Vitamin E uncouples joint destruction and clinical inflammation in a transgenic mouse model of rheumatoid arthritis.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 46, No. 2, February 2002, pp 522–532 DOI 10.1002/art.10085 © 2002, American College of Rheumatology Published by Wiley-Liss, Inc. Vitamin E Uncouples Joint Destruction and Clinical Inflammation in a Transgenic Mouse Model of Rheumatoid Arthritis Michel De Bandt,1 Maggy Grossin,2 Fathi Driss,3 Joël Pincemail,4 Catherine Babin-Chevaye,5 and Catherine Pasquier5 Objective. Reactive oxygen species are thought to play a role in rheumatoid arthritis (RA) in humans. We postulated that antioxidant treatment could have a beneficial effect in this disease. We therefore investigated the effects of vitamin E in the transgenic KRN/ NOD mouse model of RA. Methods. Mice were treated by gavage with oral vitamin E (␣-tocopherol). Clinical, histologic, and biochemical parameters were assessed for 6 weeks. Results. Vitamin E treatment did not modify the clinical features of the disease (date of onset or disease intensity, as measured by the articular index), but it did prevent joint destruction, as measured by qualitative and semiquantitative analyses. Redox status did not differ between treated and control mice. White blood cell chemiluminescence was higher in transgenic KRN/NOD mice than in controls, but vitamin E treatment attenuated this difference. Vitamin E treatment of the transgenic animals led to a significant decrease in the levels of interleukin-1␤ (IL-1␤) but not tumor necrosis factor ␣. Conclusion. Vitamin E seems to uncouple joint inflammation and joint destruction in this model of RA, with a beneficial effect on joint destruction. Since many investigations are currently in progress to evaluate the benefit of interventions targeted toward anti–IL-1␤, our findings suggest opportunities of therapeutic interest in human RA. Rheumatoid arthritis (RA) is a chronic disabling disease that affects ⬃1.5% of the Caucasian population. There is no cure for RA. Several factors play a role in the onset and clinical course of the disease. A genetic background is suspected (linkage with HLA genes, such as DR4), as well as a T cell receptor (TCR)–restricted presentation of an unknown “arthritogenic epitope.” Various immunocompetent cells (mainly, CD4 T cells) and proinflammatory cytokines are involved in the disease process. Reactive oxygen species (ROS) are thought to play a role in RA. Epidemiologic studies have shown that RA occurs in previously healthy subjects who have low levels of circulating antioxidants (␤-carotene, selenium, ␣-tocopherol, etc.). Once established, RA is characterized by ROS production within affected joints. Transient hypoxia and ischemia-reperfusion phenomena are involved in this ROS production, as well as activated polymorphonuclear neutrophils (PMNs), monocytes, and macrophages. PMNs from the joints of RA patients are locally primed and show an increased chemiluminescence response to fMLP (1,2). The hydroxyl radical seems able to modify the structure of human IgG, thereby increasing the production of rheumatoid factors, the biologic hallmark of the disease (3–5). In addition to low levels of vitamin E and selenium, RA patients also have low blood levels of vitamin C, low erythrocyte superoxide dismutase (SOD) activity, and elevated levels of thiobarbituric acid–reactive substances (3,4,6,7). Supported by institutional grants from l’Institut National de la Santé et de la Recherche Médicale, and in part by research grants from La Société Française de Rhumatologie, Le Fond d’Études et de Recherche du Corps Médical des Hôpitaux de Paris (AP-HP), France, and Cognis Nutrition and Health, Düsseldorf, Germany. 1 Michel De Bandt, MD, PhD: INSERM U479, Faculté Xavier Bichat, and Centre Hospitalo-Universitaire Xavier Bichat, Paris, France; 2Maggy Grossin, MD: Centre Hospitalo-Universitaire Xavier Bichat, Paris, France; 3Fathi Driss, MD: Centre Hospitalier Universitaire Bichat, and Centre Hospitalo-Universitaire Xavier Bichat, Paris, France; 4Joël Pincemail, MD: Centre Hospitalier Universitaire de Liège, Domaine du Sart Tilman, Liège, Belgium; 5Catherine BabinChevaye, MD, Catherine Pasquier, PhD: INSERM U479, Faculté Xavier Bichat, Paris, France. Address correspondence and reprint requests to Michel De Bandt, MD, PhD, Service de Rhumatologie, Centre HospitaloUniversitaire Xavier Bichat, 46 rue Henri Huchard, Paris 75018, France. E-mail: email@example.com. Submitted for publication May 25, 2001; accepted in revised form October 2, 2001. 522 VITAMIN E IN A TRANSGENIC MOUSE MODEL OF RA Many therapeutic trials have assessed the clinical value of antioxidants in RA (1,8–11), administered in an effort to restore a normal pool of ROS scavengers and modulate eicosanoic acid production. The results are controversial, mainly because of the heterogeneity of the patients studied (5,11). One clinical trial, testing the benefit of vitamin E combined with the nonsteroidal antiinflammatory drug (NSAID) diclofenac, showed good tolerability and analgesic effects of vitamin E, allowing the doses of the NSAID to be tapered. In a randomized placebo-controlled study recently conducted by Edmonds et al (12), a trend toward a clinical benefit of vitamin E was identified, but the study period was short, the groups were small and heterogeneous (age, sex, previous treatments, etc.), and the concomitant use of steroids was allowed. The potential long-term benefit of antioxidants in RA remains unknown (11,13), particularly in terms of clinically relevant end points, such as bone erosion, steroid sparing, disability, and survival. There is no good animal model of RA (14–17). The most widely used is the murine model of collageninduced arthritis, which was established 25 years ago. The recently established KRN/NOD mouse (also known as KBN), provides an intriguing animal model of human RA. As in humans, the disease develops on a