Kinetics of eotaxin expression and its relationship to eosinophil accumulation and activation in bronchial biopsies and bronchoalveolar lavage (BAL) of asthmatic patients after allergen inhalation

Abstract

We investigated the kinetics of allergen-induced eotaxin expression and its relationship to eosinophil accumulation and activation in the airways of patients with allergic asthma. Twenty-four patients with allergic asthma and late asthmatic responses to allergen inhalation were randomly allocated into three groups of eight patients each, who received bronchoscopy with bronchial biopsies and BAL at 2, 4 and 24 h, respectively, after the inhalation of the diluent and the allergen. The expression of eotaxin mRNA and protein and eotaxin release were evaluated by in situ hybridization, immunohistochemistry, immunocytochemistry, and radioimmunoassay. Increased transcription from the eotaxin gene preceded the appearance of the late asthmatic response and the influx of activated eosinophils in bronchial tissue and BAL fluid (BALF). This was followed by increased cell expression of eotaxin protein (P<0.001) and increased eotaxin release (P<0.001), which correlated with the numbers of total and activated eosinophils and the level of airflow obstruction at 4 h after allergen exposure (P<0.05 for all correlations). At 24 h after allergen inhalation, enhanced eotaxin expression declined without a similar reduction in the numbers of eosinophils in bronchial biopsies and when there was a further increase in the number of these cells in BALF (P<0.05). These results indicate that eotaxin contributes to the early phase of allergen-induced recruitment of activated eosinophils into the airways of patients with allergic asthma and that other factors are implicated in the persistence of eosinophil infiltration.

Identification of Circulating Fibrocytes as Precursors of Bronchial Myofibroblasts in Asthma

“…To investigate whether fibrocyte accumulation occurs in allergic asthma, we used a model from a previous study (25) that mimics the inflammatory and functional changes of an exacerbation due to natural allergen exposure in allergic asthmatics, the late asthmatic response induced by allergen inhalation in the laboratory (26,27). The study protocol and the patient characteristics have been reported in detail previously (25).…”

“…The study protocol and the patient characteristics have been reported in detail previously (25). Briefly, allergic asthmatics were randomly allocated into three groups of eight patients each.…”

“…Therefore, these cells can be identified in tissue samples by double immunohistochemistry and in situ hybridization technique using a specific Ab against CD34 and a probe for procollagen I mRNA. For this analysis we prepared serial cryosections (5 m) of each frozen bronchial tissue available from our previous study (25). Double immunohistochemistry and in situ hybridization were performed sequentially on the same sections.…”

“…To detect fibrocytes that had undergone local differentiation into myofibroblast-like cells, adjacent sections were therefore double labeled with the anti-CD34 mAb indicated above and with an anti ␣-SMA mAb (DAKO). Binding of the anti-CD34 mAb was revealed as reported above, while binding of the anti-␣-SMA mAb was visualized by an enzyme-linked indirect immunoperoxidase method (25,30,31) using a chromogenic substrate that labels positive cells blue (Fast Blue, Vector Laboratories). Omission or substitution of each primary mAb with an irrelevant, isotypematched mAb from the same species was used as a negative control.…”

“…Binding sites were revealed by the enzymelinked indirect immunoperoxidase method using 3,3Ј-diaminobenzidine as chromogenic substrate (25,30,31). Omission or substitution of the primary mAb with an irrelevant, isotype-matched mouse mAb was used as a negative control.…”