Reactive oxygen species (ROS) play an important role in a variety of pathologic conditions, such as ischemia-reperfusion, carcinogenesis, acquired immunodeficiency syndrome, and aging. In rheumatoid arthritis (RA), oxidative stress has been described as an important mechanism that underlies destructive proliferative synovitis (1, 2). In addition, oxidative stress was found to influence functional characteristics of synovial T lymphocytes, with critical implications for proximal and distal T cell receptor (TCR) signaling events (3–6). Chronic oxidative stress in synovial fluid (SF) T lymphocytes inhibits TCR-dependent phosphorylation of pivotal signaling molecules required for efficient T cell proliferation, thus contributing to severe hyporesponsiveness of these cells to antigenic stimulation. Oxidative stress in RA SF lymphocytes also plays a role in NF-κB–dependent gene transcription, resulting, for example, in the up-regulation of tumor necrosis factor α and interleukin-1 (7).
Several sources of ROS in the synovial joint that could lead to the disturbed redox homeostasis in SF T lymphocytes have been proposed. These include exposure to free radicals liberated by activated phagocytic cells at the site of inflammation (8), ischemia-reperfusion–compromised oxygen radical tension in the inflamed joint (9), and generation of hydroxyl radicals by Fe2+ released from dying cells (10). Recent evidence, however, suggests that T lymphocyte oxidative stress originates from intracellular enzyme activity controlled by the small GTPases Ras and Rap1 (11).
The specific detection of free radicals is hampered by several methodologic problems due to the high reactivity and short life of free radicals. Ongoing oxidative stress is therefore generally analyzed by measurement of secondary products, such as oxidized proteins, peroxidized lipids and their breakdown products, or oxidized DNA. These methods, however, give only limited information on the cellular source(s) of ROS production in situ. Therefore, we attempted to identify the source(s) of synovial oxidative stress using a cytochemical technique based on the principles described for the histochemical localization of ROS production in polymorphonuclear leukocytes (developed by Karnovsky 1993), using 3,3′-diaminobenzidine (DAB) and manganese ions (12). Free radicals directly react with DAB, forming an insoluble DAB polymer in a reaction catalyzed by the presence of Mn2+. This technique has been successfully used to identify ROS-producing sites (13–16).
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T lymphocytes are considered to play a key role in the pathogenesis and perpetuation of RA. Synovial T lymphocytes display features of severe oxidative stress, which results in a number of proliferative and signaling abnormalities (2–5). While there is consensus about the presence of oxidative stress in synovial T cells, the origin of this oxidative stress is unknown.
Our present data demonstrate that chronic oxidative stress observed in synovial T cells mainly originates from free radicals generated by intracellular sources. This conclusion is at odds with suggestions that exposure of synovial T cells to environmental free radicals or proximity to ROS-producing neutrophils and/or macrophages is responsible for oxidative stress in synovial T cells. Moreover, perivascular T cells, which have recently entered the synovial milieu, as well as T cells distributed in the intima, the subintima, and in lymphocyte clusters all produced ROS. This suggests that acquisition of intracellular ROS production is a very early event following extravascularization of T lymphocytes into synovial tissue and, again, indicates that in synovial T cells, free radicals originate from intracellular sources rather than environmental free radicals. Although DAB+ cells were clearly identified as T lymphocytes, not all synovial T lymphocytes contained DAB precipitate, which suggests that only a specific subpopulation of synovial T cells is under oxidative stress. The presence of DAB precipitate in more than 90% of SF T cells on cytospin preparations versus 68% on tissue sections could be due to a difference in CD3 subset composition between SF and synovial tissue, or it could be due to technical issues. Further analysis of specific T cell subpopulations within synovial fluid and tissue should provide insight into these possibilities.
The presence of ROS-producing T cells was not entirely specific for RA, since synovial tissue specimens from a proportion (5 of 15) of patients with other forms of chronic arthritis also contained DAB+ synovial T cells. No DAB precipitate was found in synovial tissue from OA patients.
During the last decade, reduction–oxidation (redox) reactions that generate ROS have been identified as important chemical mediators in the regulation of signal transduction processes (14). In particular, ROS appears to play a central role in the balance between cell growth, survival, and apoptosis. The specific cellular response is dependent on the species of oxidants produced, their subcellular source and localization, the kinetics of production, and the quantities produced. Therefore, the identification of intracellular free radicals in synovial T cells may not only provide an explanation for the altered behavior of synovial T cells, but also prove a pivotal hallmark in understanding the underlying pathophysiologic mechanisms in RA.
Jackson et al (17) recently identified 3 different ROS-producing events following T cell receptor activation: first, a rapid H2O2 production independent of Fas or NADPH oxidase; second, a sustained H2O2 production dependent on both Fas and NADPH oxidase; and third, a delayed superoxide production that was dependent on Fas ligand and Fas, yet independent of NADPH oxidase. Our results favor the first oxidase as the primary source in synovial T lymphocytes, since the intracellular ROS production was catalase-dependent and DPI-independent. Under physiologic conditions, however, intracellular ROS production is a transient phenomenon, occurring only up to 15 minutes after T cell stimulation. Therefore, the sustained intracellular ROS production in SF T cells might be a hallmark for chronic arthritis, which is not found in PB T cells isolated from RA patients or healthy controls. Identifying the oxidase responsible for the intracellular ROS in SF T cells, as well as the proteins that regulate the oxidase, could provide new therapeutic targets in RA.
There is a growing body of evidence demonstrating the critical role of ROS in the regulation of mitogenesis, differentiation, and apoptosis in physiologic and pathophysiologic conditions. Our present results indicate that the chronic oxidative stress observed in synovial T lymphocytes from RA patients originates from intracellularly generated free radicals, rather than environmental influences.