specific genetic background (NOD/Lt mice), is HLA- and TCRrestricted (the transgene encodes for a unique V␤6 TCR), and requires no immunization. By day 30 of life, 100% of the animals spontaneously develop an acute (and later chronic) bilateral, symmetric, erosive, and disabling polyarthritis that is quite similar to the human disease. The aim of the present study was to investigate the effects of oral supplementation with vitamin E (natural ␣-tocopherol) in the KRN/NOD mouse model of arthritis, which provides a very homogeneous and reproducible model of RA in humans, and to quantify the effects of vitamin E supplementation on the clinical, biochemical, and histologic indices of disease. We found that oral supplementation had no effects on clinical symptoms but reduced articular destruction, probably through a decrease in interleukin-1␤ (IL-1␤), the main cytokine involved in articular destruction. MATERIALS AND METHODS Mice. The transgenic KRN/NOD mice used in this study were obtained from the cross between KRN transgenic males (kindly provided by Dr. Christophe Benoist, Institut de Génétique et de Biologie Moléculaire, Illkirch, France) and female NOD mice (NOD/Orl Ico; purchased from IFFA Credo, l’Abresle, France). Mice were bred and maintained in our animal facility at Bichat Teaching Hospital and Medical 523 School. The animals were weighed, examined, and bled regularly before, during, and after the onset of arthritis. (KRN/ NOD)F1 offspring were bled on day 21 after birth for identification of those expressing the V␤6 TCR transgene, as determined by flow cytometry. All transgenic (KRN/NOD)F1 mice typically developed polyarthritis 27 ⫾ 2 days (mean ⫾ SD) after birth, whereas nontransgenic (KRN/NOD)F1 offspring remained normal. Mice were bled by venous puncture on day 21, and then weekly until day 60. Six blood samples (P1, P2, P3, P4, P5, and P6) were obtained from week 1 to week 6, and 3 urine samples (U0, U1, and U2) were obtained before treatment and after 3 and 6 weeks of treatment, respectively, from each mouse. Plasma and serum were divided into aliquots and stored at ⫺80°C. Flow cytometry. Flow cytometry was used to identify transgenic mice among (KRN/NOD)F1 offspring on day 21 of life. Whole blood was incubated on ice with anti– phycoerythrin–CD4 T cell (0.5 g) and anti–fluorescein isothiocyanate–V␤6 TCR chain (0.5 g) monoclonal antibodies (PharMingen, Le Pont de Claix, France). After red cell lysis and washing steps, flow cytometry was performed using a Becton Dickinson FACScan (Immunocytometry Systems, San Jose, CA) equipped with a 15-mW, 488-nm argon laser. CD4⫹,V␤6⫹ cells were considered positive for the KRN transgene. All the results were obtained with a constant photomultiplier gain. Data were analyzed using CellQuest software (Becton Dickinson, Mountain View, CA). Nonspecific antibody binding was determined on cells incubated with the same concentration of an irrelevant antibody of the same isotype. Treatment. All animals received long-term (before and after disease onset) supplementation with vitamin E administered at the same doses and according to the same regimen. Vitamin E (natural ␣-tocopherol) was dissolved in sunflower oil and administered at a dosage of 0.134 mg/day (0.171 IU/day for a 30-gm mouse), which is equivalent to a dosage of 400 IU/day for humans, as previously recommended (18). We administered vitamin E by gavage every other day (0.268 mg in 100 l), starting on day 21 of life (1 week before the onset of arthritis) and continuing for 6 weeks. The placebo group underwent the same procedure, but received sunflower oil alone. Mice were given conventional oral food and water ad libitum. A total of 140 mice (35 per group) were studied. There were 2 groups of transgenic KRN/NOD mice, one of which received vitamin E, and the other received placebo. There were 2 groups of wild-type mice, one of which received vitamin E and the other received placebo. Determination of plasma levels of vitamin E. Vitamin E was extracted from plasma with a mixture of 1 volume of ethanol containing 2 g of tocopherol acetate (internal standard) and 1 volume of n-heptane per volume of plasma. After vigorous vortexing, 750 l of the n-heptane layer was evaporated in a nitrogen atmosphere, and the pellet was dissolved in 200 l of ethanol and then subjected to high-performance liquid chromatography (Gilson, Villiers Le Bel, France) with a reverse-phase C-18 120 column (100 ⫻ 4.5 mm inner dimension). The mobile phase was a mixture of methanol and water (98:2), and the eluate was passed through an ultraviolet detector (285 nm) at a flow rate of 1.5 ml/minute. A Milton Roy (Pont Saint Pierre, France) integrator calculated the peak ratio of ␣-tocopherol:tocopherol acetate. The concentration of 524 ␣-tocopherol in plasma was determined by comparison with a standard curve obtained with a fixed concentration of tocopherol acetate and various amounts of ␣-tocopherol. Values were expressed as micrograms per milliliter. Clinical assessment of arthritis. The incidence of arthritis and its severity were assessed daily. Arthritis was quantified by measuring the thickness of each paw (a direct measure of joint swelling) with a caliper-square (precision 1/100 mm). The sum of the measurements of the 4 paws was calculated for each animal to yield an articular index (AI). Curves were established for each group of animals. Values are given as the mean ⫾ SD. Histologic examination. Mice were killed by cervical dislocation, and the joints were harvested for analysis. Joints were fixed for 12 hours in 4% paraformaldehyde, decalcified in 5% nitric acid for 12 hours, fixed again for another 12 hours in paraformaldehyde, and then embedded in paraffin. Sagittal sections 4 m thick were cut with a Microm semiautomatic microtome (model HM340E; Microm France, Francheville, France), and stained with hematoxylin–phloxine–saffron. Histologic examination included assessment of morphologic features