Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis
Article first published online: 28 JUN 2005
Copyright © 2005 by the American College of Rheumatology
Arthritis & Rheumatism
Volume 52, Issue 7, pages 2003–2009, July 2005
How to Cite
Remans, P. H. J., van Oosterhout, M., Smeets, T. J. M., Sanders, M., Frederiks, W. M., Reedquist, K. A., Tak, P. P., Breedveld, F. C. and van Laar, J. M. (2005), Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis. Arthritis & Rheumatism, 52: 2003–2009. doi: 10.1002/art.21111
- Issue published online: 28 JUN 2005
- Article first published online: 28 JUN 2005
- Manuscript Accepted: 16 MAR 2005
- Manuscript Received: 17 AUG 2004
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.
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.
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), NG-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.
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.