title: Intracellular free radical production by peripheral blood T lymphocytes from patients with systemic sclerosis: role of NADPH oxidase and ERK1/2.
abstract: BACKGROUND
  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.
authors.affiliation: Dipartimento Scienze Cliniche e Molecolari, Università Politecnica delle Marche, Via Tronto 10, 60020, Ancona, Italy. donatellaamico@libero.it.
authors.forename: Donatella
authors.initials: D
authors.lastname: Amico
authors.affiliation: Dipartimento Scienze Cliniche e Molecolari, Università Politecnica delle Marche, Via Tronto 10, 60020, Ancona, Italy. t.spadoni@univpm.it.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Spadoni
authors.affiliation: Dipartimento Scienze Cliniche e Molecolari, Università Politecnica delle Marche, Via Tronto 10, 60020, Ancona, Italy. marina.rovinelli@libero.it.
authors.forename: Marina
authors.initials: M
authors.lastname: Rovinelli
authors.affiliation: Centro Ricerca M. Tettamanti, Clinica Pediatrica Università Milano-Bicocca, Ospedale San Gerardo, Via Donizetti 106, 20900, Monza, Italy. m.serafini@hsgerardo.org.
authors.forename: Marta
authors.initials: M
authors.lastname: Serafini
authors.affiliation: Centro Ricerca M. Tettamanti, Clinica Pediatrica Università Milano-Bicocca, Ospedale San Gerardo, Via Donizetti 106, 20900, Monza, Italy. giovanna.damico@hsgerardo.org.
authors.forename: Giovanna
authors.initials: G
authors.lastname: D'Amico
authors.affiliation: Dipartimento Scienze Cliniche e Molecolari, Università Politecnica delle Marche, Via Tronto 10, 60020, Ancona, Italy. nadia.c@hotmail.it.
authors.forename: Nadia
authors.initials: N
authors.lastname: Campelli
authors.affiliation: Dipartimento Scienze Cliniche e Molecolari, Università Politecnica delle Marche, Via Tronto 10, 60020, Ancona, Italy. s.svegliati@univpm.it.
authors.forename: Silvia
authors.initials: S
authors.lastname: Svegliati Baroni
authors.affiliation: Dipartimento Scienze Cliniche e Molecolari, Università Politecnica delle Marche, Via Tronto 10, 60020, Ancona, Italy. a.gabrielli@univpm.it.
authors.forename: Armando
authors.initials: A
authors.lastname: Gabrielli
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D008875:Middle Aged; D019950:Mitogen-Activated Protein Kinase 1 / Q000096:biosynthesis / Q000235:genetics*; D019255:NADPH Oxidases / Q000096:biosynthesis / Q000235:genetics*; D018384:Oxidative Stress; D012313:RNA / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D012595:Scleroderma, Systemic / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000473:pathology
chemical_list: D017382:Reactive Oxygen Species; D012313:RNA; D019255:NADPH Oxidases; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1