Download Apocynin, a plant-derived, cartilage-saving drug, might be useful in

Rheumatology 1999;38:1088–1093
Apocynin, a plant-derived, cartilage-saving drug,
might be useful in the treatment of rheumatoid
arthritis
F. P. J. G. Lafeber, C. J. Beukelman1, E. van den Worm1,
J. L. A. M. van Roy, M. E. Vianen, J. A. G. van Roon,
H. van Dijk1 and J. W. J. Bijlsma
Department of Rheumatology and Clinical Immunology, University Medical
Centre Utrecht, PO Box 85500, 3508 GA Utrecht and 1Department of
Medicinal Chemistry, Faculty of Pharmacy, Utrecht University, The Netherlands
Abstract
Objective. To investigate whether apocynin, 1-(4-hydroxy-3-methoxyphenyl )ethanone, is
able to diminish inflammation-induced cartilage destruction in rheumatoid arthritis (RA),
studied in a human in vitro model.
Methods. Apocynin was added to cultures of RA peripheral blood mononuclear cells
(PBMNC ). Cartilage-destructive activity was determined by addition of culture supernatant
to tissue samples of human articular cartilage. In addition, the proliferation of PBMNC, their
production of tumour necrosis factor alpha ( TN-Fa), interleukin (IL)-1 and IL-10, and T-cell
production of interferon gamma (IFN-c) and IL-4, as measures for T1 and T2 cell activity,
were determined.
Results. Apocynin was able to counteract RA PBMNC-induced inhibition of cartilage
matrix proteoglycan synthesis, while no effect on inflammation-enhanced proteoglycan release
was found. The effect was accompanied by a decrease in IL-1 and TNF-a production by the
MNC. No effect on T-cell proliferation was found, but the production of IFN-c, IL-4 and
T-cell-derived IL-10 was strongly diminished. Most important, apocynin did not show any
direct adverse effects on chondrocyte metabolism; on the contrary, it diminished the release of
proteoglycans from the cartilage matrix.
Conclusion. Apocynin in vitro inhibits inflammation-mediated cartilage destruction without
having adverse effects on cartilage. The latter may be an advantage of apocynin over many
other non-steroidal anti-inflammatory drugs. Therefore, apocynin might have an added
beneficial effect in protecting RA patients from joint destruction.
K : Apocynin, Rheumatoid arthritis, Non-steroidal anti-inflammatory drugs, Cartilage.
Non-steroidal anti-inflammatory drugs (NSAIDs) are
widely used in the treatment of rheumatoid arthritis
(RA). Clinically, analgesic and anti-inflammatory effects
have been clearly demonstrated and do not deviate
much between the different NSAIDs [1, 2]. However,
during long-term use, as in chronic disorders such as
RA, unfortunately most can give rise to side-effects in
the gastrointestinal tract [3]. Moreover, several of them,
when tested in animal and in vitro models for joint
destruction, have been reported to have direct adverse
effects on cartilage homeostasis [4, 5]. Much effort is
being put into the development of NSAIDs which
exhibit less or no gastrointestinal side-effects (e.g. the
Submitted 17 March 1999; revised version accepted 30 April 1999.
Correspondence to: F. P. J. G. Lafeber, Department of Rheumatology and Clinical Immunology (F02.127), University Medical Centre
Utrecht, PO Box 85500, 3508 GA Urecht, The Netherlands.
selective cyclooxygenase inhibitors; [6 ]) and which lack
adverse effects on cartilage [7]. The latter may be most
important when such drugs are used in the treatment of
joint disorders in which the inflammatory component is
not the primary cause of cartilage damage, such as in
osteoarthritis [8]. Therefore, there is a need for analgesic
products with anti-inflammatory properties having less
or no side-effects, in particular on cartilage metabolism,
when used for long-term treatment.