and semiquantitative grading (19,20). For each joint, the following elements were analyzed to determine the intensity of inflammatory lesions: joint cavity (normal or dilated, presence or absence of liquid and/or cells), synovial membrane (normal or abnormal, edema, synovial hyperplasia, vascular congestion, cellular infiltration and/or fibrosis, type of inflammatory cells), articular surfaces (normal or presence of chondral and/or bone destruction and/or fibrosis), and tendon sheaths (normal or synovial hyperplasia, edema, vascular congestion, cellular infiltration, and fibrosis). Features were scored on a scale of 0–5, where 0 ⫽ normal, 1 ⫽ edematous arthritis, 2 ⫽ nonerosive acute arthritis, 3 ⫽ erosive acute arthritis without fibrosis, 4 ⫽ chronic erosive and fibrosing arthritis, and 5 ⫽ chronic erosive and fibrosing arthritis with persistent and progressive acute arthritis. Morphologic and semiquantitative analyses were performed by an independent examiner who was blinded to the animal’s clinical status, type of treatment, and number of days after treatment. Measurement of reduced glutathione (GSH). GSH was measured in whole blood as described elsewhere (21), with modifications. Briefly, 100 l of blood was withdrawn from the corner of an eye, and 0.9 ml of the following solution was immediately added: 1.67 gm of metaphosphoric acid (Fisher Scientific, Springfield, NJ), 200 mg of EDTA (Sigma, St. Louis, MO), 30 gm of NaCl, and up to 200 ml of distilled water. The tubes were centrifuged for 10 minutes at 3,000 revolutions per minute, and the supernatant was tested. Ten microliters of supernatant was added to each well of a 96-well plate in 200 l of GSH buffer (0.1M NaH2PO4, 5 mM EDTA, pH 8) and 10 l of o-phthalaldehyde reagent (1 mg/ml of methanol). After 15 minutes at room temperature, the plates were read on a Fluostar plate reader (BMG, Lab Technologies, Champigny sur Marne, France) at ex 350 nm and em 420 nm. The values shown were calculated from a GSH standard curve (0–50 g/100 ml). Measurement of neutrophil activation by chemiluminescence. The activation status of white blood cells (WBCs) was measured immediately after blood sampling, by means of luminol-enhanced chemiluminescence on an E&G Berthold AutoLumat (Berthold, Wildbad, Germany). Whole blood (100 l) was stimulated with opsonized zymosan (10 mg/ml) in DE BANDT ET AL Hanks’ balanced salt solution, and chemiluminescence was detected in a luminol (10 M)–enhanced reaction in the AutoLumat at 37°C for 10 minutes. The maximum counts per minute peak appeared ⬃7–8 minutes after the beginning of stimulation. WBCs were counted in capillary pipettes (Unoppet; Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ). Results are expressed as counts per minute per 106 WBCs. Measurement of isoprostanes. Isoprostanes, namely, 8-epi-prostaglandin F2␣ (8-epi-PGF2␣) were analyzed by competitive enzyme-linked immunosorbent assay (ELISA) (22), using a kit from Cayman Chemical (Ann Arbor, MI). Three 24-hour urine samples from each animal (U0 ⫽ before treatment, U1 ⫽ 3 weeks after starting treatment, and U2 ⫽ 6 weeks after starting treatment) were analyzed. Briefly, free 8-epiPGF2␣ competes with the tracer (8-epi-PGF2␣ linked to acetylcholinesterase) for specific rabbit antiserum binding sites. The rabbit antiserum–8-epi-PGF2␣ complex binds to a monoclonal mouse anti-rabbit antibody. After washing, Ellman’s reagent (acetylcholinesterase substrate) was added to the wells, and absorbance was read at 405 nm in a Fluostar microplate reader. Results are expressed as picograms per milligram of urinary creatinine. Measurement of hydroperoxides. Assay kits for plasma determination of lipid hydroperoxide were purchased from Kamiya Biomedical (Thousands Oaks, CA) (23). In this assay, hemoglobin catalyzes the reaction of lipid hydroperoxides with a methylene blue derivative, 10-N-methylcarbamoyl-3,7dimethylamino,10,H-phenothiazine (MCDP), forming an equimolar concentration of methylene blue. Lipid hydroperoxide measurement was based on the amount of methylene blue formed. We fully automated this assay using the Monarch analyzer (Instrumentation Laboratory, Lexington, MA). Absorption was measured at 675 nm, and the assay was calibrated using cumene hydroperoxide. Hydroperoxide was measured in 100 l of plasma obtained from all 4 groups of mice (samples P2–P6). ELISA determination of tumor necrosis factor ␣ (TNF␣) and IL-1␤. Vitamin E– and placebo-treated mice were bled by venous puncture on day 21, and then weekly until day 60. Plasma and sera were divided into aliquots and stored at ⫺80°C. All samples were thawed on the same day and tested in the same laboratory. ELISA test kits for TNF␣ and IL-1␤ were obtained from R&D Systems Europe (Abingdon, UK) and were used according to the manufacturer’s instructions. The detection limit of both assays was 5 pg/ml. Statistical analysis. Statistical analyses were performed using Statwork software to calculate the means and standard deviations. Group means were compared by using analysis of variance, followed by Fisher’s exact test to identify statistically significant differences (i.e., P ⬍ 0.05). RESULTS Time course of the AI in transgenic versus nontransgenic KRN/NOD mice. All of the transgenic KRN/ NOD mice and none of the nontransgenic KRN/NOD mice developed arthritis. As expected, the AI in nontransgenic mice increased after birth and stabilized on day 33 (mean ⫾ SD 1,255 ⫾ 73) (Figure 1). In transgenic KRN/NOD mice, the disease VITAMIN E IN A TRANSGENIC MOUSE MODEL OF RA 525 Figure 2. Changes in the articular index (AI) in the 4 groups of mice treated with vitamin E (Vit-E) or placebo. The AI was determined as described in Figure 1. Values are the mean (for simplicity, the SDs are not shown). Figure 1. Changes in the articular index (AI) in transgenic KRN/ NOD mice compared with controls. The thickness of each paw (a direct measure of joint swelling) was measured with a caliper-square (precision 1/100 mm). The AI was calculated as the sum of the measurements for the 4 paws of each animal. Curves were established for each group of animals (35 per group). Values are the mean ⫾ SD. started on day 27 (⫾2 days) of life, with an acute phase characterized by joint effusions and florid synovitis spreading out between days 27 and 36. The AI increased rapidly, reaching 1,587 ⫾ 96 on day 36 (P ⬍ 0.05 versus nontransgenic mice), consistent with previous results in this model (24). The disease remained active from day 36 to day 60, with a high AI (mean ⫾ SD over this period 1,596 ⫾ 103). The peak AI was observed on day 43 (1,678 ⫾ 102) and then declined from day 60 to day 110, but without reaching the values in the healthy nontransgenic mice (P ⬍ 0.05 versus nontransgenic mice). After day 110, the transgenic KRN/NOD mice had rather mild clinical expression of the disease, but obvious clinical sequelae. The AI remained stable. Statistical analysis showed a difference in the AI between transgenic KRN/NOD mice and nontransgenic mice. The difference remained statistically significant over the period from day 27 to day 170 after birth (P ⬍ 0.05). Time course of the AI in vitamin E–treated versus placebo-treated transgenic KRN/NOD mice. We sought to determine the effect of vitamin E on arthritis. Vitamin E was given orally, according to the scheduled dose (0.268 mg every other day), beginning on day 21 after birth (before the onset of disease) and continuing for 6 weeks. The onset of clinical manifestations was not delayed by vitamin E (Figure 2). In both vitamin E–treated and placebo-treated transgenic KRN/NOD mice, the disease started with an acute phase identical to that seen in placebo-treated transgenic mice (mean ⫾ SD day of onset 27 ⫾ 2), following the same pattern and reaching values consistent with previous results (peak and decline of AI). No difference between vitamin E– and placebo-treated mice was observed. For wild-type nontransgenic mice, no difference between the vitamin E–treated and the placebo-treated groups was observed. The day of arthritis onset and the evolution of clinical features in the 2 groups were similar (Figure 2). Time course of changes in weight in vitamin E–treated versus placebo-treated transgenic KRN/NOD mice. Body weight was measured with electronic scales after measuring the AI (Figure 3). Vitamin E– and placebo-treated transgenic KRN/NOD mice exhibited Figure 3. Changes in body weight in the 4 groups of mice treated with vitamin E (Vit E) or placebo. Arthritic and healthy mice were weighed daily beginning on the day of arthritis onset and continuing for 100 days. Values are the mean (for simplicity, the SDs are not shown). 526 DE BANDT ET AL Figure 4. Histologic findings in joint sections from transgenic KRN/NOD mice, which developed arthritis. A, Shoulder of a placebo-treated mouse on day 60. Cartilage is focally eroded and synovial tissue is infiltrated by inflammatory cells, resulting in acute arthritis with minor cartilaginous alterations. B, Shoulder of a vitamin E–treated mouse on day 60. Note the normal histologic appearance of all the joint structures. C, Knee of a placebo-treated mouse on day 90. The cartilage is partially altered, and a mild inflammatory infiltrate is present in the synovial tissue. D, Knee of a vitamin E–treated mouse on day 90. The inflammatory infiltrate is quite similar to that in the synovial tissue shown in C, but the cartilage remains normal. E, Elbow of a placebo-treated mouse on day 120. The joint cavity is filled with fibrin and inflammatory cells and is lined with synovitis, which covers an altered cartilage. F, Elbow of a vitamin E–treated mouse on day 120. The joint cavity is devoid of inflammatory processes and is lined with fibrous synovitis. Qualitative analysis of nontransgenic (i.e., nonarthritic) mice revealed normal joints throughout the study period (data not shown). (Hematoxylin–phloxine–saffron stained; original magnification ⫻ 10 in A and B; ⫻ 25 in C–F.) VITAMIN E IN A TRANSGENIC MOUSE MODEL OF RA weight loss at the onset of arthritis but slowly regained weight during the following weeks. From day 90 of life, body weight was similar in all 4 groups of mice studied. No difference in body weight was seen between vitamin E– and placebo-treated transgenic KRN/NOD mice, or between their nontransgenic counterparts, at any time of the study. Plasma concentrations of vitamin E. Since vitamin E was given orally, we measured plasma levels of vitamin E to verify an increase compared with the placebo-treated mice. Plasma levels of vitamin E were measured in all 4 groups of mice (12 mice in each group) for 6 weeks after disease onset, starting on day 21 after birth. Vitamin E levels were significantly higher in treated than in untreated mice. The vitamin E concentration peaked at weeks 3 and 4 after the beginning of treatment. One week before gavage, all mice had the same blood levels of vitamin E (mean ⫾ SD 1.4 ⫾ 0.4 g/ml). During gavage, the vitamin E–treated mice had higher levels of vitamin E than did the placebo-treated mice (2.7 ⫾ 0.6 and 1.4 ⫾ 0.5 g/ml, respectively). Two weeks after the end of gavage, the values normalized in all groups at 1.3 ⫾ 0.04 g/ml. Findings of histologic analyses. Morphologic analysis. Joints were prepared as described in Materials and Methods. The time course of histologic changes in the inflamed joints of placebo- and vitamin E–treated mice is shown in Figure 4 (representative of 1 experiment; 3 mice per group). Throughout the study, nontransgenic mice exhibited normal joints by qualitative analysis (data not shown). With regard to the transgenic animals, the results were as follows. On day 60, placebo-treated animals (Figure 4A) showed erosive arthritis, with pannus formation and new vessels, but without fibrosis; tendon rupture was observed, mainly in the tarsal and carpal joints. Joint spaces were filled with inflammatory material, synovial