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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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ROS production by PBL from SSc patients and healthy controls. (A) PBL from 17 healthy controls (white bar) and 34 SSc patients (black bar), isolated from peripheral blood were stained with 20 μM DCFH-DA for 20 minutes, and fluorescence was measured in a plate reader fluorimeter. Each patient and control 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 normal PBL. (B) ROS production by PBL from one healthy control (black line) and one SSc patient (grey line) was analyzed by FACS analysis. A representative histogram of three independent experiments is shown. (C and D) SSc CD14- (C) and SSc CD19- cells (D) were purified from PBL using CD14 and CD19 microbeads. The total fractions of cells (PBL, white bars) and the collected depleted fractions (black bars) were stained with 20 μM DCFH-DA for 20 minutes, and fluorescence was measured on a plate reader fluorimeter. Data are means ± SD of three independent experiments with cells from three distinct subjects. DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; PBL, peripheral blood lymphocytes; ROS, reactive oxygen species; SSc, systemic sclerosis.
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Fig1: ROS production by PBL from SSc patients and healthy controls. (A) PBL from 17 healthy controls (white bar) and 34 SSc patients (black bar), isolated from peripheral blood were stained with 20 μM DCFH-DA for 20 minutes, and fluorescence was measured in a plate reader fluorimeter. Each patient and control 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 normal PBL. (B) ROS production by PBL from one healthy control (black line) and one SSc patient (grey line) was analyzed by FACS analysis. A representative histogram of three independent experiments is shown. (C and D) SSc CD14- (C) and SSc CD19- cells (D) were purified from PBL using CD14 and CD19 microbeads. The total fractions of cells (PBL, white bars) and the collected depleted fractions (black bars) were stained with 20 μM DCFH-DA for 20 minutes, and fluorescence was measured on a plate reader fluorimeter. Data are means ± SD of three independent experiments with cells from three distinct subjects. DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; PBL, peripheral blood lymphocytes; ROS, reactive oxygen species; SSc, systemic sclerosis.

Mentions: We previously demonstrated that unstimulated monocytes isolated from SSc patients released large amount of reactive oxygen species (ROS) [25]. These data led us to investigate whether other blood cell types were involved in the oxidative burst that characterizes SSc. Peripheral blood lymphocytes (PBL) were obtained from 17 healthy individuals and 34 SSc patients (Table S1 in Additional file 1) and analyzed for ROS production. PBL from SSc patients produced a significantly larger amount of ROS compared to healthy controls as measured in a plate reader fluorimeter (150 ± 45 and 100 ± 20 respectively, Figure 1A) and by FACS analysis (100 and 46.2 ± 8 respectively, Figure 1B) (P <0.05). We then analyzed the relationship between the amount of ROS generated by PBL and the clinical features of SSc patients. No difference was detected when all patients were divided into the limited or the diffuse subset (17 limited SSc patients 132.8 ± 56 vs. 17 diffuse SSc patients 163.3 ± 48, P = 0.066), or into early (less than 5 years) or late (6 years or more) disease (17 early SSc patients 157.37 vs. 17 late SSc patients 161.25, P = 0.45). In order to identify the PB cell subpopulation responsible for the increased ROS production in SSc samples, we purified cell populations depleted of CD14 (CD14-) and CD19 (CD19-) fractions by magnetic beads. Since no difference in ROS generation between PBL and the purified cell populations was observed, we could assume that neither CD14+ nor CD19+ cells were responsible for the oxidative burst in SSc samples (Figure 1C and D respectively). To test whether ROS generation by PBL was due to T cells, we performed a negative selection procedure specific for T cell isolation in 12 controls and in 23 SSc samples and ROS production was analyzed. Figure 2A shows that T cells from SSc patients, even in absence of deliberate stimulation, produced significantly larger amount of ROS compared to T cells from healthy controls (186 ± 17 and 100 ± 15 respectively, P <0.05). Data were confirmed by FACS analysis (Figure 2A, lower panel). No difference was detected when all patients were divided into the limited or the diffuse subset (P = 0.13). The simultaneous staining of SSc PBL with DCFH-DA and anti-CD3 PerCP-conjugated antibody confirmed the implication of T cell population in ROS production. To understand which subpopulation of T cells was implicated in ROS production, we stained SSc PBL with DCFH-DA and PE-conjugated anti-CD4 (Figure S1A in Additional file 2) or anti-CD8 antibody (Figure S1B in Additional file 2). We observed that both CD4+ and CD8+ lymphocytes were involved in ROS generation. To further validate the involvement of CD3+ T cells in ROS generation, CD3+ T cells from SSc patients were treated with 10 mM N-acetylcysteine (NAC), a ROS scavenging agent, and a significant reduction in free radical production was observed (100 ± 10 and 60 ± 5 respectively, P <0.05) (Figure 2C). These results were confirmed by FACS analysis (Figure 2D).Figure 1