Plant extracts, in this respect, have been tested for
their anti-inflammatory activity [9–11] and have been
used in the treatment of experimental arthritis in
rats [12]. Apocynin, 1-(4-hydroxy-3-methoxyphenyl )ethanone, is a plant-derived drug, discovered during
activity-guided isolation of immunomodulatory constituents from Picrorhiza kurroa. The generation of
reactive oxygen species (ROS ) by serum-treated zymosan-triggered human neutrophils from healthy blood
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© 1999 British Society for Rheumatology
Apocynin in treatment of rheumatoid arthritis
donors was inhibited by apocynin in a dose-dependent
manner (50% inhibition at ~6 m [13, 14]). The mode
of action may involve (myeloperoxidase-dependent)
metabolization and inhibition of NADPH assembly by
interfering with the intracellular translocation of two
cytosolic components, p47-phox and p67-phox [15]. In
addition, apocynin is anti-inflammatory as it interferes
with arachidonic acid metabolism [16 ]. In in vivo experiments, apocynin was effective at low daily doses. Upon
oral administration ( lowest mean daily intake 24 mg/kg),
anti-arthritic activity was observed in collagen-induced
arthritis in rats [17]. An important observation was that
apocynin dose dependently inhibited tumour necrosis
factor ( TNF ) release from human adherent mononuclear cells induced by either lipopolysaccharide or
peptidoglycan [18]. In an ongoing phase I clinical study,
apocynin is being tested for the treatment of lung
emphysema. The patients received, during 4 days, four
daily dosages of 3 ml (1 mg apocynin/ml ) by inhalation;
so far, no side-effects, including gastrointestinal effects,
of this treatment have been observed (J. Stolk and
J. Brahim, Pulmonology, Leiden University Hospital,
The Netherlands, personal communication).
In the present study, the ability of apocynin to diminish inflammation-induced cartilage destruction in
rheumatoid arthritis (RA) is evaluated using a human
ex vivo model. In addition to the anti-inflammatory
properties, attention was given to direct effects of apocynin on chondrocyte metabolism.
Materials and methods
Experimental design
Apocynin was obtained from Roth GmbH ( Karlsruhe,
Germany) and was recrystallized in methanol before
use. It was added in concentrations varying from 1 to
100 mg/ml to cultures of peripheral blood mononuclear
cells (PBMNC ) obtained from RA patients. Cartilagedestructive activity was determined by the addition of
culture supernatant (2.5% v/v) to 4 day cultures of
human articular cartilage (after 1 day of pre-culture and
medium refreshment). In addition, apocynin (30 mg/ml )
was added directly to cartilage cultures without MNC
culture supernatants. Proteoglycan synthesis and release,
as measures of cartilage matrix synthesis and matrix
loss, were determined.
The effect of apocynin on T-cell proliferation was
assessed in quadruple per donor by [3H ]thymidine
incorporation during the last 18 h of culture according
to standard procedures. The production of TNF-a,
interleukin (IL)-1 and IL-10 by PBMNC was determined
in the 4 day culture supernatants of the cells. Moreover,
after additional T-cell stimulation (anti-CD3/anti-CD28;
[19]) during the last 48 h of the 4 day cell culture, the
T-cell cytokines interferon gamma (IFN-c) and IL-4,
as estimates of T1 or T2 cell activity, and T-cellstimulation-dependent IL-10 production, were measured
in supernatants.
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Mononuclear cell cultures
PBMNC were isolated from six patients with RA [21].
RA was defined by the 1987 ACR criteria; all patients
(two males and four females, aged 60.0 ± 13.0 yr) were
rheumatoid factor positive. Blood was diluted 1:1 with
Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco
074-01600) containing 0.81 m SO2 − , supplemented
with glutamine (2 m), penicillin (1004 IU/ml ) and streptomycin sulphate (100 mg/ml; DMEM+). MNC were
isolated by density centrifugation using Ficoll-Paque
(Pharmacia, Uppsala, Sweden). The viability of the
cells, checked by trypan blue exclusion, always exceeded
95%. Subsequently, MNC were cultured (0.5 × 106
cells/ml ) in 96 well plates in DMEM+ supplemented
with 10% human male AB+ serum (Red Cross Blood
Transfusion Centre, Utrecht, The Netherlands). Part of
the MNC cultures were performed in the presence of a
bacterial antigen (mycobacterial 60 kDa heat shock protein; 5 mg/ml ) to give the cells an extra stimulus ( T1 cell
directed ) in an attempt to mimic the conditions in the
rheumatoid joint [19–21].
