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)–Mn²⁺ 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 isothiocyanate–labeled 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 H₂O₂, since catalase suppressed intracellular ROS production. Superoxide dismutase, which uses superoxide as a substrate to form H₂O₂, 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 H₂O₂, although the oxidase(s) involved in its generation remains to be determined.