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)

ROS production by PBL from SSc patients and healthy controls. (A) PBL from 17 healthy controls (white bar) and 34 SSc patients (black bar), isolated from peripheral blood were stained with 20 μM DCFH-DA for 20 minutes, and fluorescence was measured in a plate reader fluorimeter. Each patient and control 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 normal PBL. (B) ROS production by PBL from one healthy control (black line) and one SSc patient (grey line) was analyzed by FACS analysis. A representative histogram of three independent experiments is shown. (C and D) SSc CD14- (C) and SSc CD19- cells (D) were purified from PBL using CD14 and CD19 microbeads. The total fractions of cells (PBL, white bars) and the collected depleted fractions (black bars) were stained with 20 μM DCFH-DA for 20 minutes, and fluorescence was measured on a plate reader fluorimeter. Data are means ± SD of three independent experiments with cells from three distinct subjects. DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; PBL, peripheral blood lymphocytes; ROS, reactive oxygen species; SSc, systemic sclerosis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: ROS production by PBL from SSc patients and healthy controls. (A) PBL from 17 healthy controls (white bar) and 34 SSc patients (black bar), isolated from peripheral blood were stained with 20 μM DCFH-DA for 20 minutes, and fluorescence was measured in a plate reader fluorimeter. Each patient and control 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 normal PBL. (B) ROS production by PBL from one healthy control (black line) and one SSc patient (grey line) was analyzed by FACS analysis. A representative histogram of three independent experiments is shown. (C and D) SSc CD14- (C) and SSc CD19- cells (D) were purified from PBL using CD14 and CD19 microbeads. The total fractions of cells (PBL, white bars) and the collected depleted fractions (black bars) were stained with 20 μM DCFH-DA for 20 minutes, and fluorescence was measured on a plate reader fluorimeter. Data are means ± SD of three independent experiments with cells from three distinct subjects. DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; PBL, peripheral blood lymphocytes; ROS, reactive oxygen species; SSc, systemic sclerosis.
Mentions: We previously demonstrated that unstimulated monocytes isolated from SSc patients released large amount of reactive oxygen species (ROS) [25]. These data led us to investigate whether other blood cell types were involved in the oxidative burst that characterizes SSc. Peripheral blood lymphocytes (PBL) were obtained from 17 healthy individuals and 34 SSc patients (Table S1 in Additional file 1) and analyzed for ROS production. PBL from SSc patients produced a significantly larger amount of ROS compared to healthy controls as measured in a plate reader fluorimeter (150 ± 45 and 100 ± 20 respectively, Figure 1A) and by FACS analysis (100 and 46.2 ± 8 respectively, Figure 1B) (P <0.05). We then analyzed the relationship between the amount of ROS generated by PBL and the clinical features of SSc patients. No difference was detected when all patients were divided into the limited or the diffuse subset (17 limited SSc patients 132.8 ± 56 vs. 17 diffuse SSc patients 163.3 ± 48, P = 0.066), or into early (less than 5 years) or late (6 years or more) disease (17 early SSc patients 157.37 vs. 17 late SSc patients 161.25, P = 0.45). In order to identify the PB cell subpopulation responsible for the increased ROS production in SSc samples, we purified cell populations depleted of CD14 (CD14-) and CD19 (CD19-) fractions by magnetic beads. Since no difference in ROS generation between PBL and the purified cell populations was observed, we could assume that neither CD14+ nor CD19+ cells were responsible for the oxidative burst in SSc samples (Figure 1C and D respectively). To test whether ROS generation by PBL was due to T cells, we performed a negative selection procedure specific for T cell isolation in 12 controls and in 23 SSc samples and ROS production was analyzed. Figure 2A shows that T cells from SSc patients, even in absence of deliberate stimulation, produced significantly larger amount of ROS compared to T cells from healthy controls (186 ± 17 and 100 ± 15 respectively, P <0.05). Data were confirmed by FACS analysis (Figure 2A, lower panel). No difference was detected when all patients were divided into the limited or the diffuse subset (P = 0.13). The simultaneous staining of SSc PBL with DCFH-DA and anti-CD3 PerCP-conjugated antibody confirmed the implication of T cell population in ROS production. To understand which subpopulation of T cells was implicated in ROS production, we stained SSc PBL with DCFH-DA and PE-conjugated anti-CD4 (Figure S1A in Additional file 2) or anti-CD8 antibody (Figure S1B in Additional file 2). We observed that both CD4+ and CD8+ lymphocytes were involved in ROS generation. To further validate the involvement of CD3+ T cells in ROS generation, CD3+ T cells from SSc patients were treated with 10 mM N-acetylcysteine (NAC), a ROS scavenging agent, and a significant reduction in free radical production was observed (100 ± 10 and 60 ± 5 respectively, P <0.05) (Figure 2C). These results were confirmed by FACS analysis (Figure 2D).Figure 1

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