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Intracellular free radical production by peripheral blood T lymphocytes from patients with systemic sclerosis: role of NADPH oxidase and ERK1/2.

Amico D, Spadoni T, Rovinelli M, Serafini M, D'Amico G, Campelli N, Svegliati Baroni S, Gabrielli A - Arthritis Res. Ther. (2015)

Bottom Line: Since NADPH oxidase complex is involved in oxidative stress in SSc and we found high levels of gp91phox in SSc T cells, SSc T cells were incubated with chemical inhibititors or specific siRNAs against gp91phox.Inhibition of NADPH oxidase partially reverted CD69 activation and proliferation rate increase, and significantly influenced cytokine production and ERK1/2 activation.SSc T lymphocityes are characterized by high levels of ROS, generated by NADPH oxidase via ERK1/2 phosphorylation, that are essential for cell activation, proliferation, and cytokine production.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento Scienze Cliniche e Molecolari, Università Politecnica delle Marche, Via Tronto 10, 60020, Ancona, Italy. donatellaamico@libero.it.

ABSTRACT

Introduction: Abnormal oxidative stress has been described in systemic sclerosis (SSc) and previous works from our laboratory demonstrated an increased generation of reactive oxygen species (ROS) by SSc fibroblasts and monocytes. This study investigated the ability of SSc T lymphocytes to produce ROS, the molecular pathway involved, and the biological effects of ROS on SSc phenotype.

Methods: Peripheral blood T lymphocytes were isolated from serum of healthy controls or SSc patients by negative selection with magnetic beads and activated either with PMA or with magnetic beads coated with anti-CD3 and anti-CD28 antibodies. Intracellular ROS generation was measured using a DCFH-DA assay in a plate reader fluorimeter or by FACS analysis. CD69 expression and cytokine production were analyzed by FACS analysis. Protein expression was studied using immunoblotting techniques and mRNA levels were quantified by real-time PCR. Cell proliferation was carried out using a BrdU incorporation assay.

Results: Peripheral blood T lymphocytes from SSc patients showed an increased ROS production compared to T cells from healthy subjects. Since NADPH oxidase complex is involved in oxidative stress in SSc and we found high levels of gp91phox in SSc T cells, SSc T cells were incubated with chemical inhibititors or specific siRNAs against gp91phox. Inhibition of NADPH oxidase partially reverted CD69 activation and proliferation rate increase, and significantly influenced cytokine production and ERK1/2 activation.

Conclusions: SSc T lymphocityes are characterized by high levels of ROS, generated by NADPH oxidase via ERK1/2 phosphorylation, that are essential for cell activation, proliferation, and cytokine production. These data confirm lymphocytes as key cellular players in the pathogenesis of systemic sclerosis and suggest a crucial link between ROS and T cell activation.

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pERK1/2 implication in ROS production. (A) T lymphocytes from five SSc patients were incubated with PD98059 (50 μM, 2 hours) or left untreated. After staining with 20 μM DCFH-DA, fluorescence was analyzed in a plate reader fluorimeter (left panel). Each treatment was tested three times and the mean value used to calculate the mean of each group. Data are means ± standard deviation (SD). *P <0.05 compared to untreated T cells (control). Representative histogram of ROS production by SSc T cells is shown in the right panel. Untreated (control, grey line) and PD98059 treated cells (black line) were stained with 2 μM DCFH-DA and analyzed by FACS analysis. (B) Representative pERK1/2 immunoblot of three independent experiments is shown (upper panel). T lymphocytes were isolated from five healthy controls and five SSc patients, treated with NAC (10 mM, 1 hour) or DPI (20 μM, 1 hour), and then analyzed by immunoblotting with specific antibodies. The lower panel shows the densitometric analysis from distinct experiments with five healthy controls and five SSc patients. Data are means ± SD. *P <0.05 compared to untreated normal T cells. **P < 0.05 compared to untreated SSc T cells. (C) Representative Ha-Ras immunoblot of three independent experiments is shown (upper panel). T lymphocytes were isolated from five healthy controls and five SSc patients, treated with NAC (10 mM, 1 hour) or DPI (20 μM, 1 hour), and then analyzed by immunoblotting with specific antibodies. The lower panel shows the densitometric analysis from distinct experiments with five healthy controls and five SSc patients. Data are means ± SD. *P <0.05 compared to untreated normal T cells. **P <0.05 compared to untreated SSc T cells. DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; DPI, diphenylene iodonium; NAC, N-acetylcysteine; ROS, reactive oxygen species; SSc, systemic sclerosis.
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Fig4: pERK1/2 implication in ROS production. (A) T lymphocytes from five SSc patients were incubated with PD98059 (50 μM, 2 hours) or left untreated. After staining with 20 μM DCFH-DA, fluorescence was analyzed in a plate reader fluorimeter (left panel). Each treatment was tested three times and the mean value used to calculate the mean of each group. Data are means ± standard deviation (SD). *P <0.05 compared to untreated T cells (control). Representative histogram of ROS production by SSc T cells is shown in the right panel. Untreated (control, grey line) and PD98059 treated cells (black line) were stained with 2 μM DCFH-DA and analyzed by FACS analysis. (B) Representative pERK1/2 immunoblot of three independent experiments is shown (upper panel). T lymphocytes were isolated from five healthy controls and five SSc patients, treated with NAC (10 mM, 1 hour) or DPI (20 μM, 1 hour), and then analyzed by immunoblotting with specific antibodies. The lower panel shows the densitometric analysis from distinct experiments with five healthy controls and five SSc patients. Data are means ± SD. *P <0.05 compared to untreated normal T cells. **P < 0.05 compared to untreated SSc T cells. (C) Representative Ha-Ras immunoblot of three independent experiments is shown (upper panel). T lymphocytes were isolated from five healthy controls and five SSc patients, treated with NAC (10 mM, 1 hour) or DPI (20 μM, 1 hour), and then analyzed by immunoblotting with specific antibodies. The lower panel shows the densitometric analysis from distinct experiments with five healthy controls and five SSc patients. Data are means ± SD. *P <0.05 compared to untreated normal T cells. **P <0.05 compared to untreated SSc T cells. DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; DPI, diphenylene iodonium; NAC, N-acetylcysteine; ROS, reactive oxygen species; SSc, systemic sclerosis.

