IL-17 is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines.

BACKGROUND: IL-17 is a cytokine that has been reported to be produced by T lymphocytes. In vitro, IL-17 activates fibro-blasts and macrophages for the secretion of GM-CSF, TNF-alpha, IL-1beta, and IL-6. A number of these cytokines are involved in the airway remodeling that is observed within the lungs of asthmatic individuals. OBJECTIVE: In this study, we investigated the expression of IL-17 in sputum and bronchoalveolar lavage specimens obtained from asthmatic subjects and from nonasthmatic control subjects. METHODS: IL-17 was detected through use of immunocytochemistry, in situ hybridization, and Western blot. Bronchial fibroblasts were stimulated with IL-17, and cytokine production and chemokine production were detected through use of ELISA and RT-PCR. RESULTS: Using immunocytochemistry, we demonstrated that the numbers of cells positive for IL-17 are significantly increased in sputum and bronchoalveolar lavage fluids of subjects with asthma in comparison with control subjects (P <.001 and P <.005, respectively). We demonstrated that in addition to T cells, eosinophils in sputum and bronchoalveolar lavage fluids expressed IL-17. Peripheral blood eosinophils were also positive for IL-17, and the level of IL-17 in eosinophils purified from peripheral blood was significantly higher in subjects with asthma than in controls (P <.01). To further investigate the mechanism of action of IL-17 in vivo, we examined the effect of this cytokine on fibroblasts isolated from bronchial biopsies of asthmatic and nonasthmatic subjects. IL-17 did enhance the production of pro-fibrotic cytokines (IL-6 and IL-11) by fibroblasts, and this was inhibited by dexamethasone. Similarly, IL-17 increased the level of other fibroblast-derived inflammatory mediators, such as the alpha-chemokines, IL-8, and growth-related oncogene-alpha. CONCLUSION: Our results, which demonstrate for the first time that eosinophils are a potential source of IL-17 within asthmatic airways, suggest that IL-17 might have the potential to amplify inflammatory responses through the release of proinflammatory mediators such as alpha-chemokines.

Functional expression of IL-12 receptor by human eosinophils: IL-12 promotes eosinophil apoptosis.

In murine models of allergic inflammation, IL-12 has been shown to decrease tissue eosinophilia, but the underlying mechanisms are not known. We evaluated the expression of IL-12R and the effect of IL-12 on eosinophil survival. In situ hybridization demonstrated the presence of mRNA and immunoreactivity for IL-12Rbeta1 and -beta2 subunits in human peripheral blood eosinophils. Surface expression of IL-12Rbeta1 and -beta2 subunits on freshly isolated human eosinophils was optimally expressed after incubation with PMA. To determine the functional significance of IL-12R studies, we studied cell viability and apoptosis. Morphological analysis and propidium iodide staining for cell cycle demonstrated that recombinant human IL-12 increased in vitro human eosinophil apoptosis in a dose-dependent manner. Addition of IL-5 together with IL-12 abrogated eosinophil apoptosis, suggesting that IL-12 and IL-5 have antagonistic effects. Our findings provide evidence for a novel role for IL-12 in regulating eosinophil function by increasing eosinophil apoptosis.

Interleukin-9 enhances interleukin-5 receptor expression, differentiation, and survival of human eosinophils.

Interleukin-9 (IL-9) has been implicated in the pathogenesis of allergic disorders. To examine the interaction between IL-9 and eosinophils, we evaluated mature peripheral blood eosinophils for their expression of the specific alpha-subunit of the IL-9 receptor (IL-9R-alpha). The expression of IL-9R-alpha by human eosinophils was detected at the messenger RNA (mRNA) and protein levels by reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry, and immunocytochemical analysis, respectively. Functional analyses demonstrated that recombinant human (rh)IL-9 inhibited in vitro peripheral blood human eosinophil apoptosis in a concentration-dependent manner. We then examined the role of IL-9 in eosinophil differentiation using the human cord blood CD34(+) cells and human promyelocytic leukemia cells (HL-60). The addition of IL-9 to CD34(+) cells cultured in IL-3 and IL-5 enhanced eosinophil development, and IL-9 alone induced the expression of IL-5R-alpha. IL-9 also up-regulated the IL-5R-alpha chain cell surface expression during terminal eosinophil differentiation of the HL-60 cell line. Our findings suggest that IL-9 may potentiate in vivo eosinophil function by increasing their survival and IL-5-mediated differentiation and maturation. Taken together, these results suggest a mechanism by which IL-9 potentiates airway and tissue eosinophilia.