Cartilage-destructive activity
Human knee cartilage, normal healthy as checked by
histology, was obtained at autopsy from six donors
(mean age 53 ± 16 yr). Cartilage was cut aseptically, as
thick as possible, excluding the subchondral bone, and
was kept in phosphate-buffered saline (PBS; pH 7.4).
Within 1 h after dissection, the slices were cut into
square pieces weighing 10–15 mg, which were cultured
for 4 days in round bottom 96 well microtitre plates
(200 ml culture medium/well, 37°C, 5% CO in air). The
culture medium was DMEM (Gibco, 2 074-01600)
containing 0.81 m SO2 − , supplemented with ascorbic
4
acid
(0.85 m),
glutamine
(2 m),
penicillin
(100 IU/ml ), streptomycin sulphate (100 mg/ml ) and
10% heat-inactivated pooled human male adult-derived
AB+ serum [22–24]. Proteoglycan synthesis rate and
release, as measures for cartilage matrix synthesis and
loss, were determined as follows.
Proteoglycan synthesis rate. As a measure of chondrocyte proteoglycan synthetic activity, the sulphate incorporation rate into proteoglycans was determined using
35SO2− as a tracer (Dupont, Nex-041-H, carrier free,
4
2 mCi/well
[22–24]). The sulphate incorporation rate
was determined during the last 4 h of a 4 day culture
and was calculated from the 35SO2− incorporation rate
4 medium, and was
and the specific activity of the culture
expressed as nanomoles of sulphate incorporated per
hour per gram wet tissue weight (nmol/h/g).
Proteoglycan release. After 4 days of culture, the
concentrations of glycosaminoglycan (GAG) in culture
media were determined as a measure of proteoglycan
release. GAG was precipitated and stained with Alcian
Blue and measured as described previously [22–24].
GAG release was expressed as milligrams of GAG per
gram wet cartilage tissue weight (mg/g).
Cytokine measurements
Supernatants of MNC cultures were harvested and freed
from cellular material by centrifugation (5 min, 450 g),
1090
F. P. J. G. Lafeber et al.
(a)
(b)
F. 1. (a) Proteoglycan synthesis of cartilage as measured by sulphate incorporation rate after 4 days of culture (n = 6). Basic
proteoglycan synthesis was 15.2 nmol/h/g; after addition of apocynin (30 mg/ml ), this was 15.1 nmol/h/g (not statistically
significantly different). The effect of culture supernatants of RA peripheral blood mononuclear cells (PBMNC ) in a concentration
of 2.5% (v/v) resulted in a synthesis of 8.1 nmol/h/g [statistically significantly lower compared to control (P ∏ 0.02)], and culture
supernatants of RA PBMNC stimulated with mycobacterial heat shock protein (5 mg/ml ) gave a synthesis of 3.7 nmol/h/g, even
lower (P ∏ 0.0003). Supernatants of RA PBMNC cultured in the presence of apocynin (1–100 mg/ml ) were less potent in this
respect, showing a partial to complete restoration of proteoglycan synthesis. This effect was dose dependent and irrespective of
stimulation of MNC (open symbols, unstimulated MNC; filled symbols, stimulated MNC; *P ∏ 0.05). Conditions in the absence
of apocynin are set at 100%. (b) Proteoglycan loss from cartilage as measured by glycosaminoglycan (GAG) release during
4 days of culture (n = 6). Background release was 1.6 mg/g; in the presence of apocynin (30 mg/ml ), this was 1.3 mg/g
[statistically significantly lower (P ∏ 0.002)]. Culture supernatants of RA peripheral blood mononuclear cells (PBMNC ) in a
concentration of 2.5% (v/v) and culture supernatants of RA PBMNC stimulated with mycobacterial heat shock protein (5 mg/ml )
resulted in proteoglycan release of 1.8 and 2.7 mg/g, respectively, the latter being elevated compared to controls (P ∏ 0.01).
