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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

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), 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.

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.

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).

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Reagents.

KCN, NaN3, and CoCl2 were obtained from Merck (Darmstadt, Germany). Polyvinyl alcohol (Mr 70,000–100,000), menadione, catalase, diphenyleneiodonium (DPI), rotenone, and MnCl2·4H2O were purchased from Sigma (St. Louis, MO). DMSO was obtained from PGM Chemicals (New Germany, South Africa), DAB from Fluka (Buchs, Switzerland), Cu/Zn superoxide dismutase (SOD), nordihydroguaiaretic acid (NDGA) from Alexis Biochemicals (Breda, The Netherlands), and NG-methyl-L-arginine, acetate salt (L-NMA) from Molecular Probes (Leiden, The Netherlands).

Patients and tissue processing.

Synovial tissue samples were obtained from patients during knee arthroscopy, which was performed under local anesthesia. These included 30 patients with established RA (mean ± SD disease duration 14 ± 11 years), 3 patients with early (joint symptoms ≤3 months), disease-modifying antirheumatic drug (DMARD)–naive RA, and 25 control patients (7 with psoriatic arthritis, 8 with reactive arthritis, and 10 with osteoarthritis [OA]). Tissue fragments were snap frozen by immersion in methylbutane (−80°C) and stored in liquid nitrogen until used.

Peripheral blood (PB) and SF T cells from 5 RA patients (mean ± SD disease duration 6 ± 16 years) were purified from mononuclear cells using a negative isolation procedure (T Cell Negative Isolation kit; Dynal, Oslo, Norway), which resulted in a >90% CD3+ cell population. Purified T cells were subsequently spun onto slides using a cytospin centrifuge (Shandon, Frankfurt, Germany) for histochemical ROS staining or for ROS detection using a FACScan (Becton Dickinson, Bilthoven, The Netherlands).

Histochemical procedures.

Cryostat sections (8 μm thick) were freshly cut at a cabinet temperature of −25°C. The sections were placed on Star Frost adhesive slides (Optic Labor, Friedrichsdorf, Germany) and immediately used for staining. Before use, sections were air dried for 3 minutes at room temperature. The incubation medium contained 10% weight/volume polyvinyl alcohol (PVA), dissolved in 100 mM Tris maleate buffer (pH 8.0). Sodium azide (5 mM) was added to inhibit endogenous myeloperoxidase activity. The following components were added shortly before incubation of the cryostat sections: 0–12.5 mM DAB, 0–6.5 mM MnCl2, and 0–100 mM CoCl2 (1). All the compounds were added in strict order and thoroughly mixed from stock solutions into the PVA-containing medium. After incubation for 60 minutes at 37°C, sections were washed in distilled water to stop the reaction and then mounted in glycerol for light microscopy.

To further characterize DAB+ cells, immunohistochemical staining was performed using a double-staining immunofluorescence procedure following incubation in DAB-Mn2+ solution. The synovial cryostat sections were subsequently washed, air dried, and fixed with 4% paraformaldehyde. Sections were then incubated for 30 minutes at 4°C with unconjugated mouse anti-human monoclonal antibodies against CD3, CD15, CD19, and CD68 (Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands), or the appropriate isotype control antibodies. Fluorescein isothiocyanate (FITC)–labeled sheep anti-mouse secondary antibodies (PharMingen, Erembodegem, Belgium; 1:1,000 dilution) were used for visualization. Tissue sections were scored independently by 2 observers (PHJR and TJMS) for the presence of DAB precipitate in 50–200 FITC-positive cells. Data were expressed as the mean ± SD.

ROS detection by FACScan.

Purified T cells were resuspended at 5 × 106 cells/ml in phenol red–free Dulbecco's modified Eagle's medium and loaded with 28 μM 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate-di(acetoxymethyl ester) (6-carboxy-DCF) (Molecular Probes) for 20 minutes at 37°C. Cells were analyzed on a FACScan for the mean fluorescence intensity of oxidated 6-carboxy-DCF in the fluorescence channel 1 at 3 different time points (0, 10, and 20 minutes). Thirty minutes before addition of 6-carboxy-DCF, the inhibitors of the different enzymes were added: SOD (1,000 units/ml), catalase (1,000 units/ml), DPI (5 μM), L-NMA (5 μM), and NDGA (10 μM). Rotenone (5 μM) was added 60 minutes before ROS measurement.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Production of free radicals in rheumatoid synovium.