IL-9 expression by human eosinophils: regulation by IL-1beta and TNF-alpha.

BACKGROUND: IL-9 is a pleiotropic cytokine that exhibits biologic activity on cells of diverse hemopoietic lineage. IL-9 stimulates the proliferation of activated T cells, enhances the production of IgE from B cells, and promotes the proliferation and differentiation of mast cells and hematopoietic progenitors. OBJECTIVE: In this study we evaluated the expression of IL-9 messenger (m)RNA and protein by human peripheral blood eosinophils. We also investigated the role of IL-1beta and TNF-alpha in the release of IL-9 from human peripheral blood eosinophils. METHODS: RT-PCR, in situ hybridization, and immunocytochemistry were used to investigate the presence of IL-9 mRNA and protein in human peripheral blood eosinophils from asthmatic patients and normal control subjects. Furthermore, biologic assay was used to investigate the release of IL-9 protein from IL-1beta- or TNF-alpha-stimulated eosinophils in vitro. RESULTS: RT-PCR analysis showed the presence of IL-9 mRNA in human peripheral blood eosinophil RNA preparations from subjects with atopic asthma, as well as in the eosinophil-differentiated HL-60 cell line. By using in situ hybridization, a significant difference (P <.01) in IL-9 mRNA expression was detected in human peripheral blood eosinophils freshly isolated from asthmatic subjects compared with those isolated from normal control subjects. Furthermore, the percentage of IL-9 immunoreactive eosinophils from asthmatic patients was increased compared with that found in normal control subjects (P <.01). We also demonstrate that cultured human peripheral blood eosinophils from asthmatic subjects synthesize and release IL-9 protein, which is upregulated on stimulation with TNF-alpha and IL-1beta. CONCLUSION: Human eosinophils express biologically active IL-9, which suggests that these cells may influence the recruitment and activation of effector cells linked to the pathogenesis of allergic disease. These observations provide further evidence for the role of eosinophils in regulating airway immune responses.

Evidence for expression of eosinophil-associated IL-12 messenger RNA and immunoreactivity in bronchial asthma.

BACKGROUND: Eosinophils are a source of cytokines within the airways of asthmatic individuals that may exert an important immunoregulatory influence. OBJECTIVES: We examined IL-12 messenger (m)RNA and protein expression in eosinophils from peripheral blood and bronchoalveolar lavage (BAL) fluid obtained from subjects with atopic asthma (n = 7), patients with chronic bronchitis (n = 5), and nonatopic healthy control subjects (n = 7). To further define this IL-12(+) population of eosinophils for the expression of other cytokines, we colocalized IL-12 and IL-5 within the peripheral blood eosinophils. METHODS: To detect IL-12 mRNA and protein expression, we used in situ hybridization and immunocytochemistry techniques. The double-immunocytochemistry technique was used to localize IL-12 protein to BAL eosinophils and to colocalize IL-5 and IL-12 in peripheral blood eosinophils. RESULTS: IL-12 mRNA and immunoreactive protein were localized to peripheral blood eosinophils. BAL fluid-derived eosinophils from asthmatic subjects were also reactive to IL-12. The percentage of peripheral blood eosinophils expressing mRNA for IL-12 was significantly lower in asthmatic subjects compared with that found in eosinophils obtained from patients with chronic bronchitis (P<.001) and control patients (P <.05). Colocalization studies demonstrated that the percentages of IL-12(+) eosinophils that are also IL-5(+) were 72% in asthmatic subjects and only 11% in control subjects (P<.001). CONCLUSION: These results suggest that eosinophils are a potential source of IL-12. Eosinophil-derived IL-12 may contribute and modulate the local allergic inflammatory responses.