 2 in the presence or absence of a combination of ionomycin (300 n M , Sigma) and PMA (5 n M , Sigma), or 1 µg/ml Df antigen (asthma patients only) [26,27]. The purity of lymphocytes was >90% in all experiments.…”

Section: Methodsmentioning

confidence: 99%

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

Yamaguchi¹,

Soma²,

Takaku³

et al. 2010

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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.

“…PBMC were isolated by the Ficoll-Hypaque density gradient technique, adjusted to 2 × 10 6 cells/ml in an RPMI-complete culture medium (RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 m M L -glutamine), and cultured for 96 h at 37°C in 5% CO 2 in the presence or absence of 1 µg/ml Df antigen (Torii Pharmaceutical Co., Japan) as previously described [9, 10]. The purity of lymphocytes was more than 90% in all experiments.…”

Section: Methodsmentioning

confidence: 99%

Eosinophil Transmigration across VCAM-1-Expressing Endothelial Cells Is Upregulated by Antigen-Stimulated Mononuclear Cells

Nagata

Yamamoto²,

Tabe³

et al. 2001

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Background: Adhesion to and transmigration across endothelial cells expressing vascular cell adhesion molecule 1 (VCAM-1) may be key steps in the development of selective eosinophil accumulation at the allergic inflammation sites. There is evidence that cytokines/chemokines produced by CD4+ T cells play a prominent role in these processes. Objective: The objective of this study was to evaluate whether eosinophil migration across human pulmonary microvascular endothelial cells (HPMEC) expressing VCAM-1 is modulated by the supernatants of antigen-stimulated mononuclear cells obtained from atopic asthmatics. Methods: Peripheral blood mononuclear cells (PBMC) were isolated from Dermatophagoides farinae(Df)-sensitive asthmatic subjects and cultured for 96 h at 37°C in the presence or absence of 1 µg/ml Df antigen. Eosinophils were isolated from blood of healthy subjects and placed on the HPMEC monolayers cultured on a transwell filter (3-µm pore size) stimulated with IL-4 plus TNF-α (both at 100 pM, 24 h) The supernatants of PBMC were then applied to the lower compartment and the transmigration of eosinophils was examined. Results: The supernatants of PBMC stimulated with Df significantly enhanced the eosinophil transmigration across VCAM-1-expressing HPMEC (% migration: 7.6 ± 0.6 by the supernatants of PBMC cultured without Df vs. 12.3 ± 1.2 by the PBMC cultured with Df, p < 0.01, n = 8). The enhanced migration, but not spontaneous migration, was blocked by the anti-α₄ integrin antibody. Moreover, the enhanced transmigration was blocked by anti-CCR3 antibody. Conclusion: The antigen-stimulated PBMC from atopic asthmatics produce an activity to induce eosinophil migration across VCAM-1-expressing endothelial cells. This activity appears to involve CCR3 as an essential molecule.