DAB precipitates in the presence of free radicals to form an insoluble final reaction product that is visible as a brown DAB polymer. Manganese ions can be added to catalyze DAB polymerization, since Mn2+ is oxidized to Mn3+ by O2, enabling Mn3+ to oxidize DAB. In synovial tissue from the 30 patients with established RA, a large number of DAB+ cells were detectable perivascularly, in leukocyte aggregates, and in the intimal and subintimal synovial lining (Figure 1). The presence of DAB in the incubation medium led to strong formation of the insoluble final reaction product (Figure 1A). Addition of Mn2+ (0.5–5 mM) only marginally enhanced the amount of DAB precipitate, without affecting the localization pattern of the precipitate (Figure 1B). The highest amounts of final reaction product were found when the incubation medium contained 12.5 mM DAB and 2.5 mM MnCl2. This composition was then used for all further experiments. Addition of cobalt ions to DAB- or DAB-Mn2+–containing media led to the formation of a blue final reaction product, but this also did not affect the amount or the localization pattern of the precipitate (Figure 1C).

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Figure 1. Staining of rheumatoid arthritis synovial tissue with 3,3′- diaminobenzidine (DAB). DAB precipitates in the presence of free radicals to form an insoluble final reaction product, which is visible as a brown DAB polymer. A, A large number of DAB+ cells are seen. B, Addition of Mn2+ only marginally enhances the amount of DAB precipitate, without affecting the localization pattern of the precipitate. C, Addition of Co2+ to DAB- or DAB-Mn2+–containing media leads to the formation of a blue final reaction product. (Original magnification × 200.)

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Strong staining intensity did not allow recognition of the shape of the nuclei when counterstained with hematoxylin or Giemsa (results not shown) for the differentiation between polymorphonuclear cells and mononuclear cells. To further characterize the cellular sources of ROS, cryostat sections of synovium were incubated with monoclonal antibodies against CD3, CD15, CD19, and CD68. Within the synovial CD15+ population (neutrophils), 36 ± 19% (mean ± SD) of the cells contained intracellular DAB final reaction product. Additionally, 67 ± 22% of the CD3+ cells (T lymphocytes) also contained high levels of intracellular DAB precipitate (Figure 2). No DAB precipitate was found in B cells (CD19) or macrophages (CD68) (results not shown).

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Figure 2. Combined 3,3′-diaminobenzidine (DAB) and CD3 immunofluorescence of rheumatoid arthritis synovial tissue. CD3+ cells contained high levels of intracellular DAB precipitate. Left, Fluorescence microscopy image. Center, Corresponding light microscopy image. Right, Combined fluorescence and brightfield image. (Original magnification × 400.)

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To eliminate possible effects of DMARDs and to investigate whether intracellular oxidation of DAB in T cells also occurs in early arthritis, we performed the DAB staining on sections from 3 patients with recent-onset, DMARD-naive RA. ROS staining of cryostat sections from these patients (Figure 3B) was indistinguishable from that of sections from patients with established RA (Figure 3C).

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Figure 3. Staining of synovial tissue sections from A, osteoarthritis, B, early rheumatoid arthritis (RA), and C, established RA patients with 3,3′-diaminobenzidine (DAB). Tissue sections from patients with early RA and established RA both stained equally positive for DAB. (Original magnification × 200.)

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To examine the disease specificity of ROS production by synovial T lymphocytes, cryostat sections of RA and non-RA synovial tissue were compared for DAB precipitation products. No polymerization of DAB was observed in any of the T cells in OA tissue samples (n = 10) (Figure 4, top). A minimum of 50 CD3+,DAB– cells were detected in each of the OA tissue samples. Rare DAB+ cells were identified as CD15+ neutrophils (Figure 4, middle). In 1 of the 8 patients with reactive arthritis and 3 of the 7 patients with psoriatic arthritis, DAB precipitation was observed in 73 ± 3% (mean ± SD) and 48 ± 12% of the CD3+ cells, respectively (Figure 4, bottom). There was no correlation between DAB precipitation and disease activity, disease duration, rheumatoid factor positivity, disease pattern, or erosiveness.

