Dual role of interleukin-1alpha in delayed-type hypersensitivity and airway hyperresponsiveness.

BACKGROUND: Using a T helper (Th)1/Th2 disease model, we previously showed that genetically determined Th development depends on dendritic cell-derived interleukin (IL)-1alpha. In Leishmania major infections, Th1 immunity develops if IL-1alpha is present during T cell priming, whereas at later time points, IL-1alpha worsens disease outcome. In the present study, we determined the role of IL-1alpha in other Th2-mediated diseases. METHODS: BALB/c mice were subjected to delayed-type hypersensitivity (DTH) or ovalbumin (OVA)/alum-induced allergic asthma in the presence or absence of IL-1alpha. RESULTS: In DTH, mice treated with IL-1alpha during sensitization with keyhole limpet hemocyanin (KLH)/alum developed decreased footpad swelling associated with elevated KLH-specific interferon-gamma levels. In asthma, significantly decreased airway hypersensitivity responses (AHRs) were detected upon treatment with IL-1alpha during T cell priming. In contrast to control mice, IL-1alpha-treated mice showed reduced peribronchial inflammatory infiltrates. The bronchoalveolar lavage (BAL) fluid contained significantly decreased eosinophil numbers (approximately 50%), but 4 times more neutrophils. The BAL fluid of IL-1alpha-treated BALB/c exhibited reduced amounts of IL-5 and OVA-specific IgE serum levels. In contrast, IL-1alpha treatment at later time points after sensitization or during allergen challenge worsened AHR, had no effect on lung inflammation and BAL fluid cell composition. Furthermore, cytokine levels (IL-5, IL-13) and antigen-specific IgE were increased or unaltered under these conditions. CONCLUSION: Similarly to leishmaniasis, IL-1alpha administration during sensitization of Th2-mediated allergic reactions suppresses the course of disease by shifting the immune response towards Th1, whereas later treatments worsen disease outcome. Future studies will elucidate the therapeutic value of IL-1alpha in asthmatic patients.

A key regulatory role of the transcription factor NFATc2 in bronchial adenocarcinoma via CD8+ T lymphocytes.

The Ca(2+)-regulated calcineurin/nuclear factor of activated T cells (NFAT) cascade controls alternative pathways of T-cell activation and peripheral tolerance. Here, we describe reduction of NFATc2 mRNA expression in the lungs of patients with bronchial adenocarcinoma. In a murine model of bronchoalveolar adenocarcinoma, mice lacking NFATc2 developed more and larger solid tumors than wild-type littermates. The extent of central tumor necrosis was decreased in the tumors in NFATc2((-/-)) mice, and this finding was associated with reduced tumor necrosis factor-alpha and interleukin-2 (IL-2) production by CD8(+) T cells. Adoptive transfer of CD8(+) T cells of NFATc2((-/-)) mice induced transforming growth factor-beta(1) in the airways of recipient mice, thus supporting CD4(+)CD25(+)Foxp-3(+)glucocorticoid-induced tumor necrosis factor receptor (GITR)(+) regulatory T (T(reg)) cell survival. Finally, engagement of GITR in NFATc2((-/-)) mice induced IFN-gamma levels in the airways, reversed the suppression by T(reg) cells, and costimulated effector CD4(+)CD25(+) (IL-2Ralpha) and memory CD4(+)CD127(+) (IL-7Ralpha) T cells, resulting in abrogation of carcinoma progression. Agonistic signaling through GITR, in the absence of NFATc2, thus emerges as a novel possible strategy for the treatment of human bronchial adenocarcinoma in the absence of NFATc2 by enhancing IL-2Ralpha(+) effector and IL-7Ralpha(+) memory-expressing T cells.

Coexpression of TGF-beta1 and IL-10 enables regulatory T cells to completely suppress airway hyperreactivity.

In allergic airway disease, Treg may play an important role in the modulation of airway hyperreactivity (AHR) and inflammation. We therefore investigated the therapeutic potential of Treg in an Ag-dependent murine asthma model. We here describe that AHR can be completely suppressed by adoptive transfer of Treg overexpressing active TGF-beta1. Using mice with impaired TGF-beta signaling in T cells, we could demonstrate that TGF-beta signaling in recipient effector T cells or transferred Treg themselves is not required for the protective effects on AHR. However, the expression of IL-10 by Treg was found to be essential for the suppression of AHR, since Treg overexpressing active TGF-beta1 but deficient in IL-10 lacked protective effects. Airway inflammation could not be significantly suppressed by wild-type or transgenic Treg. In conclusion, modulation of cytokine expression by Treg may have therapeutic potential for the treatment of AHR in asthma. The mechanisms of the effects of Treg on airway inflammation require further clarification.

