Jun Atsuta

Chemokine production by the BEAS-2B human bronchial epithelial cells: Differential regulation of eotaxin, IL-8, and RANTES by TH2- and TH1-derived cytokines

BACKGROUND Bronchial epithelial cells produce many types of chemokines and may contribute to lung inflammation by recruiting inflammatory cells. The CC chemokine eotaxin is a potent, eosinophil-specific chemoattractant that has been detected in the bronchial epithelium of patients with asthma. OBJECTIVES The aim of this study was to investigate the regulatory mechanisms of chemokine production from bronchial epithelium by inflammatory cytokines, especially T(H)2- and T(H)1-derived cytokines, in bronchial asthma. METHODS BEAS-2B human bronchial epithelial cells were cultured with TNF-alpha, IL-4, IL-13, and IFN-gamma alone or in combination, after which supernatants were assayed for eotaxin, IL-8, and RANTES proteins with ELISA. Reverse transcription-PCR was also performed. RESULTS TNF-alpha induced production of eotaxin, IL-8, and RANTES in a concentration-dependent manner. Both IL-4 and IL-13 synergistically enhanced TNF-alpha-induced eotaxin production, whereas IL-8 production induced by TNF-alpha was significantly down-regulated by the T(H)2-derived cytokines. IFN-gamma, a T(H)1 cytokine, counteracted the enhancing effects of IL-4 and IL-13 on eotaxin production. RANTES production by TNF-alpha was not affected by IL-4 and IL-13 but was markedly enhanced by IFN-gamma. CONCLUSIONS These results suggest that T(H)2 cytokines are involved in preferential recruitment of eosinophils in bronchial asthma by enhancing eotaxin and reducing IL-8 production from bronchial epithelial cells and that T(H)1 cytokines counteract the effects of T(H)2 cytokines by reducing eotaxin production.

An in vitro comparison of commonly used topical glucocorticoid preparations

BACKGROUND Glucocorticoids (GC) are potent inhibitors of peripheral blood eosinophil, basophil, and airway epithelial cell function. OBJECTIVES We compared in vitro the inhibitory activity of synthetic GC used for topical treatment in asthma and rhinitis on basophil histamine release (HR), eosinophil viability, and expression of vascular cell adhesion molecule-1 (VCAM-1) in the human bronchial epithelial cell line BEAS-2B. METHODS Cells were treated for 24 hours with increasing concentrations (range 10(-13) to 10(-6) mol/L) of fluticasone propionate (FP), mometasone furoate (MF), budesonide (BUD), beclomethasone dipropionate (BDP), triamcinolone acetonide (TAA), hydrocortisone (HC), or dimethyl sulfoxide diluent before challenge. HR was measured by a fluorometric assay, viability of purified eosinophils was assessed by erythrosin B dye exclusion, and expression of VCAM-1 was measured by flow cytometry. RESULTS GC induced a concentration-dependent inhibition of anti-IgE-induced HR. Maximum inhibition ranged from 59. 7% to 81%, with a rank order of GC potency of FP > MF > BUD > BDP congruent with TAA >> HC. Three-day treatment of eosinophils with GC concentration-dependently inhibited IL-5-induced eosinophil viability, with a rank of potency almost identical to that observed with basophil HR. The rank order of potency of GC for inhibition of the expression of VCAM-1 in BEAS-2B cells was MF congruent with FP >> BUD > TAA > HC congruent with BDP. Inhibitory concentration of 50% values revealed that epithelial cells were the most sensitive and eosinophils were the least sensitive. CONCLUSIONS These data, combined with information on pharmacodynamics of these drugs in vivo, may be useful in estimating GC local anti-inflammatory effects.

High prevalence and young onset of allergic rhinitis in children with bronchial asthma

Bronchial asthma and allergic rhinitis often co‐exist, and rhinitis is a major risk factor for the development of asthma. However, the reported incidence of allergic rhinitis in asthmatic children varies widely. The aim of this study was to elucidate the incidence of allergic rhinitis, the onset age of chronic upper and lower airway symptoms, and the correlation of these two symptoms in asthmatic children. A cohort of 130 consecutive children (ages 2–10) with asthma was evaluated. A questionnaire regarding upper and lower airway symptoms was filled out by the parents. Objective diagnosis of allergic rhinitis was also made on the basis of rhinoscopy, nasal cytology, nasal challenge, and specific serum IgE (CAP‐RAST). Persistent nasal symptoms were present in 83.8% of the asthmatic children. The incidence of allergic rhinitis was 77.7% based on the objective findings. The mean onset age of asthma was 2.8 yr, and that of rhinitis was 2.9 yr. Nasal symptoms started as early as the first year of life in 8.9% of the children. In children with comorbid asthma and allergic rhinitis, rhinitis preceded in 33.7%, asthma preceded in 31.7%, and both started in the same year in 26.7%. In 7.9%, rhinitis was asymptomatic. Concomitant exacerbation of the upper and lower airways occurred in 34.6% of the total 130 children. These results demonstrate that allergic rhinitis manifested early in life in the majority of the asthmatic children. Persistent nasal symptoms in infancy may point to subsequent development of asthma and possible early intervention.