membranes were invaded by inflammatory cells, and tendon sheaths were inflamed. Cartilage was focally eroded, and synovial tissue was infiltrated by inflammatory cells, resulting in acute arthritis with cartilaginous alterations. Figure 4B shows a joint from a vitamin E–treated transgenic KRN/NOD mouse on day 60, with nearly normal histologic findings in the joint structures. On days 90 and 120 of life, histologic features were grossly similar in vitamin E– and placebo-treated mice in terms of inflammation (i.e., pannus proliferation and cell invasion). In contrast, sequelae (intense bone or cartilage destruction, together with fibrosis and fusion) were noted in placebo-treated mice but not in vitamin 527 Table 1. Findings of semiquantitative histologic analysis in transgenic KRN/NOD mice* Day 60 66 70 90 100 120 Vitamin E 10 ⫾ 2 10 ⫾ 2.5 17.5 ⫾ 1.9 15 ⫾ 4 30 ⫾ 2.1 30 ⫾ 1.2 Placebo 18 ⫾ 2.1 20 ⫾ 3 22.5 ⫾ 3.1 30 ⫾ 2.5 33 ⫾ 2.9 33 ⫾ 4.1 P ⬍0.5 ⬍0.5 ⬍0.5 ⬍0.5 NS NS * Mice were given vitamin E or placebo treatment, and on days 60, 66, 70, 90, 100, or 120 (i.e., from the end of treatment until 2 months later), mice were killed (n ⫽ 3 per group) and their joints were examined. Histologic findings were scored semiquantitatively by an independent, blinded examiner using a 0–5 scale, where 0 ⫽ normal, 1 ⫽ edematous arthritis, 2 ⫽ nonerosive acute arthritis, 3 ⫽ erosive acute arthritis without fibrosis, 4 ⫽ chronic erosive and fibrosing arthritis, and 5 ⫽ chronic erosive and fibrosing arthritis with persistent and progressive acute arthritis. Values are the mean ⫾ SD. Throughout the study period, nontransgenic mice exhibited normal joints, as demonstrated by a score of zero on semiquantitative analysis. Therefore, the values shown here are only for the transgenic KRN/NOD mice. NS ⫽ not significant. E–treated mice. Figures 4C and D show representative joints on day 90. In placebo-treated transgenic KRN/ NOD mice (Figure 4C), the cartilage was partially altered, and a mild inflammatory infiltrate was seen in synovial tissue. In vitamin E–treated transgenic KRN/ NOD mice (Figure 4D), the inflammatory infiltrate in the synovial tissue was quite similar to that in the placebo-treated mice, but the cartilage remained normal. Figures 4E and 4F show representative joints on day 120. In placebo-treated transgenic KRN/NOD mice (Figure 4E), the joint cavity was filled with fibrin and inflammatory cells and was lined with synovitis, which covered the altered cartilage. In vitamin E–treated transgenic KRN/NOD mice (Figure 4D), the joint cavity was devoid of inflammatory markers and lined with fibrous synovitis. Semiquantitative analysis. Vitamin E treatment improved the semiquantitative histologic arthritis score in the transgenic KRN/NOD mice (Table 1). Throughout the entire study period, nontransgenic mice exhibited normal joints, as demonstrated by a score of zero on semiquantitative analysis (data not shown). Regarding transgenic animals, results were as follows: Histologic scoring of the lesions (19,20) was performed on mice treated at the scheduled dose of vitamin E (3 mice in each group). Mice were given vitamin E or placebo treatment and killed on days 60, 66, 70, 90, 100, or 120 (i.e., from the end of treatment until 2 months later). One anterior and 1 posterior limb from 528 DE BANDT ET AL mice was not different from that in healthy nontransgenic mice (Figure 5). Isoprostane. Isoprostane levels in urine were measured in all groups of mice. We found no statistically significant difference in urinary isoprostane levels, regardless of the treatment or sampling time (data not shown). Hydroperoxide. Hydroperoxide levels were measured in plasma samples obtained at weeks 2–6 (samples P2–P6) from transgenic and control mice (6–9 mice per group). Values were always below the detection limit (data not shown). Levels of proinflammatory mediators. The inflammation induced in this mouse model of RA was measured as the activation of WBCs and the release of cytokines in blood. Chemiluminescence of WBCs and ELISAs for TNF␣ and IL-1␤ were used as described in Materials and Methods. Chemiluminescence of zymosan-activated WBCs. Chemiluminescence was measured in samples of whole blood obtained at the beginning of vitamin E treatment (21–25 days of life) and for 6 weeks thereafter. WBCs were counted, and the intensity of chemiluminescence was expressed as the cpm/106 WBCs. Values were 5–145 ⫻ 106 cpm/106 WBCs. Table 2 gives the values for vitamin E– and placebo-treated transgenic KRN/NOD mice. In KRN/NOD mice, chemiluminescence values were high in the P1 samples from both vitamin E– and placebo-treated mice. The values then fell at P2–P5, and were lower in vitamin E–treated mice than in placebotreated mice. At P6, we observed an increase in both groups, but values remained significantly lower in vitamin E–treated mice than in placebo-treated transgenic mice. No significant difference was observed between the corresponding groups of healthy nontransgenic mice. No statistically significant difference was observed at P1 Figure 5. Levels of reduced glutathione in whole blood samples obtained at baseline (P0) and during weeks 1–6 (P1–P6) of vitamin E (vit E) or placebo treatment in the 4 groups of mice (n ⫽ 7, 6, 7, and 9 mice of groups 1–4, respectively). Values are the mean ⫾ SD. each animal was examined histologically, and each joint (anterior: shoulder, elbow, wrist, and metacarpophalangeal joints; posterior: hip, knee, ankle, and metatarsophalangeal joints) was analyzed and scored. The results are given in Table 1. Treatment with vitamin E reduced the intensity of bone and joint destruction. Redox status of the mice. Oxygen radicals have been said to be involved in RA. We therefore measured the redox status of blood and urine in the KRN/NOD mouse model as markers of oxidation. Glutathione. GSH was measured in whole blood samples obtained from all mice. To avoid GSH oxidation, protein was immediately