No abstract

SUMMARYInterleukin-13 ( IL-13) shares many, but not all, of the properties of the prototypic T-helper type 2 (Th2 ) cytokine IL-4, but its role in allergen-driven T-cell responses remains poorly defined. We hypothesized that allergen stimulation of peripheral blood T cells from patients with atopic disease compared with non-atopic controls results in elevated IL-13 synthesis in the context of a 'Th2-type' pattern. Freshly isolated peripheral blood mononuclear cells ( PBMC) obtained from sensitized atopic patients with allergic disease, and non-atopic control subjects, were cultured with the allergens Phleum pratense ( Timothy grass pollen) or Dermatophagoides pteronyssinus (house dust mite) and the non-allergenic recall antigen Mycobacterium tuberculosis purified protein derivative (PPD). Supernatant concentrations of IL-13, along with IL-5 and interferon-c ( IFN-c) ( Th2-and Th1-type cytokines, respectively) were determined by enzyme-linked immunosorbent assay ( ELISA). Allergen-induced IL-13 and IL-5 production by T cells from patients with allergic disease was markedly elevated (P=0·0075 and P=0·0004, respectively) compared with non-atopic controls, whereas IFN-c production was not significantly diÂerent. In contrast to allergen, the prototypic Th1-type antigen M. tuberculosis PPD induced an excess of IFN-c over IL-13 and IL-5 production, and absolute concentrations of cytokines were not aÂected by the presence or absence of atopic disease. Addition of exogenous recombinant IFN-c or IL-12, cytokines known to inhibit Th2-type responses, significantly inhibited allergen-driven production of both IL-13 and IL-5, but not T-cell proliferation, whereas exogenous IL-4 did not significantly aÂect production of IL-13 or IL-5. We conclude that allergen-specific T cells from atopic subjects secrete elevated quantities of IL-13 compared with non-atopic controls, in the context of a Th2-type pattern of cytokine production.

In order to detect and characterize allergen-specific T cells in the airways of atopic asthmatics, we measured proliferation and cytokine production by bronchoalveolar lavage (BAL) T cells isolated from Dermatophagoides pteronyssinus (Der p)-sensitive asthmatics and nonatopic control subjects, and compared the results with those generated using peripheral blood (PB) T cells. BAL and PB mononuclear cells were collected 24 h after segmental allergen challenge by fibreoptic bronchoscopy and venepuncture, respectively. T cells purified from BAL and PB were stimulated with autologous, irradiated antigen-presenting cells and D. pteronyssinus extract or a control, nonallergen antigen (M. tuberculosis purified protein derivative [PPD]). IL-5 and IFN-gamma concentrations were measured in culture supernatants by ELISA, and T-cell proliferation by 3H-thymidine uptake. D. pteronyssinus-induced proliferation of T cells derived from both BAL and PB was elevated in asthmatics when compared with control subjects (p < 0.05), whereas PPD-induced proliferation was equivalent in both compartments. In the asthmatics, D. pteronyssinus-induced proliferative responses of equivalent numbers of BAL and PB T cells obtained after allergen challenge were statistically equivalent. Nevertheless, BAL T cells stimulated with D. pteronyssinus produced significantly greater amounts of IL-5 than did PB T cells (p < 0.05). Allergen-induced proliferation and IL-5 production by BAL T cells in the asthmatics after segmental allergen challenge correlated with the percentages of eosinophils in the BAL fluid (p < 0.01). Further, BAL T cells from asthmatic patients produced significantly higher amounts of IL-5 than did the same number of cells from nonatopic control subjects (p < 0.05). We conclude that, in D. pteronyssinus-sensitive asthmatics, allergen-specific T cells can be detected in the bronchial lumen after allergen challenge and that allergen-induced proliferation and IL-5 production by these cells correlates with local eosinophil influx. Although bronchial luminal T cells show an equivalent proliferative response to allergen stimulation as compared with PB T cells, they do produce more IL-5, consistent with the hypothesis that local differentiation or priming of these cells within the bronchial mucosal environment results in upregulation of allergen-induced IL-5 secretion.

Specific eosinophil accumulation and activation within the asthmatic bronchial mucosa are thought to occur at least partly through the actions of cytokines, including interleukin (IL)-5, IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF). Although mRNA encoding some of these cytokines has been demonstrated in bronchoalveolar lavage (BAL) fluid cells and bronchial biopsies from asthmatics, it has yet to be established whether these cells produce the translated products and whether expression is associated with CD4+ T helper or CD8+ cytotoxic T cells. We addressed this problem by raising polyclonal CD4+ and CD8+ T cell lines from the BAL fluid of six atopic asthmatics, five atopic non-asthmatics and seven non-atopic non-asthmatic controls. BAL fluid cells obtained at fiberoptic bronchoscopy were depleted of adherent cells, and then T lymphocytes expanded by stimulation with monoclonal anti-CD3 antibody and recombinant human IL-2. When lymphocytes had expanded to sufficient numbers, CD4+ and CD8+ cells were separated by positive selection with magnetic beads coated with anti-CD4 or anti-CD8 monoclonal antibodies and further expanded. Cytokine secretion by standardized cell numbers was measured by enzyme-linked immunosorbent assays. BAL CD4+ T cell lines from the asthmatics secreted significantly elevated quantities of both IL-5 and GM-CSF as compared with lines from the atopic and non-atopic controls (p = 0.023-0.003). In contrast, IL-3 secretion did not significantly differ between the groups. In some subjects, CD8+ T cell lines also secreted significant quantities of these cytokines and there was a trend for IL-5 secretion by these cells to be higher in asthmatics than non-atopic controls (p = 0.035). These data are consistent with the hypothesis that activated T lymphocytes from asthmatics, particularly of the CD4+ subset, are predisposed to release elevated quantities of cytokines relevant to the accumulation and activation of eosinophils.

Interleukin-5 (IL-5) specifically induces the differentiation of eosinophils, which are important in host defence and the pathogenesis of allergies and asthma. Structurally, IL-5 is a unique member of the short-chain helical-bundle subfamily of cytokines whose canonical motif contains four helices (A-D) arranged in an up-up-down-down topology. In contrast to other subfamily members, which fold unimolecularly into a single helical bundle, IL-5 forms a pair of helical bundles by the interdigitation of two identical monomers that contribute a D helix to the other's A-C helices. We predicted that the lack of bioactivity by an IL-5 monomer was due to a short loop between helices C and D which physically prevents unimolecular folding of helix D into a functionally obligate structural motif. Here we report that, by lengthening this loop, we have engineered an insertional mutant of IL-5 that was expressed as a monomer with biological activity similar to that of native IL-5. These studies demonstrate that all of the structural features necessary for IL-5 to function are contained within a single helical bundle.