The histopathology of bronchial asthma is associated with structural changes within the airways, including subepithelial fibrosis, as well as chronic eosinophilic inflammation. The mechanisms responsible for this tissue remodeling, and in particular the role of inflammatory cells, remain to be established. Transforming growth factor-beta (TGF-beta) is a potent profibrotic cytokine which may contribute to the thickening of the reticular lamina by the deposition of collagen fibers. To investigate the molecular mechanisms underlying these structural changes, we have investigated the expression of TGF-beta1 mRNA and immunoreactivity within the bronchial mucosa of mild to severe asthmatic individuals and normal control subjects using the techniques of in situ hybridization and immunocytochemistry. As eosinophils are prominent within the asthmatic airway and are known to synthesize pro-inflammatory cytokines, the presence of TGF-beta1 mRNA and immunoreactive protein in eosinophils was also examined. Asthmatic individuals exhibited a greater expression of TGF-beta1 mRNA and immunoreactivity in the airways submucosa than normal control subjects (P < 0.05), and these increases were directly related to the severity of the disorder. The extent of airways fibrosis, as detected histochemically, was also increased in asthmatics compared with normal control subjects (P < 0.005). In asthmatic subjects, the presence of subepithelial fibrosis was associated with the severity of the disease and correlated with the decline in forced expiratory volume in 1 s (r2 = 0.78; P < 0.05). Within the asthmatic airways, EG2-positive eosinophils represented the major source of TGF-beta1 mRNA and immunoreactivity. These results provide evidence that TGF-beta1 may play a role in the fibrotic changes occurring within asthmatic airways and that activated eosinophils are a major source of this cytokine.

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In many chronic inflammatory disorders, glucocorticoid (GC) insensitivity is a challenging clinical problem associated with life-threatening disease progression. The molecular basis of GC insensitivity, however, is unknown. Alternative splicing of the GC receptor (R) pre–messenger RNA generates a second GCR, termed GCR-β, which does not bind GCs but antagonizes the transactivating activity of the classic GCR, termed GCR-α. In the current study, we demonstrate that GC-insensitive asthma is associated with a significantly higher number of GCR-β–immunoreactive cells in peripheral blood than GC-sensitive asthmatics or normal controls. Furthermore, we show that patients with GC-insensitive asthma have cytokine-induced abnormalities in the DNA binding capability of the GCR. These abnormalities can be reproduced by transfection of cell lines with the GCR-β gene resulting in significant reduction of their GCR-α DNA binding capacity. We conclude that increased expression of GCR-β is cytokine inducible and may account for GC insensitivity in this common inflammatory condition.

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Interleukin-12 (IL-12) and IL-13 are two recently characterized cytokines which play an important role in the induction of T helper cell type 1 (Th1-) and Th2-like cells, respectively. Using the technique of in situ hybridization, we have investigated the expression of these cytokines in bronchial biopsies from nine allergic asthmatics and nine normal control subjects. To determine the effect of steroid therapy on the expression of IL-12 and IL-13 in asthma, the numbers of cells expressing these cytokine mRNA before and after a 1-wk course of oral prednisone in six steroid-sensitive (SS) and five steroid-resistant (SR) moderately severe asthmatics were also examined. There was an increased number of IL-13, and a decreased number of IL-12 mRNA positive cells in asthmatic subjects compared with normal control subjects (p < 0.001). After steroid treatment, the increase in FEV1 values observed in SS asthmatics was accompanied by a significant decrease in cells expressing IL-13 mRNA (p < 0.05) and an increase in the numbers of cells expressing IL-12 mRNA (p < 0.05). In contrast, steroid therapy in SR asthmatics was not associated with significant changes in IL-12 and IL-13 mRNA expression. Thus, allergic asthma is associated with a downregulation of IL-12 mRNA expression and an upregulation of IL-13 mRNA expression. These results suggest an in vivo role for IL-12 and IL-13 in modulating allergic responses and support the notion that the clinical effects of glucocorticoids are at least partially mediated through the modulation of cytokine production.

Eotaxin is an eosinophil-specific chemokine associated with the recruitment of eosinophils to the site of allergic inflammation. The aims of this study were to determine the expression of eotaxin in nasal biopsies from allergic and nonallergic individuals with chronic severe sinusitis, and to examine whether the expression of this chemokine is upregulated following allergen challenge in the nasal mucosa of patients with allergic rhinitis. We also undertook to phenotype of inflammatory cells within the submucosa expressing eotaxin mRNA. Nasal turbinate tissue from 16 individuals with allergic or nonallergic chronic sinusitis and 10 normal controls were examined for the presence of eotaxin mRNA and immunoreactivity by in situ hybridization and immunocytochemistry. The numbers of cells expressing eotaxin mRNA were also determined after either allergen or diluent challenge in atopic subjects with a history of allergic rhinitis. There was a constitutive expression of eotaxin-immunoreactivity and the presence of eotaxin mRNA-positive cells in nasal biopsies from normal individuals. Compared with normal controls, the numbers of cells expressing eotaxin mRNA and protein were significantly increased in both allergic and nonallergic sinusitis (P < 0.001). Eotaxin mRNA was expressed by nasal epithelial cells and primarily colocalized to CD68-positive macrophages within the subepithelium. In subjects with allergic rhinitis, allergen challenge markedly increased the numbers of cells expressing eotaxin mRNA and immunoreactivity in the epithelial and subepithelial cell layers (P < 0.05). This could be largely attributed to a local increase in eotaxin production within the nasal tissues. The results of this study demonstrate the constitutive expression of eotaxin and show that the numbers of cells expressing eotaxin mRNA are increased within the epithelial and subepithelial layers of the nasal mucosa in individuals with chronic sinusitis. Furthermore, allergen challenge of the nasal mucosa in atopic subjects results in a local upregulation of eotaxin expression. These data suggest a potential role for this chemokine in the pathogenesis of allergic and nonallergic eosinophilic inflammation characterizing chronic sinusitis and allergic rhinitis.

Eosinophil differentiation occurs within the bone marrow in response to eosinopoietic cytokines, particularly IL-5. Recently, however, eosinophil precursors (CD34/IL-5Rα+ cells) and IL-5 mRNA+ cells have been identified within the lungs of asthmatics, indicating that a population of eosinophils may differentiate in situ. In this report, we examined the presence of eosinophil precursors within allergic nasal mucosa and examined whether they undergo local differentiation following ex vivo stimulation. We cultured human nasal mucosa obtained from individuals with seasonal allergic rhinitis with either specific allergen, recombinant human IL-5 (rhIL-5), or allergen + soluble IL-5Rα (sIL-5Rα), shown to antagonize IL-5 function. Simultaneous immunocytochemistry and in situ hybridization demonstrated that there were fewer cells coexpressing CD34 immunoreactivity and IL-5Rα mRNA following culture with allergen or rhIL-5, compared with medium alone. Immunostaining revealed that the number of major basic protein (MBP) immunoreactive cells (eosinophils) was higher within tissue stimulated with allergen or rhIL-5, compared with unstimulated tissue. In situ hybridization detected an increase in IL-5 mRNA+ cells in sections from tissue cultured with allergen, compared with medium alone. These effects were not observed in tissue cultured with a combination of allergen and sIL-5Rα. Colocalization analysis indicated this expression to be mainly, but not exclusively, T cell (44%) and eosinophil (10%) derived. Our findings suggest that a subset of eosinophils may differentiate locally within allergic nasal mucosa, in what appears to be a highly IL-5-dependent fashion, and imply that this process might be regulated in vivo by endogenous production of sIL-5Rα.

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