precipitated from the blood samples. The amount of GSH in vitamin E–treated and placebo-treated transgenic KRN/NOD Table 2. Chemiluminescence of zymosan-activated WBCs from transgenic KRN/NOD mice* Blood sample Vitamin E Placebo P P1 P2 P3 P4 P5 P6 84.2 ⫾ 28 82.6 ⫾ 42 NS 10.6 ⫾ 1.1 16.5 ⫾ 0.7 0.01 21.4 ⫾ 10.6 45.2 ⫾ 13.4 0.001 39.3 ⫾ 12.3 55.4 ⫾ 15.9 0.01 17.5 ⫾ 0.7 49.4 ⫾ 4.9 0.001 82.6 ⫾ 21 139.7 ⫾ 40.2 0.01 * Samples of whole blood were obtained during weeks 1–6 (P1–P6) of vitamin E or placebo treatment (beginning at age 21–25 days). A mean of 6 mice were tested at each time point. White blood cells (WBCs) were counted, and the intensity of chemiluminescence was expressed as the cpm/106 WBCs. Values are the mean ⫾ SD ⫻106 cpm/106 WBCs. The values in the nontransgenic mice were not statistically significantly different between treatment groups. Therefore, the values shown here are only for the transgenic KRN/NOD mice. NS ⫽ not significant. VITAMIN E IN A TRANSGENIC MOUSE MODEL OF RA Figure 6. Levels of interleukin-1␤ (IL-1␤) in plasma samples obtained during weeks 1–6 (P1–P6) of vitamin E (vit E) or placebo treatment in the 4 groups of mice. IL-1␤ was measured with an enzyme-linked immunosorbent assay kit. Means and SDs were calculated with Statwork software. For simplicity, only the means are shown. The differences between vitamin E and placebo treatment were statistically significant after 2 weeks of treatment, at P3, P4, and P6, but not at P2 and P5. between vitamin E– and placebo-treated transgenic KRN/NOD mice, whereas at the following measurements, values were significantly lower in vitamin E–treated transgenic KRN/NOD mice than in their placebo-treated counterparts (Table 2). Plasma levels of TNF␣ and IL-1␤. Figures 6 and 7 show that the amounts of IL-1␤ and TNF␣ were markedly higher in transgenic KRN/NOD mice than in the Figure 7. Levels of tumor necrosis factor ␣ (TNF␣) in plasma samples obtained during weeks 1–6 (P1–P6) of vitamin E (vit E) or placebo treatment in the 4 groups of mice. TNF␣ was measured with an enzyme-linked immunosorbent assay kit. Means and SDs were calculated with Statwork software. For simplicity, only the means are shown. The differences between vitamin E and placebo treatment were not statistically significant. 529 nontransgenic control mice (n ⫽ 16 mice per group for TNF␣ and for IL-1␤). IL-1␤ was statistically significantly increased in all the samples from placebo-treated transgenic mice compared with the nontransgenic mice. In vitamin E–treated KRN/NOD mice, this level was significantly decreased compared with that in the placebotreated KRN/NOD mice. The highest levels of IL-1␤ in the placebo-treated transgenic mice were obtained at P3 and P4, which corresponded to the highest AI in these mice (Figure 6). TNF␣ was clearly higher in transgenic compared with nontransgenic mice (Figure 7). TNF␣ levels were slightly lower in transgenic mice receiving vitamin E than in their placebo-treated counterparts, although the difference was not significant. TNF␣ values were always higher in transgenic KRN/NOD mice than in the healthy nontransgenic mice, but the differences were not significant. DISCUSSION In this study, we determined the effects of vitamin E supplementation on clinical, histologic, and biochemical parameters in a transgenic KRN/NOD mouse model of RA. The mice were treated orally with natural ␣-tocopherol, at a dosage of 0.171 IU every other day, which is equivalent to the recommended dose of 400 IU/day for adult humans. Vitamin E treatment did not modify the clinical characteristics of the disease (AI and body weight), but it did prevent joint destruction, suggesting an uncoupling of the 2 phenomena. TNF␣ and IL-1␤ values, which were low in nontransgenic control mice, were increased in the transgenic KRN/NOD mice. Vitamin E–treated arthritic mice had significantly lower IL-1␤ levels than their placebo-treated counterparts. Blood levels of GSH, urinary isoprostane values, and plasma hydroperoxide values did not differ between vitamin E–treated transgenic KRN/NOD mice and placebo-treated controls. WBC chemiluminescence was higher in transgenic KRN/NOD mice than in the nontransgenic nonarthritic controls, and was decreased by treatment with vitamin E. Transgenic KRN/NOD mice exhibited an increase in the AI on day 27 after birth, corresponding to the onset of arthritis. The AI peaked on day 50 and remained high until the end of the experiment (day 170). These mice also showed concomitant weight loss. Both the AI and weight loss (see below) were directly related to joint effusion and synovitis, i.e., to the clinical inflammatory aspect of the disease (24). Vitamin E treatment did not modify the date of onset or the intensity of 530 arthritis, as shown by the AI and the evolution of the disease. Transgenic KRN/NOD mice lost weight regardless of treatment (vitamin E or placebo), and the 2 groups regained weight at the same rate. Both the AI and weight loss reflect the inflammatory process. Our data suggest that vitamin E does not modify the clinical inflammatory component of RA in this model. The KRN/NOD mouse develops early and persistently severe joint destruction (invading pannus, numerous inflammatory cells, cartilage destruction, articular fibrosis, and joint fusion). This is in fact the main problem to solve in the human disease. Our histologic analysis clearly showed that joint inflammation (i.e., pannus proliferation and invasion) was very similar regardless of the treatment throughout the course of the disease. However, bone lesions (bone and cartilage destruction, fibrosis, and fusion) were far more intense in placebo-treated mice than in vitamin E–treated mice. Semiquantitative analysis also showed that treatment with vitamin E reduced the intensity of bone and joint destruction. The histologic benefit appeared to be durable, despite continuing overt clinical disease. We also measured levels of the proinflammatory