Immunosurveillance of lung melanoma metastasis in EBI-3-deficient mice mediated by CD8+ T cells.

EBV-induced gene 3 (EBI-3) codes for a soluble type I receptor homologous to the p40 subunit of IL-12 that is expressed by APCs following activation. In this study, we assessed the role of EBI-3 in a model of lung melanoma metastasis. Intravenous injection of the B16-F10 cell line resulted in a significant reduction of lung tumor metastasis in EBI-3(-/-) recipient mice compared with wild-type mice. The immunological finding accompanying this effect was the expansion of a newly described cell subset called IFN-gamma producing killer dendritic cells associated with CD8(+) T cell responses in the lung of EBI-3(-/-) mice including IFN-gamma release and TNF-alpha-induced programmed tumor cell death. Depletion of CD8(+) T cells as well as targeting T-bet abrogated the protective effects of EBI-3 deficiency on lung melanoma metastases. Finally, adoptive transfer of EBI-3(-/-) CD8(+) T cells into tumor bearing wild-type mice inhibited lung metastasis in recipient mice. Taken together, these data demonstrate that targeting EBI-3 leads to a T-bet-mediated antitumor CD8(+) T cell responses in the lung.

IL-2 receptor beta-chain signaling controls immunosuppressive CD4+ T cells in the draining lymph nodes and lung during allergic airway inflammation in vivo.

IL-2 influences both survival and differentiation of CD4(+) T effector and regulatory T cells. We studied the effect of i.n. administration of Abs against the alpha- and the beta-chains of the IL-2R in a murine model of allergic asthma. Blockade of the beta- but not the alpha-chain of the IL-2R after allergen challenge led to a significant reduction of airway hyperresponsiveness. Although both treatments led to reduction of lung inflammation, IL-2 signaling, STAT-5 phosphorylation, and Th2-type cytokine production (IL-4 and IL-5) by lung T cells, IL-13 production and CD4(+) T cell survival were solely inhibited by the blockade of the IL-2R beta-chain. Moreover, local blockade of the common IL-2R/IL-15R beta-chain reduced NK cell number and IL-2 production by lung CD4(+)CD25(+) and CD4(+)CD25(-) T cells while inducing IL-10- and TGF-beta-producing CD4(+) T cells in the lung. This cytokine milieu was associated with reduced CD4(+) T cell proliferation in the draining lymph nodes. Thus, local blockade of the beta-chain of the IL-2R restored an immunosuppressive cytokine milieu in the lung that ameliorated both inflammation and airway hyperresponsiveness in experimental allergic asthma. These findings provide novel insights into the functional role of IL-2 signaling in experimental asthma and suggest that blockade of the IL-2R beta-chain might be useful for therapy of allergic asthma in humans.

Transgenic models in allergic responses.

The immunoresponses are mediated by cells presenting the antigen to T cells. The transcription factors involved in the differentiation of T helper cells enclose T-bet (Th1), c-maf (Th2), GATA-3 (Th2), Foxp3 (T reg) and RORgammaT (Th17). They are regulated in allergic asthma. The use of murine models either as germline or as tissue specific transgenic mice has given decisive immunological tools to understand the importance of selected transcription factors or cytokines. Tissue specific transgenic lines have been generated into the Clara Cell or CD2 promoter directing tissue- and immune cells specific expression of the gene of interest. We identified T cell transcription factors important for asthma - such as T-bet, c-maf, GATA-3. Transgenic and knockout murine models of these transcription factors provided very important information for the human disease. Regarding to the pathogenesis of chronic asthma, we generated transgenic lines overexpressing IL-18 and analyzed a dominant negative mutant of the TGF-betareceptor II. These models will offer to us a great input for the understanding of the T cell memory and the processes like airway remodelling. Beside DNA microinjection and stem cell transfer the On/Off systems like Cre-lox models have helped to understand the role of selected genes in different steps of experimental disease. Moreover, the transgenic model provide reliable models for the pre-clinical approval of therapy for allergic asthma to develop more efficient compounds and functional antibodies.