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Figure 4. Staining of osteoarthritis (OA) and psoriatic arthritis synovial tissue sections with 3,3′-diaminobenzidine (DAB). DAB precipitate was observed rarely and only in CD15+ neutrophils. Although a minimum of 50 CD3+ cells per patient were counted, no DAB+ T lymphocytes were found in OA patients. Top, OA tissue with a significant number of CD3+ lymphocytes (arrow). Middle, All DAB+ cells in OA tissue corresponded to CD15+ neutrophils. Bottom, CD3+,DAB+ lymphocytes in tissue from a psoriatic arthritis patient. Left, Fluorescence microscopy images. Center, Corresponding light microscopy images. Right, Combined fluorescence and brightfield images. (Original magnification × 400.)

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ROS production in RA SF T cells.

When DAB staining was performed on cytospin preparations of purified paired PB and SF T cells, DAB precipitate was not found in any of the PB T lymphocytes (Figure 5, top), but was easily detectable in T cells from SF (Figure 5, middle and bottom). The formation of ROS-dependent DAB polymerization in synovial T lymphocytes is likely due to intracellular enzymatic activity. First, DAB polymerization was not affected by preincubation of cytospin preparations or cryostat sections in aqueous medium at 37°C, but was completely inhibited by preincubation for 10 minutes at 100°C. Second, prefixation with 4% paraformaldehyde also abolished the formation of final reaction product, as did overnight air-drying at room temperature. Third, the amount of DAB polymerization increased with increasing incubation times, from 15 minutes to 60 minutes. On cytospin preparations, the initial DAB precipitate (15 minutes) in T lymphocytes was detected in the cytoplasm but not the nucleus of the SF T cells. With increasing incubation periods, DAB precipitate could be detected throughout the cell.

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Figure 5. Staining of cytospin preparations of purified peripheral blood (PB) T cells and synovial fluid (SF) T cells with 3,3′-diaminobenzidine (DAB). PB T cells (PBTC) and SF T cells (SFTC) were incubated for various periods, as indicated. As shown in the SF samples incubated for 15 minutes, DAB precipitation in SF T cells originates in the cytoplasm. After longer incubation periods (30–60 minutes), there is a time-dependent increase in intracellular DAB final reaction product. (Original magnifications are indicated at left.)

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Catalase-sensitive generation of H2O2 in synovial T lymphocytes.

Because it is difficult to quantitate DAB precipitation, we measured intracellular ROS production in purified SF and PB T cells from 5 RA patients, using FACS analysis of the ROS-reactive dye 6-carboxy-DCF. SF T cells displayed a higher basal rate of ROS production than did PB T cells (Figure 6A). To determine which oxidants and enzymes are responsible for the free radical production, inhibitors of the different enzyme systems proposed to regulate ROS production in T lymphocytes were tested (Figure 6B).

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Figure 6. A, Constitutive up-regulation of reactive oxygen species (ROS) production in synovial fluid (SF) T cells compared with peripheral blood (PB) T cells from 5 rheumatoid arthritis (RA) patients. Mean fluorescence intensity (MFI) of 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate-di(acetoxymethyl ester) (6-carboxy-DCF) in PB T cells (PBTC) and SF T cells (SFTC). After a 20-minute preincubation at 37°C with 6-carboxy-DCF, the MFI was measured at 3 different time points. Values are the mean ± SD of 3 independent experiments. B, Catalase-induced reduction of basal ROS production in SF T cells to levels observed in PB T cells. No significant inhibition of intracellular ROS production was seen after preincubation with superoxide dismutase (SOD), NG-methyl-L-arginine, acetate salt (NMMA), nordihydroguaiaretic acid (NDGA), rotenone, and diphenyleneiodonium (DPI). ROS generation is expressed as the fold increase in ROS production under the different conditions relative to that in PB T cells. Values are the mean and SD of 3 independent experiments (n = 5 RA patients). ∗ = P < 0.01 versus SF T cells, by Student's t-test.

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The addition of exogenous catalase, which uses H2O2 as substrate, clearly diminished the DAB polymerization in SF T cells, reducing basal ROS production in SF T cells to near the levels observed in PB T cells. A majority of intracellular H2O2 originates from the dismutation of O2 by SODs. However, when SOD was added to the incubation medium, no significant inhibition of ROS production was observed. Also, addition of DPI (an NADPH oxidase inhibitor), L-NMA (a nitric oxide synthetase inhibitor), or NDGA (a lipoxygenase inhibitor) did not result in significant inhibition of intracellular ROS production. Addition of rotenone (the mitochondrial complex I inhibitor) also did not inhibit increased ROS production in SF T cells.

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

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.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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