ABSTRACT
<p><b>BACKGROUND</b>Allergic asthma is associated with airway inflammation and hyperresponsiveness caused by dysregulated production of cytokines secreted by allergen-specific helper T-type 2 (Th2) cells. The linker for activation of T cells (LAT) is a membrane-associated adaptor protein, which has been shown to take part in regulating T cell receptor (TCR) signaling and T cell homeostasis. In this study, we established an asthmatic mouse model to examine the changes in LAT levels during allergic airway disease and the effects of LAT transgenic expression on airway inflammation.</p><p><b>METHODS</b>T cells from mouse lung tissues were isolated from allergen challenged (ovalbumin (OVA)) and control mice, and the purity of these isolated T cells was examined by fluorescence-activated cell sorter (FACS). Semi-quantitative RT-PCR and Western blotting were used to detect the expression of the LAT gene and LAT protein, respectively. After an intranasally administered mixture of pCMV-HA-LAT plasmid and Lipofectamine 2000, 24 hours before and 72 hours after allergen challenge, the BALF cell count and the differential cytologies were studied. In addition, IL-4 and IFN-γ levels in the BALF were determined by ELISA, and pathological changes in lung tissues were observed.</p><p><b>RESULTS</b>LAT protein and mRNA expression were decreased in lung T cells in a mouse model of allergen-induced airway disease. After intranasal administration of pCMV-HA-LAT, histopathological examination of the lungs showed that intervention with LAT overexpression prevented mice from developing airway inflammation, and the number of total cells, eosinophils, neutrophils, and lymphocytes in the BALF was reduced significantly compared with the OVA sensitized and challenged group. In addition, the Th2 cytokine IL-4 decreased, while the Th1 cytokine IFN-γ increased compared to the OVA sensitized and challenged group or the OVA sensitized group plus pCMV-HA treatment.</p><p><b>CONCLUSION</b>This study demonstrates that LAT might effectively diminish Th2 cytokine responses, lung histopathological changes and lung inflammation to allergen challenge in a model of experimentally induced asthma.</p>
Subject(s)
Animals , Female , Mice , Asthma , Allergy and Immunology , Metabolism , Blotting, Western , Bronchoalveolar Lavage Fluid , Allergy and Immunology , Cells, Cultured , Cytokines , Metabolism , Inflammation , Allergy and Immunology , Mice, Inbred BALB C , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes , Allergy and Immunology , MetabolismABSTRACT
<p><b>BACKGROUND</b>The immunologic response to allergens mediated by T lymphocytes is an incipient key element in the pathogenesis of asthma, and Th1/Th2 balance is regarded as the core of asthma pathogenesis. Notch is a single-pass transmembrane receptor protein that regulates differentiation, proliferation and apoptosis in a broad range of cells. It is considered that the Notch signal pathway works in every stage of T cell development and differentiation. Whether the pathway of asthma pathogenesis is related to Notch1 remains unknown. This study is aimed to investigate whether the pathway of asthma pathogenesis is related to Notch1 by examining the effect of knockdown of the Notch1 gene by small interfering RNA on T cell differentiation.</p><p><b>METHODS</b>An OVA-induced asthma mouse model was established. The expression of Notch1 in the tissue and T cells of the lung from asthmatic mice was detected by RT-PCR and Western blotting. The expression of Notch1 and cytokine interleukin (IL)-4 and interferon (IFN)-gamma in activated lung T cells was detected by RT-PCR and enzyme-linked immunosorbent assay after blocking Notch1 by small interfering RNA.</p><p><b>RESULTS</b>The mRNA and protein expression of Notch1 increased significantly both in the lung tissue and lung T cells of asthmatic mice (both P < 0.05). IL-4 decreased and IFN-gamma increased significantly in active lung T cells after Notch1 was blocked by Notch1-specific small interfering RNA (IL-4: (2.51 +/- 0.51) pg/ml vs 0.64 +/- 0.27) pg/ml protein; IFN-gamma: (21.72 +/- 4.24) pg/ml vs (39.79 +/- 4.09) pg/ml protein, P < 0.05).</p><p><b>CONCLUSION</b>This study demonstrated that the Notch1 signal might play a role in the pathogenesis of asthma by its involvement in Th1/Th2 differentiation.</p>
Subject(s)
Animals , Female , Male , Mice , Blotting, Western , Interferon-gamma , Metabolism , Interleukin-4 , Metabolism , Lung , Metabolism , Mice, Inbred BALB C , RNA, Small Interfering , Genetics , Metabolism , Receptor, Notch1 , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes , MetabolismABSTRACT
Objective To construct the eukaryotic expression vector of linker for activation of T cells(LAT) gene in mouse. MethodsLAT cDNA of mouse mononuclear cells was amplified with polymerase chain reaction.The fragment was inserted into the eukaryotic expression vector of pCMV-HA plasmid.The recombinant plasmid was verified by DNA sequencing and used to transfect lymphocytes of the asthmatic model. Results The length of amplified fragment was 729 bp.The sequence of LAT gene was confirmed by Genbank.The LAT protein could be detected in the lymphocytes of asthmatic model.Conclusion The recombinant eukaryotic expression vector pCMV-HA-LAT can be successfully constructed.