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Txk, a nonreceptor tyrosine kinase of the Tec family, is expressed in T helper type 1 cells and regulates interferon gamma production in human T lymphocytes.

Kashiwakura J, Suzuki N, Nagafuchi H, Takeno M, Takeba Y, Shimoyama Y, Sakane T - J. Exp. Med. (1999)

Bottom Line: We found that Txk expression is restricted to Th1/Th0 cells with IFN-gamma producing potential.Txk transfection did not affect IL-2 and IL-4 promoter activities.These results indicate that Txk expression is intimately associated with development of Th1/Th0 cells and is significantly involved in the IFN-gamma production by the cells through Th1 cell-specific positive transcriptional regulation of the IFN-gamma gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and the Department of Medicine, St. Marianna University School of Medicine, Kawasaki, Kanagawa 216-8511, Japan.

ABSTRACT
Differentiation of human T cells into T helper (Th)1 and Th2 cells is vital for the development of cell-mediated and humoral immunity, respectively. However, the precise mechanism responsible for the Th1 cell differentiation is not fully clarified. We have studied the expression and function of Txk, a member of the Tec family of nonreceptor tyrosine kinases. We found that Txk expression is restricted to Th1/Th0 cells with IFN-gamma producing potential. Txk transfection of Jurkat T cells resulted in a several-fold increase of IFN-gamma mRNA expression and protein production; interleukin (IL)-2 and IL-4 production were unaffected. Antisense oligodeoxynucleotide of Txk specifically inhibited IFN-gamma production of normal peripheral blood lymphocytes, antigen-specific Th1 clones, and Th0 clones; IL-2 and IL-4 production by the T cells was unaffected. Txk cotransfection led to the enhanced luciferase activity of plasmid (p)IFN-gamma promoter/enhancer (pIFN-gamma[-538])-luciferase-transfected Jurkat cells upon mitogen activation. Txk transfection did not affect IL-2 and IL-4 promoter activities. Thus, Txk specifically upregulates IFN-gamma gene transcription. In fact, Txk translocated from cytoplasm into nuclei upon activation and transfection with a mutant Txk expression plasmid that lacked a nuclear localization signal sequence did not enhance IFN-gamma production by the cells, indicating that nuclear localization of Txk is obligatory for the enhanced IFN-gamma production. In addition, IL-12 treatment of peripheral blood CD4(+) T cells enhanced the Txk expression, whereas IL-4 treatment completely inhibited it. These results indicate that Txk expression is intimately associated with development of Th1/Th0 cells and is significantly involved in the IFN-gamma production by the cells through Th1 cell-specific positive transcriptional regulation of the IFN-gamma gene.

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Effects of Txk antisense ODN on cytokine production by human T cells. (a) Txk expression of normal peripheral blood T cells treated with Txk antisense ODN was analyzed. Txk expression was specifically reduced when treated with Txk antisense ODN, but not sense ODN (both 10 μM), for 12 h. The result shown (magnification 250) is representative of three independent experiments with essentially the same results. (b) Normal peripheral blood T cells were cultured in the presence of Txk antisense ODN for 12 h to reduce Txk expression. Thereafter, the T cells were stimulated with PHA. The result shown is representative of three independent experiments with essentially the same results. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted. (c) PPD- and Crj-1-specific T cell clones pretreated with the ODNs (5 μM, which inhibits Txk expression of the clones) were stimulated with the relevant Ag plus irradiated autologous PBMCs. The similar results were reproduced in the experiments using cells from different donors and different Ag-specific T cell clones. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted.
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Figure 4: Effects of Txk antisense ODN on cytokine production by human T cells. (a) Txk expression of normal peripheral blood T cells treated with Txk antisense ODN was analyzed. Txk expression was specifically reduced when treated with Txk antisense ODN, but not sense ODN (both 10 μM), for 12 h. The result shown (magnification 250) is representative of three independent experiments with essentially the same results. (b) Normal peripheral blood T cells were cultured in the presence of Txk antisense ODN for 12 h to reduce Txk expression. Thereafter, the T cells were stimulated with PHA. The result shown is representative of three independent experiments with essentially the same results. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted. (c) PPD- and Crj-1-specific T cell clones pretreated with the ODNs (5 μM, which inhibits Txk expression of the clones) were stimulated with the relevant Ag plus irradiated autologous PBMCs. The similar results were reproduced in the experiments using cells from different donors and different Ag-specific T cell clones. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted.

Mentions: To confirm the involvement of Txk in IFN-γ production by human T cells, we tested inhibition of IFN-γ production by Txk antisense ODN. Peripheral blood T lymphocytes were cultured in the presence of sense or antisense ODN corresponding to the original translation start site of Txk 16 for several hours and were then stimulated with PHA. Antisense ODN, but not sense ODN, specifically inhibited cytoplasmic expression of Txk (Fig. 4 a); IFN-γ production of T cells was specifically inhibited by the antisense ODN (Fig. 4 b). IL-2 and IL-4 production were not modulated by either the sense or antisense ODNs (Fig. 4 b). To further confirm that Txk antisense ODN inhibits IFN-γ production, Ag-specific T cell clones were cultured with ODNs and then stimulated with Ag plus irradiated autologous PBMCs. Again, IFN-γ production by the Ag-specific Th1 and Th0 clones (Fig. 4 c), but not IL-4 production by the Ag-specific Th0 (Fig. 4 c) and Th2 clones (data not shown) was inhibited by the Txk antisense ODN.


