Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis

Abstract

Objective. To investigate the cellular and molecular sources of oxidative stress in patients with rheumatoid arthritis (RA) through analysis of the production of reactive oxygen species (ROS) in synovium.Methods. Cytochemical procedures based on the 3,3-diaminobenzidine (DAB)-Mn 2؉ deposition technique were used on unfixed cryostat sections of synovium from RA patients and rheumatic disease controls. For immunophenotyping, sections were incubated, fixed, and stained with fluorescein isothiocyanatelabeled antibodies. Fluorescence-activated cell sorter analysis of the ROS-reactive dye 6-carboxy-2,7-dichlorodihydrofluorescein diacetate-di(acetoxymethyl ester) was used to measure intracellular ROS in T lymphocytes from peripheral blood and synovial fluid. To determine which enzymes produced ROS, different inhibitors were tested.Results. Large quantities of DAB precipitated in the majority of RA synovial T lymphocytes, indicative of intracellular ROS production. These ROS-producing T lymphocytes were observed throughout the synovium. Polymerization of DAB was observed to a lesser extent in other forms of chronic arthritis, but was absent in osteoarthritis. DAB staining of cytospin preparations of purified RA synovial fluid T cells confirmed the presence of ROS-producing cells. One of the ROS involved appeared to be H 2 O 2 , since catalase suppressed intracellular ROS production. Superoxide dismutase, which uses superoxide as a substrate to form H 2 O 2 , diphenyleneiodonium (an inhibitor of NADPH oxidase), N G -monomethyl-L-arginine (an inhibitor of nitric oxide synthesis), nordihydroguaiaretic acid (an inhibitor of lipoxygenase), and rotenone (an inhibitor of mitochondrial ROS production) failed to suppress ROS production.Conclusion. Our findings show that chronic oxidative stress observed in synovial T lymphocytes is not secondary to exposure to environmental free radicals, but originates from intracellularly produced ROS. Additionally, our data suggest that one of the intracellularly generated ROS is H 2 O 2 , although the oxidase(s) involved in its generation remains to be determined.

T cell surface redox levels determine T cell reactivity and arthritis susceptibility

“…Although others reported expression of a functional NADPH oxidase complex in T cells (21,22), we could detect neither Ncf1 expression nor oxidative burst in T cells from our rats or mice. We do not exclude the occurrence of constitutive low levels of ROS in T cells (23), but the very low background ROS levels we observed did not differ between T cells derived from the different animal strains and thus seem to be NADPH oxidase independent. This observation leads to the hypothesis that the number of T cell surface ϪSH groups is determined by other cells that do produce ROS, such as APCs or neutrophils (24).…”

CTLA‐4IG suppresses reactive oxygen species by preventing synovial adherent cell–induced inactivation of rap1, a ras family GTPASE mediator of oxidative stress in rheumatoid arthritis T cells

“…We have recently demonstrated that oxidative stress in T lymphocytes from the synovial fluid (SF) (24) and synovial tissue (25) of RA patients is a result of T cell-intrinsic intracellular ROS production. Persistent ROS production in RA SF T cells correlates with constitutive activation of the small GTPase Ras, and blocks activation of the related GTPase Rap1 (24).…”

Efficacy of Methotrexate in Combination with Antioxidant Vitamins (A, C & E) versus Methotrexate alone in the Treatment of Rheumatoid Arthritis

“…It is known that neutrophil granulocytes, macrophages and lymphocytes are activated, so that reactive oxygen and nitrogen species (RS) are produced. These RS can react with lipid, protein and nucleic acids and are thought to be of importance for the etiology and chronicity of the inflammatory rheumatic diseases [8]. One approach to counteract this oxidative stress situation is the use of antioxidants as therapeutic agents.…”