Mechanism of eosinophil infiltration in the patient with subcutaneous angioblastic lymphoid hyperplasia with eosinophilia (Kimura's disease). Mechanism of eosinophil chemotaxis mediated by candida antigen and IL-5.

Kimuras disease is a chronic granulomatous disease of unknown etiology. Although eosinophilia is one of the characteristic features in this disease, little is known about the mechanism of eosinophilia. In the present study it was demonstrated that interleukin-5 (IL-5) was produced and released from the site of a granuloma and lymph nodes after stimulation with candida antigen. It was also shown that peripheral blood eosinophils from patients with Kimuras disease contained a large proportion of hypodense eosinophils and that their viability was prolonged. These results strongly suggest that locally produced IL-5 induced by candida antigen contributes to the eosinophilia in this disease.

IL-5 as a Strong Secretagogue for Human Eosinophils

The ability of IL-5 to induce eosinophil degranulation was investigated. Peripheral blood eosinophils from patients with mildly allergic individuals were isolated with CD16- selection method. Eosinophils were then incubated with interleukin-5 (IL-5) (0.1-100 ng/ml) for 1-48 h and EPX in the supernatants were measured with RIA. We found that IL-5 induced significant amount of eosinophil protein X in a concentration-dependent manner at 24 h. Eosinophil viability was about 90% either in the presence or absence of IL-5 at 24 h. Eosinophils stimulated with IL-5 adhered to the plate. Anti-CD18 mAb blocked adhesion and degranulation induced by IL-5. Dexamethasone and TGFbeta significantly inhibited degranulation in a concentration-dependent manner. These results suggest that IL-5 may be a strong secretagogue for eosinophils, that adhesion via beta2 integrin is a requisite for degranulation, and that the anti-inflammatory effect of corticosteroids and TGFbeta may be exerted at least in part, through the inhibition of eosinophil degranulation.

Leukotriene D4 induces production of transforming growth factor-β1 by eosinophils

Eosinophils may play an important role in the pathogenesis of airway remodeling in asthma through the production of various fibrogenic cytokines such as transforming growth factor-β1 (TGF-β1). Cysteinyl leukotrienes are also suggested to be involved in remodeling with their potential to induce proliferation of airway smooth muscle cells. Since massive eosinophil infiltration and the release of cysteinyl leukotrienes in airway secretions are often seen in asthma, we hypothesized that cysteinyl leukotrienes may be involved in airway remodeling through induction of TGF-β1 from eosinophils. Peripheral blood eosinophils were cultured with leukotriene D4 (LTD4) and/or interleukin-5 (IL-5) or granulocyte colony-stimulating factor (GM-CSF) for 16 h and gene expression of TGF-β1 was quantified with real-time PCR. A combination of LTD4 and IL-5 or LTD4 and GM-CSF synergistically induced TGF-β1 expression in eosinophils although stimulation with single factor, LTD4, IL-5 or GM-CSF did not induce the gene expression. LTD4 also induced significant gene expression in eosinophils cultured in an intercellular adhesion molecule-1-coated plate. The results suggested that CysLTs stimulate eosinophils to induce TGF-β1 production in allergic inflammation where IL-5 and GM-CSF are abundant and may be involved in the pathogenesis of airway remodeling.

Inhibitory Effect of Transforming Growth Factor β1 on Cytokine-Enhanced Eosinophil Survival and Degranulation

The effects of transforming growth factor (TGF) beta 1 on cytokine-enhanced eosinophil survival and degranulation were investigated in vitro to determine whether it is an inhibitory regulator of allergic inflammation. Peripheral blood eosinophils purified by Percoll density gradient centrifugation and the CD16 negative selection technique were incubated in the presence of eosinophil-activating cytokines (interleukin-5 (IL-5), IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-gamma) with and without TFG-beta 1 for 1-3 days. On day 1, eosinophil protein X release was measured by radioimmunoassay. Eosinophil viability on day 3 was determined by staining the cells with fluorescein diacetate and propidium iodide, and on the same day DNA was extracted and subjected to gel electrophoresis to test for fragmentation. TGF-beta 1 significantly inhibited eosinophil survival enhanced by IL-5, IL-3, GM-CSF and IFN-gamma in a dose-dependent manner. The inhibitory effects of TGF-beta 1 on IL-5-enhanced survival was partially reversed by high concentrations of IL-5 and was completely neutralized with anti-TGF-beta antibody. IL-5 inhibited DNA fragmentation of eosinophils in vitro. TGF-beta reversed the effect of IL-5, indicating that TGF-beta 1 activates the pathway of apoptosis. TGF-beta 1 significantly suppressed eosinophil protein X release induced by IL-5. These results suggest that TGF-beta 1 may play a role in the modulation of allergic inflammation.