Cells, whether stimulated or not, were not affected by apocynin (1–100 mg/ml ) in this respect: culture supernatants did not
change their effect on proteoglycan release whether or not cells had been treated with apocynin. Conditions in the absence of
apocynin are set at 100%.
frozen in liquid nitrogen, and stored at −20°C for no
longer than 3 months. All cytokines were determined
by ELISA (Medgenix, Flerus, Belgium). Detection limits
were 30, 10, 50, 10 and 10 pg/ml for IL-1, TNF-a, IL-10,
IFN-c and IL-4, respectively.
Calculations and statistical analysis
Median values of six donors are given. For statistical
evaluation, a paired non-parametric test of the absolute
values was used. P values of ∏0.05 were considered
statistically significant.
Results
Effects of apocynin on cartilage and RA MNC-induced
cartilage damage
Culture supernatants of RA PBMNC, at a concentration
of 2.5% v/v, were able to inhibit proteoglycan synthesis
by >43% (P ∏ 0.02; from 15.2 to 8.1 nmol/h/g).
Stimulation of MNC during culture with a bacterial
antigen resulted in culture supernatants which were even
more potent in this respect, reducing synthesis from 15.2
to 3.7 nmol/h/g (P ∏ 0.0003), representing 75% inhibition. When MNC were cultured in the presence of
apocynin, the inhibition was less pronounced. This
cartilage-saving effect in the case of inflammation
appeared to be dose dependent and was independent of
activation of the cells (Fig. 1a, open and solid symbols
for non-stimulated and stimulated cells, respectively).
Apocynin, tested in an effective concentration of
30 mg/ml, directly on human articular cartilage, did not
change proteoglycan synthesis (15.1 nmol/h/g).
With respect to cartilage matrix loss, there appeared
to be a direct protective effect of apocynin (30 mg/ml ).
Release of GAGs was statistically significantly inhibited
in the presence of apocynin, on average by >35%
(P ∏ 0.002); background release was 1.6 mg/g, with
apocynin this was 1.3 mg/g. After addition of RA MNC
supernatants, GAG release was enhanced only when the
cells had been stimulated (P ∏ 0.01); release unstimulated 1.8 mg/g and stimulated 2.7 mg/g. Apocynin was
unable to change the potential of the cells, activated or
not, to induce cartilage proteoglycan release ( Fig. 1b,
open and solid symbols for non-stimulated and stimulated cells, respectively).
Apocynin in treatment of rheumatoid arthritis
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Effects on pro-inflammatory cytokine production
The stimulation of RA PBMNC strongly enhanced the
production of pro-inflammatory cytokines IL-1 (8 and
460 pg/ml, respectively) and TNF-a (20 and 1230 pg/ml,
respectively). The production of IL-10, even after antigen stimulation, remained below the detection limit
(data not shown). The production of IL-1 and TNF-a
by bacterial antigen-stimulated MNC could be suppressed in a dose-dependent fashion by the addition of
apocynin. At a concentration of 100 mg/ml, inhibition
exceeded 50% for both IL-1 (triangles) and TNF-a
(dots; Fig. 2).
Effects on T-cell cytokines
The T1 and T2 cell cytokines IFN-c and IL-4 were
below the detection limit when estimated in culture
supernatants of both unstimulated and bacterial antigenstimulated RA PBMNC after 4 days of culture (data
not shown). A mitogenic T-cell stimulus (anti-CD3/antiCD28 antibodies) added during the last 48 h of the
MNC culture made IFN-c and IL-4 activity detectable
(6 and 3 ng/ml, respectively). When cells had been
stimulated with bacterial antigen, the production of
IFN-c was significantly enhanced (19 ng/ml; P ∏ 0.01),
whereas IL-4 was statistically unchanged (9 ng/ml ).
