ABSTRACT
The Src-family tyrosine kinase Lck is an enzyme associated with the CD4 and CD8 co-receptors and promoting signaling through the T cell receptor (TCR) complex. The levels of Lck expression and activity change during the development and differentiation of T cells. Here we show that Lck expression is higher in Th1 cells as compared to Th2 cells. Ectopic overexpression of Lck in Th2 cells results in increased expression of CD4 co-receptor and enhanced S73 phosphorylation of transcription factor c-Jun. Our findings indicate that TCR-mediated signaling in Th2 cells may be directly attenuated by Lck protein expression level.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Receptors, Antigen, T-Cell/metabolism , Th2 Cells/metabolism , Animals , Cells, Cultured , Gene Expression Profiling , HEK293 Cells , Humans , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Mice, Inbred C57BL , Phosphorylation , Serine/metabolism , Signal Transduction , Th1 Cells/metabolismABSTRACT
This chapter provides protocols for in vitro and in vivo analysis of TNF-producing cells from a novel TNF reporter mouse. In these transgenic mice, genetic sequence encoding far-red reporter protein Katyushka (FRFPK) was placed under control of the same regulatory elements as TNF, thus providing the basis for detection, isolation, and visualization of TNF-producing cells.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/pathology , Genes, Reporter , Green Fluorescent Proteins/metabolism , Luminescent Proteins/metabolism , Macrophages/pathology , Tumor Necrosis Factor-alpha/physiology , Animals , Encephalomyelitis, Autoimmune, Experimental/metabolism , Flow Cytometry , Green Fluorescent Proteins/genetics , Image Processing, Computer-Assisted , Indicators and Reagents/metabolism , Luminescent Proteins/genetics , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Promoter Regions, Genetic/genetics , Recombination, Genetic , Thy-1 Antigens/genetics , Red Fluorescent ProteinABSTRACT
Tumor necrosis factor (TNF) is one of the key primary response genes in the immune system that can be activated by a variety of stimuli. Previous analysis of chromatin accessibility to DNaseI demonstrated open chromatin conformation of the TNF proximal promoter in T cells. Here, using chromatin probing with restriction enzyme EcoNI and micrococcal nuclease we show that in contrast to the proximal promoter, the TNF transcription start site remains in a closed chromatin configuration in primary T helper (Th) cells, but acquires an open state after activation or polarization under Th1 and Th17 conditions. We further demonstrate that transcription factor c-Jun plays a pivotal role in the maintenance of open chromatin conformation at the transcription start site of the TNF gene.