Mentions: The implication of extracellular signal-regulated kinase (ERK)1/2 signaling in ROS production has been assessed in different cell types, including T lymphocytes [18], and in a previous work, we described an intracellular loop that involves Ha-Ras, ERK1/2, and ROS, leading to increased collagen gene expression in SSc fibroblasts [30]. To evaluate the involvement of ERK1/2 signaling in ROS production in SSc T cells, we treated T lymphocytes from SSc patients with PD98059, an ERK1/2 inhibitor. As shown in Figure 4A, PD98059 significantly reduced DCFH-DA fluorescence intensity compared to untreated cells (100 ± 15 and 75 ± 8, P <0.05), confirming ERK1/2 implication in ROS production in SSc T cells.Figure 4


Intracellular free radical production by peripheral blood T lymphocytes from patients with systemic sclerosis: role of NADPH oxidase and ERK1/2.

Amico D, Spadoni T, Rovinelli M, Serafini M, D'Amico G, Campelli N, Svegliati Baroni S, Gabrielli A - Arthritis Res. Ther. (2015)

pERK1/2 implication in ROS production. (A) T lymphocytes from five SSc patients were incubated with PD98059 (50 μM, 2 hours) or left untreated. After staining with 20 μM DCFH-DA, fluorescence was analyzed in a plate reader fluorimeter (left panel). Each treatment was tested three times and the mean value used to calculate the mean of each group. Data are means ± standard deviation (SD). *P <0.05 compared to untreated T cells (control). Representative histogram of ROS production by SSc T cells is shown in the right panel. Untreated (control, grey line) and PD98059 treated cells (black line) were stained with 2 μM DCFH-DA and analyzed by FACS analysis. (B) Representative pERK1/2 immunoblot of three independent experiments is shown (upper panel). T lymphocytes were isolated from five healthy controls and five SSc patients, treated with NAC (10 mM, 1 hour) or DPI (20 μM, 1 hour), and then analyzed by immunoblotting with specific antibodies. The lower panel shows the densitometric analysis from distinct experiments with five healthy controls and five SSc patients. Data are means ± SD. *P <0.05 compared to untreated normal T cells. **P < 0.05 compared to untreated SSc T cells. (C) Representative Ha-Ras immunoblot of three independent experiments is shown (upper panel). T lymphocytes were isolated from five healthy controls and five SSc patients, treated with NAC (10 mM, 1 hour) or DPI (20 μM, 1 hour), and then analyzed by immunoblotting with specific antibodies. The lower panel shows the densitometric analysis from distinct experiments with five healthy controls and five SSc patients. Data are means ± SD. *P <0.05 compared to untreated normal T cells. **P <0.05 compared to untreated SSc T cells. DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; DPI, diphenylene iodonium; NAC, N-acetylcysteine; ROS, reactive oxygen species; SSc, systemic sclerosis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: pERK1/2 implication in ROS production. (A) T lymphocytes from five SSc patients were incubated with PD98059 (50 μM, 2 hours) or left untreated. After staining with 20 μM DCFH-DA, fluorescence was analyzed in a plate reader fluorimeter (left panel). Each treatment was tested three times and the mean value used to calculate the mean of each group. Data are means ± standard deviation (SD). *P <0.05 compared to untreated T cells (control). Representative histogram of ROS production by SSc T cells is shown in the right panel. Untreated (control, grey line) and PD98059 treated cells (black line) were stained with 2 μM DCFH-DA and analyzed by FACS analysis. (B) Representative pERK1/2 immunoblot of three independent experiments is shown (upper panel). T lymphocytes were isolated from five healthy controls and five SSc patients, treated with NAC (10 mM, 1 hour) or DPI (20 μM, 1 hour), and then analyzed by immunoblotting with specific antibodies. The lower panel shows the densitometric analysis from distinct experiments with five healthy controls and five SSc patients. Data are means ± SD. *P <0.05 compared to untreated normal T cells. **P < 0.05 compared to untreated SSc T