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Discrete generation of superoxide and hydrogen peroxide by T cell receptor stimulation: selective regulation of mitogen-activated protein kinase activation and fas ligand expression.

Devadas S, Zaritskaya L, Rhee SG, Oberley L, Williams MS - J. Exp. Med. (2002)

Bottom Line: In this study, we have shown for the first time that TCR cross-linking induced rapid (within 15 min) generation of both hydrogen peroxide and superoxide anion, as defined with oxidation-sensitive dyes, selective pharmacologic antioxidants, and overexpression of specific antioxidant enzymes.Anti-CD3 induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 required hydrogen peroxide generation but was unaffected by superoxide anion.Thus, antigen receptor signaling induces generation of discrete species of oxidants that selectively regulate two distinct redox sensitive pathways, a proapoptotic (FasL) and a proliferative pathway (ERK).

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Holland Laboratory, American Red Cross, Rockville, MD 20855, USA.

ABSTRACT
Receptor-stimulated generation of reactive oxygen species (ROS) has been shown to regulate signal transduction, and previous studies have suggested that T cell receptor (TCR) signals may involve or be sensitive to ROS. In this study, we have shown for the first time that TCR cross-linking induced rapid (within 15 min) generation of both hydrogen peroxide and superoxide anion, as defined with oxidation-sensitive dyes, selective pharmacologic antioxidants, and overexpression of specific antioxidant enzymes. Furthermore, the data suggest the novel observation that superoxide anion and hydrogen peroxide are produced separately by distinct TCR-stimulated pathways. Unexpectedly, TCR-stimulated activation of the Fas ligand (FasL) promoter and subsequent cell death was dependent upon superoxide anion, but independent of hydrogen peroxide, while nuclear factor of activated T cells (NFAT) activation or interleukin 2 transcription was independent of all ROS. Anti-CD3 induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 required hydrogen peroxide generation but was unaffected by superoxide anion. Thus, antigen receptor signaling induces generation of discrete species of oxidants that selectively regulate two distinct redox sensitive pathways, a proapoptotic (FasL) and a proliferative pathway (ERK).

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Anti-CD3–induced ROS generation in human T blasts. (A). Kinetics of anti-CD3–stimulated DCFDA/DHE oxidation human T blasts. Human T blasts were stimulated with anti-CD3 as described in Materials and Methods and the oxidation of DCFDA (○) and DHE (▪) was determined by FACS® analysis. The data are expressed as the percent stimulated increase in mean channel fluorescence of DCFDA/DHE over unstimulated controls and represent the average of at least five separate experiments (± SEM). (B) Effect of protein overexpression on DCFDA/DHE oxidation in human T blasts. Human T blasts were transfected with empty vector (Vector) or expression vectors encoding BCl-xL, catalase, CuZnSOD, MnSOD, or TPx at a 2:1 ratio with one encoding GFP or RFP as a transfection marker. After 16 h incubation, anti-CD3–induced DCFDA (hatched bars) or DHE (closed bars) oxidation was measured as described in Materials and Methods and the legend to Fig. 3. DHE oxidation was measured in GFP+ cells while DCFDA oxidation was measured in RFP-transfected cells, both after 15 min anti-CD3 stimulation and was normalized to Vector control. The data represent the average of at least four separate experiments (± SEM). *Significantly different from that in Vector transfected samples (P < 0.05). #Significantly different from that in Vector transfected samples (P < 0.05). (C) Western blot confirmation of protein overexpression. Human T blasts were transiently transfected with the indicated mammalian expression vectors in twofold excess over a marker plasmid expressing a chimeric CD8-Igγ2a protein. After 16 h incubation, viable CD8+ cells were purified by magnetic bead selection. Protein overexpression was determined in positively selected cells by Western blot as described in Materials and Methods.
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fig4: Anti-CD3–induced ROS generation in human T blasts. (A). Kinetics of anti-CD3–stimulated DCFDA/DHE oxidation human T blasts. Human T blasts were stimulated with anti-CD3 as described in Materials and Methods and the oxidation of DCFDA (○) and DHE (▪) was determined by FACS® analysis. The data are expressed as the percent stimulated increase in mean channel fluorescence of DCFDA/DHE over unstimulated controls and represent the average of at least five separate experiments (± SEM). (B) Effect of protein overexpression on DCFDA/DHE oxidation in human T blasts. Human T blasts were transfected with empty vector (Vector) or expression vectors encoding BCl-xL, catalase, CuZnSOD, MnSOD, or TPx at a 2:1 ratio with one encoding GFP or RFP as a transfection marker. After 16 h incubation, anti-CD3–induced DCFDA (hatched bars) or DHE (closed bars) oxidation was measured as described in Materials and Methods and the legend to Fig. 3. DHE oxidation was measured in GFP+ cells while DCFDA oxidation was measured in RFP-transfected cells, both after 15 min anti-CD3 stimulation and was normalized to Vector control. The data represent the average of at least four separate experiments (± SEM). *Significantly different from that in Vector transfected samples (P < 0.05). #Significantly different from that in Vector transfected samples (P < 0.05). (C) Western blot confirmation of protein overexpression. Human T blasts were transiently transfected with the indicated mammalian expression vectors in twofold excess over a marker plasmid expressing a chimeric CD8-Igγ2a protein. After 16 h incubation, viable CD8+ cells were purified by magnetic bead selection. Protein overexpression was determined in positively selected cells by Western blot as described in Materials and Methods.