Apocynin diminished the production of both T-cell
cytokines significantly in a dose-dependent manner. The
effect was irrespective of antigen stimulation and comparable for IL-4 and IFN-c (Fig. 3a). The production
(a)
(b)
F. 2. Pro-inflammatory cytokine (IL-1 and TNF-a) production by RA peripheral blood mononuclear cells. Production
of both cytokines was relatively low when cells were not
stimulated: 8 and 20 pg/ml, respectively. Upon stimulation,
however, significant amounts of IL-1 and TNF-a were released
(460 and 1230 pg/ml, respectively). Apocynin significantly
inhibited the release of both TNF-a (dots) and IL-1 (triangles)
in a dose-dependent manner (*P ∏ 0.05). Conditions in the
absence of apocynin are set at 100%.
F. 3. Cytokine [(a) IFN-c and IL-4; (b) IL-10] production upon anti-CD3/CD28 stimulation, giving rise to T-cell
cytokine production. Production of IFN-c and IL-4 by
unstimulated RA peripheral blood mononuclear cells
(PBMNC ) was 6 and 3 ng/ml, respectively. Bacterial antigen
stimulation of RA PBMNC resulted in 9 and 19 ng/ml of the
cytokines, respectively [statistically significantly enhanced for
IFN-c (P ∏ 0.01)]. IL-10 production was 646 and 447 pg/ml
for unstimulated and stimulated cells, respectively (not statistically significantly different). Apocynin significantly and similarly inhibited the production of IFN-c [circles; (a)], IL-4
[triangles; (a)] and IL-10 [circles; (b)] in a dose-dependent
manner (*P ∏ 0.05). The inhibition of these cytokines was
independent of the antigen stimulation of MNC (unstimulated and stimulated cells open and filled symbols, respectively). T-cell-derived IL-10 production was almost completely
inhibited. Conditions in the absence of apocynin are set
at 100%.
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F. P. J. G. Lafeber et al.
of IL-10 upon additional T-cell stimulation was slightly
lower when cells were stimulated with bacterial antigen
(646 and 447 pg/ml; not statistically significant).
Apocynin dose-dependently inhibited IL-10 production
similarly in the case of unstimulated and antigenstimulated RA PBMNC (Fig. 3b).
Discussion
From a clinical point of view, NSAIDs have proven to
be useful analgetic and anti-inflammatory drugs in the
treatment of inflammation-mediated joint damage such
as RA, but also for other forms of joint damage in
which inflammation is a secondary phenomenon, like in
osteoarthritis [8]. However, it has been suggested that
the symptomatic benefits of NSAIDs may occur at the
expense of joint cartilage integrity [25]. Moreover,
adverse effects of NSAIDs on degenerated cartilage due
to inflammatory processes or osteoarthritis may evolve
more rapidly than on normal cartilage [26 ]. Damaged
cartilage may be particularly vulnerable to catabolic
elements. Besides clinical outcome, it is therefore important to ascertain that NSAIDs have no adverse effects
on articular cartilage itself.
The potential NSAID studied here, apocynin, obviously meets a number of important criteria. In the first
place, it was able to counteract the inflammation-related
inhibition of cartilage matrix synthesis. The observed
inhibition of IL-1 and TNF-a production is fully in line
with the described anti-inflammatory activities [17, 18].
Interestingly, although T-cell cytokine production was
strongly inhibited by apocynin, T-cell proliferation,
which was mildly enhanced upon bacterial antigen
stimulation (on average ~3 times), was slightly stimulated by apocynin: an increase of only 55% in
[3H ]thymidine incorporation was observed (not statistically significant; data not shown). Dissociation of T-cell
proliferation and cytokine production upon antigen
stimulation have been described by others as well [28].