cytokines involved in human RA, namely, TNF␣ and IL-1␤. Levels of the 2 cytokines were higher in arthritic mice than in healthy controls, as in human RA. In vitamin E–treated transgenic KRN/NOD mice, we observed no significant difference in TNF␣ values between vitamin E– and placebo-treated mice, suggesting that vitamin E supplementation has no effect on the key cytokine in the inflammatory process associated with RA. However, circulating levels of IL-1␤ were significantly lower in vitamin E–treated than in placebotreated arthritic mice, suggesting that vitamin E reduces the circulating level of IL-1␤, which is involved in joint destruction (25,26). To investigate the antioxidant effect of vitamin E, we examined markers of the redox status of cells and tissues. Vitamin E is generally thought to act by inhibiting lipid peroxidation through blockade of the oxidation chain reaction. Vitamin E is then regenerated by hydrosoluble ascorbate and by glutathione (27,28). Glutathione, which is mainly present in reduced form (GSH) in cells, is oxidized during inflammatory processes by ROS released by leukocytes. There is also substantial evidence to support the use of urinary isoprostane as a noninvasive index of lipid peroxidation in vivo (29). In our model, no differences in blood levels of GSH in vitamin E–treated and placebo-treated mice were found. Urinary excretion of isoprostane and DE BANDT ET AL plasma levels of hydroperoxide were similar in all groups of mice, indicating that neither disease status nor vitamin E treatment influenced lipid peroxidation. Thus, in our transgenic mouse model of RA, the disease does not appear to be associated with depletion of endogenous antioxidants, contrary to observations in human RA (30). Local production of ROS in the joints during RA would probably not be reflected in the general circulation, and this is a possible explanation of our negative results for GSH, isoprostane, and hydroperoxide in blood and urine. Another marker of inflammation is leukocyte activation. WBC activation status, as measured by a chemiluminescence method, was higher in KRN/NOD mice than in normal mice. Such activated leukocytes could release ROS and thereby participate in the tissue destruction of RA. Vitamin E treatment was associated with lower ROS release relative to that in placebotreated controls. Based on the chemiluminescence values, it seems that vitamin E begins to act after 3 weeks of treatment, since values in vitamin E–treated KRN/ NOD mice began to fall after this time point. These results are consistent with a vitamin E–induced attenuation of leukocyte activation. The decrease in tissue destruction despite the lack of change in lipid peroxidation in vitamin E–treated mice is intriguing. Indeed, vitamin E could inhibit ROS release by neutrophils and monocytes by blocking the activity of protein kinase C, which is involved in the activation of NADPH oxidase (31–33). The decrease in WBC chemiluminescence in KRN/NOD mice treated with vitamin E could be explained by this mechanism. Moreover, in vitro studies have shown that vitamin E decreases IL-1␤ release by lipopolysaccharidestimulated human monocytes by inhibiting 5lipoxygenase (5-LO) (34,35). In PMNs, NADPH oxidase, which is responsible for ROS release, can also be activated by leukotriene B4, the production of which is catalyzed by 5-LO. Since vitamin E inhibits WBC activation (see above), it might act on leukotriene B4 release. If so, vitamin E might act on both protein kinase C activity and 5-LO activity to decrease the amount of ROS released by PMNs, providing 2 different pathways by which vitamin E may decrease WBC activity. At the same time, decreased IL-1␤ release could be due to the 5-LO inhibition by vitamin E. Recently, Zimmer et al (36) cloned and sequenced a new cytosolic high-affinity receptor for vitamin E (human tocopherol-associated protein [TAP]) from the cytosol of bovine liver, brain, and prostate. TAP, which binds vitamin E, has a CRAL motif. This VITAMIN E IN A TRANSGENIC MOUSE MODEL OF RA protein belongs to a family of hydrophobic ligand proteins, all containing a cis-retinal binding motif with potent nuclear functions. Interaction of TAP with DNA via gene interaction might also account for the effects of vitamin E in our model and should be interesting to investigate. In conclusion, this study, using a homogeneous and reproducible animal model of arthritis, shows that vitamin E supplementation reduced circulating levels of IL-1␤, the main cytokine involved in the joint destruction associated with this disease. However, the precise mode of action of vitamin E is unclear. In particular, we found no evidence of altered oxidation status in the general circulation during treatment. In contrast, we found no effect of vitamin E on the inflammatory component of the disease (including TNF␣ level, AI, and weight loss). Our results emphasize the potential interest of vitamin E in arthritis and deserve further evaluation in order to fully understand its precise mechanism of action and its therapeutic interest in articular destruction. 531 9. 10. 11. 12. 13. 14. 15. 16. 17. ACKNOWLEDGMENTS We thank Charlotte Delarche and Liliane Louedec for their excellent technical assistance, Christophe Benoist and Diane Mathis (Institut de Génétique et de Biologie Moléculaire, Illkirch, France) for providing the male transgenic KRN male mice, and Dr. Christine Gaertner for providing the vitamin E. 18. 19. 20. REFERENCES 1. Babior B. Phagocytes and oxidative stress. Am J Med 2000;109: 33–44. 2. Nurcombe H, Bucknall R, Edwards S. Neutrophils isolated from the synovial fluid of patients with rheumatoid arthritis: priming and activation in vivo. Ann Rheum Dis 1991;50:147–53. 3. Mulherin D, Thurnam D, Situnayake D. Glutathione reductase activity, riboflavin status and disease activity in rheumatoid arthritis. Ann Rheum Dis 1996;55:837–41. 