Mentions: To extend these observations to primary T cells, anti-CD3–induced ROS generation was also measured in activated peripheral human T blasts. Kinetics of anti-CD3 stimulated oxidant production demonstrated DCFDA oxidation as early as 15 min and, as in 9C127 cells, was sustained for all time points assayed (Fig. 4 A). In contrast to the results using the T cell hybridoma, anti-CD3–induced DHE oxidation was also observed after 15 min stimulation and this was maintained throughout the assay. Thus, anti-CD3 stimulation of primary activated human T cell blasts also resulted in rapid oxidation of both DHE and DCFDA, albeit at slightly different kinetics than in the murine hybridoma.


Discrete generation of superoxide and hydrogen peroxide by T cell receptor stimulation: selective regulation of mitogen-activated protein kinase activation and fas ligand expression.

Devadas S, Zaritskaya L, Rhee SG, Oberley L, Williams MS - J. Exp. Med. (2002)

Anti-CD3–induced ROS generation in human T blasts. (A). Kinetics of anti-CD3–stimulated DCFDA/DHE oxidation human T blasts. Human T blasts were stimulated with anti-CD3 as described in Materials and Methods and the oxidation of DCFDA (○) and DHE (▪) was determined by FACS® analysis. The data are expressed as the percent stimulated increase in mean channel fluorescence of DCFDA/DHE over unstimulated controls and represent the average of at least five separate experiments (± SEM). (B) Effect of protein overexpression on DCFDA/DHE oxidation in human T blasts. Human T blasts were transfected with empty vector (Vector) or expression vectors encoding BCl-xL, catalase, CuZnSOD, MnSOD, or TPx at a 2:1 ratio with one encoding GFP or RFP as a transfection marker. After 16 h incubation, anti-CD3–induced DCFDA (hatched bars) or DHE (closed bars) oxidation was measured as described in Materials and Methods and the legend to Fig. 3. DHE oxidation was measured in GFP+ cells while DCFDA oxidation was measured in RFP-transfected cells, both after 15 min anti-CD3 stimulation and was normalized to Vector control. The data represent the average of at least four separate experiments (± SEM). *Significantly different from that in Vector transfected samples (P < 0.05). #Significantly different from that in Vector transfected samples (P < 0.05). (C) Western blot confirmation of protein overexpression. Human T blasts were transiently transfected with the indicated mammalian expression vectors in twofold excess over a marker plasmid expressing a chimeric CD8-Igγ2a protein. After 16 h incubation, viable CD8+ cells were purified by magnetic bead selection. Protein overexpression was determined in positively selected cells by Western blot as described in Materials and Methods.
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fig4: Anti-CD3–induced ROS generation in human T blasts. (A). Kinetics of anti-CD3–stimulated DCFDA/DHE oxidation human T blasts. Human T blasts were stimulated with anti-CD3 as described in Materials and Methods and the oxidation of DCFDA (○) and DHE (▪) was determined by FACS® analysis. The data are expressed as the percent stimulated increase in mean channel fluorescence of DCFDA/DHE over unstimulated controls and represent the average of at least five separate experiments (± SEM). (B) Effect of protein overexpression on DCFDA/DHE oxidation in human T blasts. Human T blasts were transfected with empty vector (Vector) or expression vectors encoding BCl-xL, catalase, CuZnSOD, MnSOD, or TPx at a 2:1 ratio with one encoding GFP or RFP as a transfection marker. After 16 h incubation, anti-CD3–induced DCFDA (hatched bars) or DHE (closed bars) oxidation was measured as described in Materials and Methods