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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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Modulation of T cell activation by ROS. (A) One representative flow cytometry analysis of CD69 expression is shown. PMA activated normal T cells (upper panels) or SSc T cells (lower panels) were treated with DPI (20 μM, 1 hour). Plots represent live cells gated for CD4+ cells. (B) Percentage of CD69+ cells in three healthy controls (upper panel) and three SSc patients (lower panel) are presented. Cells were treated as in A. Data are means ± standard deviation (SD). *P <0.05 compared to untreated T cells. (C) One representative flow cytometry analysis of CD69 expression is shown. Normal T cells (upper panels) or SSc T cells (lower panels) were activated with CD3/CD28 magnetic beads and treated with DPI (20 μM, 1 hour). Plots represent live cells gated for CD4+ cells. (D) Percentage of CD69+ cells in three healthy controls (upper panel) and three SSc patients (lower panel) are presented. Cells were treated as in C. Data are means ± SD. *P <0.05 compared to untreated T cells. DPI, diphenylene iodonium; PMA, phorbol myristate acetate; ROS, reactive oxygen species; SSc, systemic sclerosis.
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Fig6: Modulation of T cell activation by ROS. (A) One representative flow cytometry analysis of CD69 expression is shown. PMA activated normal T cells (upper panels) or SSc T cells (lower panels) were treated with DPI (20 μM, 1 hour). Plots represent live cells gated for CD4+ cells. (B) Percentage of CD69+ cells in three healthy controls (upper panel) and three SSc patients (lower panel) are presented. Cells were treated as in A. Data are means ± standard deviation (SD). *P <0.05 compared to untreated T cells. (C) One representative flow cytometry analysis of CD69 expression is shown. Normal T cells (upper panels) or SSc T cells (lower panels) were activated with CD3/CD28 magnetic beads and treated with DPI (20 μM, 1 hour). Plots represent live cells gated for CD4+ cells. (D) Percentage of CD69+ cells in three healthy controls (upper panel) and three SSc patients (lower panel) are presented. Cells were treated as in C. Data are means ± SD. *P <0.05 compared to untreated T cells. DPI, diphenylene iodonium; PMA, phorbol myristate acetate; ROS, reactive oxygen species; SSc, systemic sclerosis.

Mentions: PMA activation of CD4+ T cells was then assessed by monitoring the expression of CD69, an early activation marker on the surface of antigen-specific activated lymphocytes in vitro [32]. As shown in the upper panels of Figure 6A and B, incubation of normal T cells with PMA led to an increase of the activation rate on CD4+ cells (47% increase compared to basal, P <0.05), that was partially reverted by treatment with DPI (30% increase compared to basal). On the other hand, CD69 was upregulated in SSc T cells compared to normal controls even in basal conditions (15% increase compared to normal cells, Figure 6B), and NADPH oxidase inhibition reverted the activated state of SSc T lymphocytes (lower panels of Figure 6A and B).Figure 6


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)

Modulation of T cell activation by ROS. (A) One representative flow cytometry analysis of CD69 expression is shown. PMA activated normal T cells (upper panels) or SSc T cells (lower panels) were treated with DPI (20 μM, 1 hour). Plots represent live cells gated for CD4+ cells. (B) Percentage of CD69+ cells in three healthy controls (upper panel) and three SSc patients (lower panel) are presented. Cells were treated as in A. Data are means ± standard deviation (SD). *P <0.05 compared to untreated T cells. (C) One representative flow cytometry analysis of CD69 expression is shown. Normal T cells (upper panels) or SSc T cells (lower panels) were activated with CD3/CD28 magnetic beads and treated with DPI (20 μM, 1 hour). Plots represent live cells gated for CD4+ cells. (D) Percentage of CD69+ cells in three healthy controls (upper panel) and three SSc patients (lower panel) are presented. Cells were treated as in C. Data are means ± SD. *P <0.05 compared to untreated T cells. DPI, diphenylene iodonium; PMA, phorbol myristate acetate; ROS, reactive oxygen species; SSc, systemic sclerosis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384301&req=5

Fig6: Modulation of T cell activation by ROS. (A) One representative flow cytometry analysis of CD69 expression is shown. PMA activated normal T cells (upper panels) or SSc T cells (lower panels) were treated with DPI (20 μM, 1 hour). Plots represent live cells gated for CD4+ cells. (B) Percentage of CD69+ cells in three healthy controls (upper panel) and three SSc patients (lower panel) are presented. Cells were treated as in A. Data are means ± standard deviation (SD). *P <0.05 compared to untreated T cells. (C) One representative flow cytometry analysis of CD69 expression is shown. Normal T cells (upper panels) or SSc T cells (lower panels) were activated with CD3/CD28 magnetic beads and treated with DPI (20 μM, 1 hour). Plots represent live cells gated for CD4+ cells. (D) Percentage of CD69+ cells in three healthy controls (upper panel) and three SSc patients (lower panel) are presented. Cells were treated as in C. Data are means ± SD. *P <0.05 compared to untreated T cells. DPI, diphenylene iodonium; PMA, phorbol myristate acetate; ROS, reactive oxygen species; SSc, systemic sclerosis.
Mentions: PMA activation of CD4+ T cells was then assessed by monitoring the expression of CD69, an early activation marker on the surface of antigen-specific activated lymphocytes in vitro [32]. As shown in the upper panels of Figure 6A and B, incubation of normal T cells with PMA led to an increase of the activation rate on CD4+ cells (47% increase compared to basal, P <0.05), that was partially reverted by treatment with DPI (30% increase compared to basal). On the other hand, CD69 was upregulated in SSc T cells compared to normal controls even in basal conditions (15% increase compared to normal cells, Figure 6B), and NADPH oxidase inhibition reverted the activated state of SSc T lymphocytes (lower panels of Figure 6A and B).Figure 6

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