close

Вход

Забыли?

вход по аккаунту

?

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: debandt@bichat.inserm.fr.
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 I␬B␣ 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.
Документ
Категория
Без категории
Просмотров
5
Размер файла
1 856 Кб
Теги
mode, vitamins, destruction, inflammation, transgenic, clinical, joint, mouse, arthritis, uncoupled, rheumatoid
1/--страниц
Пожаловаться на содержимое документа