4. Sklodoswka M, Gromadzinska J, Biernacka M, Wasowicz W, Wolkanin P, Marszalek A, et al. Vitamin E, thiobarbituric acid reactive substance concentrations and superoxide dismutase activity in the blood of children with juvenile rheumatoid arthritis. Clin Exp Rheumatol 1996;14:433–9. 5. Wickens D, Norden A, Lunec J, Dormandy T. Fluorescence changes in human gamma globulin induced by free radical activity. Biochim Biophys Acta 1983;742:607–14. 6. Biemond P, Swaak A, Penders J, Beindorff C, Koster JF. Superoxide production by polymorphonuclear leucocytes in rheumatoid arthritis: in vivo inhibition by the antirheumatic drug piroxicam due to interference with the activation of NADPH-oxidase. Ann Rheum Dis 1986;45:249–55. 7. Azzini M, Girelli D, Olivieri D, Guarini P, Stanzial A, Frigo A, et al. Fatty acids and anti-oxidant micronutrients in psoriatic arthritis. J Rheumatol 1995;22:103–8. 8. Harth M, Keown P, Orange J. Monocyte dependent excited 21. 22. 23. 24. 25. 26. 27. oxygen radical generation in rheumatoid arthritis: inhibition by gold sodium thiomalate. J Rheumatol 1983;10:701–7. Bendich A, Cohen M. Vitamin E, rheumatoid arthritis and other arthritic disorders. J Nutr Immunol 1996;4:47–65. McCord JM. Free radicals and inflammation protection of synovial fluid by superoxide dismutase. Science 1974;185:529–31. Darlington L, Ramsey N. Review of dietary therapy for rheumatoid arthritis. Br J Rheumatol 1993;32:507–14. Edmonds SE, Winyard P, Guo R, Kidd B, Merry P, LangrishSmith A, et al. Putative analgesic activity of repeated oral doses of vitamin E in the treatment of rheumatoid arthritis: results of a prospective placebo controlled double blind trial. Ann Rheum Dis 1997;56:649–55. Aruoma O, Halliwell B, Hoey B, Butler J. The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide and hypochlorous acid. Free Radic Biol Med 1989;6:593–7. Iwakura Y, Tosu M, Yoshida E, Takiguchi M, Sato K, Kitajima I, et al. Induction of inflammatory arthropathy resembling rheumatoid arthritis in mice transgenic for HTLV-1. Science 1991;253: 1026–8. Keffer J, Probert L, Cazlaris H, Georgopoulos S, Kaslaris E, Kioussis D, et al. Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis. EMBO J 1991;10:4025–31. Santos L, Tipping P. Attenuation of adjuvant arthritis in rats by treatment with oxygen radical scavengers. Immunol Cell Biol 1994;72:406–14. Trentham D. Autoimmunity to type II collagen: an experimental model of arthritis. J Exp Med 1977;146:857–68. Blumberg JB. Considerations of the recommended dietary allowances for older adults. Clin Appl Nutr 1991;1:9–16. Belmatoug N, Cremieux A, Bleton R, Volk A, Saleh-Mghir A, Grossin M, et al. A new model of experimental prosthetic joint infection due to methicillin-resistant Staphylococcus aureus: a microbiologic, histopathologic, and magnetic resonance imaging characterization. J Infect Dis 1996;174:414–7. De Bandt M, Grossin M, Weber A, Chopin M, Elbim C, Pla M, et al. Suppression of arthritis and protection from bone destruction by treatment with TNF-470/AGM-1470 in a transgenic mouse model of rheumatoid arthritis. Arthritis Rheum 2000;43:2056–63. Browne RW, Armstrong D. Reduced glutathione and glutathione disulfide. Methods Mol Biol 1998;108:347–52. Morrow J, Hill K, Burk R, Nammour T, Badr KF, Roberts L. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc Natl Acad Sci U S A 1990;87:9383–7. McLemore J, Beeley P, Thorton K, Morrisroe K, Blackwell W, Dasgupta A. Rapid automated determination of lipid hydroperoxide concentrations and total antioxidant status of serum samples from patients infected with HIV: elevated lipid hydroperoxide concentrations and depleted total antioxidant capacity of serum samples. Am J Clin Pathol 1998;109:268–73. Kouskoff V, Korganoff A, Duchatelle V, Degott C, Benoist C, Mathis D. Organ specific disease provoked by systemic autoimmunity. Cell 1996;87:811–22. Dinarello CA. The role of Interleukin-1-receptor antagonist in blocking inflammation mediated by interleukin-1. N Engl J Med 2000;343:732–4. Bondenson J, Brennan F, Foxwell B, Feldmann M. Effective transfer of IB␣ into human fibroblast and chondrosarcoma cells reveals that the induction of matrix metalloproteases and proinflammatory cytokines is nuclear factor-B dependent. J Rheumatol 2000;27:2078–89. Brigelius-Flohe R, Traber MG. Vitamin E: function and metabolism. FASEB J 1999;13:1145–55. 532 28. Burton GW, Ingold K. Vitamin E as an in vitro and in vivo antioxidant. Ann N Y Acad Sci 1989;570:7–22. 29. Pratico D, Tangirala RK, Rader D, Rokach J, FitzGerald G. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in Apo E–deficient mice. Nat Med 1998;4:1189–92. 30. Comstock G, Burke A, Hoffman S, Helzlsouer K, Bendich A, Masi A, et al. Serum concentrations of ␣-tocopherol, ␤-carotene and retinol binding protein in the diagnosis of RA and systemic lupus erythematosus. Ann Rheum Dis 1997;56:323–5. 31. Cachia O, El Benna J, Pedruzzi E, Descomps B, GougerotPocidalo MA, Leger C. Alpha-tocopherol inhibits the respiratory burst in human monocytes: attenuation of p47phox membrane translocation and phosphorylation. J Biol Chem 1998;273: 32801–5. 32. Azzi A, Aratri E, Boscoboinik D, Clement S, Ozer NK, Ricciarelli DE BANDT ET AL 33. 34. 35. 36. R, et al. Molecular basis of ␣-tocopherol control: role of protein kinase C. Biofactors 1998;7:3–14. Boscoboinik D, Szewczyk A, Hensey C, Azzi A. Inhibition of cell proliferation by ␣-tocopherol: role of protein kinase C. J Biol Chem 1991;266:6188–94. Devaraj S, Li D, Jialal I. The effects of ␣-tocopherol supplementation on monocyte function: decreased lipid oxidation, interleukin-1␤ secretion, and monocyte adhesion to endothelium. J Clin Invest 1996;98:756–63. Devaraj S, Jialal I. Alpha-tocopherol decreases interleukin-1␤ release from activated human monocytes by inhibition of 5-lipoxygenase. Arterioscler Thromb Vasc Biol 1999;19:1125–33. Zimmer S, Stocker A, Sarbolouki ML, Spycher S, Sassoon J, Azzi A. A novel human tocopherol-associated protein. J Biol Chem 2000;275:25672–80.