Although allergic asthma was described to be associated with the presence of mucosal T helper (Th)2 cells, it is not entirely clear which factors are responsible for priming of T cells to differentiate into Th2 effector cells in this disease. Interleukin (IL)-6 has been recognized as important because it is secreted by cells of the innate immunity and induces the expansion of the Th2 effector cells, which are major players of the adaptive immune responses. Additionally, IL-6 released by dendritic cells (DCs) inhibits the suppressive function of CD4+CD25+ T regulatory cells, thus inhibiting the peripheral tolerance. The signal transduction of IL-6 has recently taught us how this cytokine influences different aspects of the immune response, especially under pathological conditions. IL-6 can bind to the soluble IL-6R, increased after allergen challenge in asthmatic patients, and, through a mechanism called trans-signaling, induces proliferation of cells expressing the cognate receptor gp130. This mechanism appears to be used for proliferation by developed Th2 cells in the airways. In contrast, through the membrane-bound IL-6R, IL-6 controls CD4+CD25+ survival, as well as the initial stages of the Th2 cells development in the lung. These findings impact the establishment of new therapies for allergic diseases; indeed, blockade of the soluble IL-6R through the fusion protein gp130Fc reduces Th2 cells in the lung, and by blocking the membrane-bound IL-6R, anti-IL-6R antibody treatment induces the number of T-regulatory cells in the lung, thereby reducing the local number of CD4+ T-effector cells in experimental asthma.
The regulation of the cellular immune response in lung diseases is not yet fully understood. Isolating different subsets of immune cells directly from the lung is therefore an indispensable method of gaining detailed knowledge on the function of these cells in this organ. This protocol describes a method of isolating and magnetically labeling CD4+ lung T cells, which are then loaded and retained on the column while all other cells run through it (positive selection). The yield of this isolation is approximately 5 x 10(5) to 1.5 x 10(6) CD4+ cells from a murine lung. These cells can be further investigated by several methods such as flow cytometry, western blot analysis, RT-PCR, immunostaining and ELISA. In addition, lung CD4+ T cells alone or along with other immunologically important cells such as CD8+ T cells and T regulatory cells can be adoptively transferred into immune-deficient mice, and can influence important local parameters. This protocol can be completed in approximately 4 h 20 min.
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-γ producing killer dendritic cells associated with CD8+ T cell responses in the lung of EBI-3−/− mice including IFN-γ release and TNF-α-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.
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.
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.
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