cells. (C) Representative Ha-Ras immunoblot of three independent experiments is shown (upper panel). T lymphocytes were isolated from five healthy controls and five SSc patients, treated with NAC (10 mM, 1 hour) or DPI (20 μM, 1 hour), and then analyzed by immunoblotting with specific antibodies. The lower panel shows the densitometric analysis from distinct experiments with five healthy controls and five SSc patients. Data are means ± SD. *P <0.05 compared to untreated normal T cells. **P <0.05 compared to untreated SSc T cells. DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; DPI, diphenylene iodonium; NAC, N-acetylcysteine; ROS, reactive oxygen species; SSc, systemic sclerosis.
Mentions: The implication of extracellular signal-regulated kinase (ERK)1/2 signaling in ROS production has been assessed in different cell types, including T lymphocytes [18], and in a previous work, we described an intracellular loop that involves Ha-Ras, ERK1/2, and ROS, leading to increased collagen gene expression in SSc fibroblasts [30]. To evaluate the involvement of ERK1/2 signaling in ROS production in SSc T cells, we treated T lymphocytes from SSc patients with PD98059, an ERK1/2 inhibitor. As shown in Figure 4A, PD98059 significantly reduced DCFH-DA fluorescence intensity compared to untreated cells (100 ± 15 and 75 ± 8, P <0.05), confirming ERK1/2 implication in ROS production in SSc T cells.Figure 4

Bottom Line: Since NADPH oxidase complex is involved in oxidative stress in SSc and we found high levels of gp91phox in SSc T cells, SSc T cells were incubated with chemical inhibititors or specific siRNAs against gp91phox.Inhibition of NADPH oxidase partially reverted CD69 activation and proliferation rate increase, and significantly influenced cytokine production and ERK1/2 activation.SSc T lymphocityes are characterized by high levels of ROS, generated by NADPH oxidase via ERK1/2 phosphorylation, that are essential for cell activation, proliferation, and cytokine production.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento Scienze Cliniche e Molecolari, Università Politecnica delle Marche, Via Tronto 10, 60020, Ancona, Italy. donatellaamico@libero.it.

ABSTRACT

Introduction: Abnormal oxidative stress has been described in systemic sclerosis (SSc) and previous works from our laboratory demonstrated an increased generation of reactive oxygen species (ROS) by SSc fibroblasts and monocytes. This study investigated the ability of SSc T lymphocytes to produce ROS, the molecular pathway involved, and the biological effects of ROS on SSc phenotype.

Methods: Peripheral blood T lymphocytes were isolated from serum of healthy controls or SSc patients by negative selection with magnetic beads and activated either with PMA or with magnetic beads coated with anti-CD3 and anti-CD28 antibodies. Intracellular ROS generation was measured using a DCFH-DA assay in a plate reader fluorimeter or by FACS analysis. CD69 expression and cytokine production were analyzed by FACS analysis. Protein expression was studied using immunoblotting techniques and mRNA levels were quantified by real-time PCR. Cell proliferation was carried out using a BrdU incorporation assay.

Results: Peripheral blood T lymphocytes from SSc patients showed an increased ROS production compared to T cells from healthy subjects. Since NADPH oxidase complex is involved in oxidative stress in SSc and we found high levels of gp91phox in SSc T cells, SSc T cells were incubated with chemical inhibititors or specific siRNAs against gp91phox. Inhibition of NADPH oxidase partially reverted CD69 activation and proliferation rate increase, and significantly influenced cytokine production and ERK1/2 activation.

Conclusions: SSc T lymphocityes are characterized by high levels of ROS, generated by NADPH oxidase via ERK1/2 phosphorylation, that are essential for cell activation, proliferation, and cytokine production. These data confirm lymphocytes as key cellular players in the pathogenesis of systemic sclerosis and suggest a crucial link between ROS and T cell activation.

Show MeSH
Related in: MedlinePlus