| Objective:The expression pattern of murine Immune responsive gene 1 (Irg1) has been documented in many cases of macrophages and dendritic cells (DCs), which were stimulated with lipopolysaccharide (LPS), M. avium ssp. Paratuberculosis (MAP), and pro-inflammatory cytokines such as IFN-γand TNF, respectively, since the first observation of Irg1 gene expression in LPS-challenged macrophages RAW 264.7 in 1995. Moreover, the expression profile of Irg1 mRNA has a sudden increase in the luminal epithelium of pregnant uterus during blastocyst implantation, and Irg1 specially expressed in neurotoxitic CCR9+ microglia when stimulated CCL25. These expression profiles suggested that murine Irg1 gene, as an immune responsive gene, plays a important role in resistance of Gram-negative bacteria infection and in inflammatory reaction.Importantly, the expression pattern of the human IRG1 gene has not yet established, and the predicted gene sequence has been revised several times according to computed expressed sequence tags (ESTs). Based on the EST profile (Hs.160789). the predicted human IRG1 gene is differentially expressed in blood and/or lung of patient with germ cell tumors or leukemia during different health states and developmental stages; however, whether EST counts provide a true indication of gene activity and its real function and regulation mechanism is still unknown.On the other hand, thymus is the primary place for differentiation, proliferation, and maturation of T cells by positive and/or negative selection. Thymus continues to grow between birth and puberty and then begins to atrophy, a process directed by the high levels of circulating hormones. Proportional to thymic size, thymic activity (T-cell output) is most active before puberty. Type 1 help T (Th1) cells and Th2 cells are main subsets of effector CD4+ T cells to response to diverse pathogens through secretion of distinct cytokines. Moreover, immunostimulatory Th17 cells enriched the adaptive immunity as well as effector regulatory T cells (Tregs).Methods:To determine the human IRG1 gene expression profile, human fetal tissue samples, peripheral blood mononuclear cells (PBMCs) from normal healthy subjects, and the human leukemia cell lines THP-1 and K-562 challenged with LPS were subjected to RT-PCR using degenerate primers. Based on the sequence of human IRG1 gene fragment, the amplified gene fragment was cloned into the pET32a(+) vector and fusion-expressed with a His-tag in a prokaryotic system. After affinity chromatography, human IRGls fusion proteins were isolated by SDS-PAGE and identified by mass spectrometric analysis for use as an immunogen to immunize rabbits.On the other hand, we detected the murine Thl7 cells and associated cytokines in the thymocytes culture in vitro stimulated with LPS by the means of RT-PCR, ELISA, and flow cytometry.Results:The results indicated that IRG1 gene is differentially expressed in human fetal PBMCs and LPS-stimulated normal adult PBMCs. The titer and specificity of the purified rabbit antiserum were sufficient to measure the expression of human IRG1 gene in various tissues and cultures.Th17 cells differentiated from thymocytes in thymus organ culture stimulated with LPS. It is mainly dependent on the high-dose of stimuli and the duration of LPS-stimulation. Murine Irg1 gene expressed in thymocytes when stimulated with LPS. but it is not completely consistent with the change of Th17 cell development associated molecules.Significance:This is the first observation of IRG1 gene expression pattern in humans. The purified polyclonal antiserum will allow us to initiate the study to elucidate the biological roles of human IRG1 gene.The differentiation of murine thymic Th17 cells upon LPS-stimulation demonstrated the existence of precursor cells and T-cell output in the thymus of adult mouse, which possibly plays a important role in the anti-infection immunity to prevent against severe infection mediated by Gram-negative pathogens. |
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