and the legend to Fig. 3. DHE oxidation was measured in GFP+ cells while DCFDA oxidation was measured in RFP-transfected cells, both after 15 min anti-CD3 stimulation and was normalized to Vector control. The data represent the average of at least four separate experiments (± SEM). *Significantly different from that in Vector transfected samples (P < 0.05). #Significantly different from that in Vector transfected samples (P < 0.05). (C) Western blot confirmation of protein overexpression. Human T blasts were transiently transfected with the indicated mammalian expression vectors in twofold excess over a marker plasmid expressing a chimeric CD8-Igγ2a protein. After 16 h incubation, viable CD8+ cells were purified by magnetic bead selection. Protein overexpression was determined in positively selected cells by Western blot as described in Materials and Methods.
Mentions: To extend these observations to primary T cells, anti-CD3–induced ROS generation was also measured in activated peripheral human T blasts. Kinetics of anti-CD3 stimulated oxidant production demonstrated DCFDA oxidation as early as 15 min and, as in 9C127 cells, was sustained for all time points assayed (Fig. 4 A). In contrast to the results using the T cell hybridoma, anti-CD3–induced DHE oxidation was also observed after 15 min stimulation and this was maintained throughout the assay. Thus, anti-CD3 stimulation of primary activated human T cell blasts also resulted in rapid oxidation of both DHE and DCFDA, albeit at slightly different kinetics than in the murine hybridoma.

Bottom Line: In this study, we have shown for the first time that TCR cross-linking induced rapid (within 15 min) generation of both hydrogen peroxide and superoxide anion, as defined with oxidation-sensitive dyes, selective pharmacologic antioxidants, and overexpression of specific antioxidant enzymes.Anti-CD3 induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 required hydrogen peroxide generation but was unaffected by superoxide anion.Thus, antigen receptor signaling induces generation of discrete species of oxidants that selectively regulate two distinct redox sensitive pathways, a proapoptotic (FasL) and a proliferative pathway (ERK).

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Holland Laboratory, American Red Cross, Rockville, MD 20855, USA.

ABSTRACT
Receptor-stimulated generation of reactive oxygen species (ROS) has been shown to regulate signal transduction, and previous studies have suggested that T cell receptor (TCR) signals may involve or be sensitive to ROS. In this study, we have shown for the first time that TCR cross-linking induced rapid (within 15 min) generation of both hydrogen peroxide and superoxide anion, as defined with oxidation-sensitive dyes, selective pharmacologic antioxidants, and overexpression of specific antioxidant enzymes. Furthermore, the data suggest the novel observation that superoxide anion and hydrogen peroxide are produced separately by distinct TCR-stimulated pathways. Unexpectedly, TCR-stimulated activation of the Fas ligand (FasL) promoter and subsequent cell death was dependent upon superoxide anion, but independent of hydrogen peroxide, while nuclear factor of activated T cells (NFAT) activation or interleukin 2 transcription was independent of all ROS. Anti-CD3 induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 required hydrogen peroxide generation but was unaffected by superoxide anion. Thus, antigen receptor signaling induces generation of discrete species of oxidants that selectively regulate two distinct redox sensitive pathways, a proapoptotic (FasL) and a proliferative pathway (ERK).

Show MeSH
Related in: MedlinePlus