Protective role of nuclear factor of activated T cells 2 in CD8+ long-lived memory T cells in an allergy model.

BACKGROUND: The transcriptional regulation of cytokines released and controlled by memory T cells is not well understood. Defective IFN-gamma production in allergic asthma correlates in human beings with the risk of wheezing in childhood. OBJECTIVE: To understand the role of the transcription factor nuclear factor of activated T cells 2 (NFATc2) in memory and effector T cells in the airways in experimental allergic asthma. METHODS: We used murine models of allergic asthma and adoptive cell transfer of fluorescence-activated sorted cells in a disease model. RESULTS: Mice lacking NFATc2 developed an increase in airwayhyperresponsiveness (AHR), remodeling, and serum IgE levelson ovalbumin sensitization. This phenotype was associated withCD81CD1222 T cells deficient in IFN-g production in theairways. The origin of this phenotype in NFATc2(2/2) mice wasrelated to an expanded population of lung CD81CD1221(IL-2Rb chain) CD127hi (IL-7 receptor [R] a chain1) long-livedmemory cells. Adoptive transfer of ovalbumin-specific CD81NFATc2(2/2) T cells enhanced the AHR generated byNFATc2(2/2) CD41 T cells in immunodeficient mice, increasedIL-17, and reduced IFN-g production in the reconstituted mice. Depletion of the memory CD81CD1221IL-7Rhigh T-cellpopulation corrected the defect in IFN-g production by lungNFATc2(2/2) CD81CD1222 cells and abrogated the increasedAHR observed in NFATc2(2/2) CD81 T-cell-reconstituted micewith a severe combined immunodeficiency disorder. CONCLUSION: Taken together, our results suggest that NFATc2 expression in long-lived memory CD8+ T cells controls IL-2 and IFN-gamma production in lung CD8+ T cells, which then limits TH17 and TH2 development in the airways during allergen challenge.

A method to enable the investigation of murine bronchial immune cells, their cytokines and mediators.

Innovative therapies for severe lung diseases (such as allergic and chronic asthma, chronic obstructive pulmonary disease or any type of lung cancer) require a detailed understanding of the cellular and immune processes in the lung. This protocol details a method to obtain the immune cells of the bronchi as well as the cytokines and mediators produced by these cells for further investigation. The broncho-alveolar lavage fluid (BALF) is taken by injecting physiological solution through the tracheal tube into the murine airways and carefully regained by winding up the connected syringe. After centrifugation, the resulting BALF supernatant can be stored for detection of cytokines or other mediators by enzyme-linked immunosorbent assay or other methods; the resuspended cell pellet can also be used for flow cytometric analyses, to check cell viability and the level of apoptosis, as well as other applications. In addition, CD4+ T cells isolated from wild-type and genetically modified mice alone or along with other immunologically important cells such as T regulatory cells, which can be used to reconstitute immunodeficient mice, may be retrieved from the airways with this method. This protocol can be completed within 35 min.

Asthmatic changes in mice lacking T-bet are mediated by IL-13.

Mice with a targeted deletion of the T-bet gene exhibit spontaneous airway hyperresponsiveness (AHR), airway inflammation, enhanced recovery of T(h)2 cytokines from bronchoalveolar lavage fluid, sub-epithelial collagen deposition and myofibroblast transformation. Here we analyze the mechanisms responsible for the chronic airway remodeling observed in these mice. CD4+ T cells isolated from the lung of T-bet-deficient mice were spontaneously activated CD44(high)CD69(high) memory T cells, with a typical T(h)2 cytokine profile. Neutralization of IL-13 but not IL-4 resulted in amelioration of AHR in airways of mice lacking T-bet. IL-13 blockade also led to reduced eosinophilia and decreased vimentin, transforming growth factor beta (TGF-beta) and alpha smooth muscle actin (alphaSMA) levels. T-bet(-/-) lung fibroblasts proliferated very rapidly and released increased amounts of TGF-beta. Interestingly, neutralization of TGF-beta ameliorated aspects of the chronic airway remodeling phenotype but did not reduce AHR. These data highlight a T-bet-directed function for IL-13 in controlling lung remodeling that is both dependent on and independent of its interaction with TGF-beta in the asthmatic airway.