Txk, a nonreceptor tyrosine kinase of the Tec family, is expressed in T helper type 1 cells and regulates interferon gamma production in human T lymphocytes.

Kashiwakura J, Suzuki N, Nagafuchi H, Takeno M, Takeba Y, Shimoyama Y, Sakane T - J. Exp. Med. (1999)

Effects of Txk antisense ODN on cytokine production by human T cells. (a) Txk expression of normal peripheral blood T cells treated with Txk antisense ODN was analyzed. Txk expression was specifically reduced when treated with Txk antisense ODN, but not sense ODN (both 10 μM), for 12 h. The result shown (magnification 250) is representative of three independent experiments with essentially the same results. (b) Normal peripheral blood T cells were cultured in the presence of Txk antisense ODN for 12 h to reduce Txk expression. Thereafter, the T cells were stimulated with PHA. The result shown is representative of three independent experiments with essentially the same results. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted. (c) PPD- and Crj-1-specific T cell clones pretreated with the ODNs (5 μM, which inhibits Txk expression of the clones) were stimulated with the relevant Ag plus irradiated autologous PBMCs. The similar results were reproduced in the experiments using cells from different donors and different Ag-specific T cell clones. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195662&req=5

Figure 4: Effects of Txk antisense ODN on cytokine production by human T cells. (a) Txk expression of normal peripheral blood T cells treated with Txk antisense ODN was analyzed. Txk expression was specifically reduced when treated with Txk antisense ODN, but not sense ODN (both 10 μM), for 12 h. The result shown (magnification 250) is representative of three independent experiments with essentially the same results. (b) Normal peripheral blood T cells were cultured in the presence of Txk antisense ODN for 12 h to reduce Txk expression. Thereafter, the T cells were stimulated with PHA. The result shown is representative of three independent experiments with essentially the same results. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted. (c) PPD- and Crj-1-specific T cell clones pretreated with the ODNs (5 μM, which inhibits Txk expression of the clones) were stimulated with the relevant Ag plus irradiated autologous PBMCs. The similar results were reproduced in the experiments using cells from different donors and different Ag-specific T cell clones. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted.
Mentions: To confirm the involvement of Txk in IFN-γ production by human T cells, we tested inhibition of IFN-γ production by Txk antisense ODN. Peripheral blood T lymphocytes were cultured in the presence of sense or antisense ODN corresponding to the original translation start site of Txk 16 for several hours and were then stimulated with PHA. Antisense ODN, but not sense ODN, specifically inhibited cytoplasmic expression of Txk (Fig. 4 a); IFN-γ production of T cells was specifically inhibited by the antisense ODN (Fig. 4 b). IL-2 and IL-4 production were not modulated by either the sense or antisense ODNs (Fig. 4 b). To further confirm that Txk antisense ODN inhibits IFN-γ production, Ag-specific T cell clones were cultured with ODNs and then stimulated with Ag plus irradiated autologous PBMCs. Again, IFN-γ production by the Ag-specific Th1 and Th0 clones (Fig. 4 c), but not IL-4 production by the Ag-specific Th0 (Fig. 4 c) and Th2 clones (data not shown) was inhibited by the Txk antisense ODN.

Bottom Line: We found that Txk expression is restricted to Th1/Th0 cells with IFN-gamma producing potential.Txk transfection did not affect IL-2 and IL-4 promoter activities.These results indicate that Txk expression is intimately associated with development of Th1/Th0 cells and is significantly involved in the IFN-gamma production by the cells through Th1 cell-specific positive transcriptional regulation of the IFN-gamma gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and the Department of Medicine, St. Marianna University School of Medicine, Kawasaki, Kanagawa 216-8511, Japan.

ABSTRACT
Differentiation of human T cells into T helper (Th)1 and Th2 cells is vital for the development of cell-mediated and humoral immunity, respectively. However, the precise mechanism responsible for the Th1 cell differentiation is not fully clarified. We have studied the expression and function of Txk, a member of the Tec family of nonreceptor tyrosine kinases. We found that Txk expression is restricted to Th1/Th0 cells with IFN-gamma producing potential. Txk transfection of Jurkat T cells resulted in a several-fold increase of IFN-gamma mRNA expression and protein production; interleukin (IL)-2 and IL-4 production were unaffected. Antisense oligodeoxynucleotide of Txk specifically inhibited IFN-gamma production of normal peripheral blood lymphocytes, antigen-specific Th1 clones, and Th0 clones; IL-2 and IL-4 production by the T cells was unaffected. Txk cotransfection led to the enhanced luciferase activity of plasmid (p)IFN-gamma promoter/enhancer (pIFN-gamma[-538])-luciferase-transfected Jurkat cells upon mitogen activation. Txk transfection did not affect IL-2 and IL-4 promoter activities. Thus, Txk specifically upregulates IFN-gamma gene transcription. In fact, Txk translocated from cytoplasm into nuclei upon activation and transfection with a mutant Txk expression plasmid that lacked a nuclear localization signal sequence did not enhance IFN-gamma production by the cells, indicating that nuclear localization of Txk is obligatory for the enhanced IFN-gamma production. In addition, IL-12 treatment of peripheral blood CD4(+) T cells enhanced the Txk expression, whereas IL-4 treatment completely inhibited it. These results indicate that Txk expression is intimately associated with development of Th1/Th0 cells and is significantly involved in the IFN-gamma production by the cells through Th1 cell-specific positive transcriptional regulation of the IFN-gamma gene.

Show MeSH
Related in: MedlinePlus