The bacterial antigen stimulation caused elevated IFN-c
production without affecting IL-4 and decreased IL-10
production, changes to be expected for T1 cell-directed
bacterial antigen stimulation [20]. Studies of adverse
effects of NSAIDs on cartilage metabolism up to now
have not included effects on the balance between T1
and T2 cells. This balance seems to be important with
respect to the degree of joint damage occurring in RA
[20, 21]. On the basis of the IFN-c/IL-4 ratio, it is
concluded that the balance between T1 and T2 cell
activity remained unchanged. This effect was irrespective
of bacterial antigen stimulation. The inhibitory effect on
T-cell cytokine production, including T-cell-dependent
IL-10 production, without interference with T-cell proliferation, might be caused by induction of regulatory
T cells producing suppressive cytokines such as transforming growth beta ( Th3 cells). The anti-inflammatory
activity of such T cells has been described [27].
Involvement of other suppressive cytokines such as
IL-13 and IL-16 cannot be excluded in this respect.
Independent of the possible effects on T-cell activity,
apocynin directly affects monocyte activity [18], which
may influence T-cell activity in the way observed.
Most important, apocynin in vitro does not adversely
affect chondrocyte metabolism (as based on proteoglycan turnover). On the contrary, it has a cartilagesaving effect in reducing matrix loss from normal cartilage without affecting synthesis. Apart from in vitro and
animal experiments [4, 5], a clinical study including
patients with knee osteoarthritis also showed negative
effects of NSAID use on cartilage [28]. Taking into
account that in (emphysema) patients no side-effects of
apocynin treatment were observed (see Introduction),
we conclude that apocynin, with its anti-inflammatory
and cartilage-protecting properties, is a drug which
deserves further in vivo study to test its suitability for
long-term treatment of chronic inflammatory and degenerative joint diseases.
Acknowledgement
The authors are indebted to their colleagues at the
Department of Pathology of the University Hospital of
Utrecht for their skilful cartilage supply.
References
1. McCormak K, Urquhart E. Correlation between nonsteroidal anti-inflammatory drug efficacy in a clinical pain
model and dissociation of their anti-inflammatory and
analgesic properties in animal models. Clin Drug Invest
1995;9:88–97.
2. Wright V. Historical overview of non-steroidal antiinflammatory drugs. Br J Rheumatol 1995;34(suppl. 1):
2–4.
3. Simon LS. Actions and toxicity of non-steroidal antiinflammatory drugs. Curr Opin Rheumatol 1996;8:169–75.
4. Bassleer C, Magotteaux J, Geenen V, Malais M. Effects
of meloxicam compared to acetylsalicylic acid in human
articular chondrocytes. Pharmacology 1997;54:49–56.
5. Rainsford KD, Yung C, Smith FC. Effects of meloxicam
compared with NSAIDs, on cartilage proteoglycan metabolism, synovial prostaglandin E and production of
2
interleukins 1, 6 and 8, in human and porcine explants in
organ culture. J Pharm Pharmacol 1997;49:991–8.
6. Simon LS. Biology and toxic effects of nonsteroidal antiinflammatory drugs. Curr Opin Rheumatol 1998;10:153–8.
7. Mauviel A, Redini F, Loyan G, Pujol JP. Modulation
of extracellular matrix metabolism in rabbit articular
chondrocytes and human rheumatoid synovial cells by the
non-steroidal anti-inflammatory drug etodolac. I collagen
synthesis. Agents Actions 1990;31:345–52.
8. Oddis CV. New perspectives on osteoarthritis. Am J Med
1996;100:10S–5S.
9. Duwiejua M, Zeitlein JJ, Waterman PG, Chapman J,
Mhango GJ, Provan GJ. Anti-inflammatory activity of
resins of some species of the plant family Burseraceae.
Planta Med 1993;59:12–6.
10. Perez RM, Perez S, Zavala MA, Salaza M. Antiinflammatory activity of the bark of Hipporatea excela.
J Echnopharmacol 1995; 47:85–90.
11. Shinde UA, Phadke AS, Nair AM, Mungantiwar AA,
Dikshit VJ, Saraf MN. Studies on the anti-inflammatory
Apocynin in treatment of rheumatoid arthritis
12.
13.
14.
15.
16.
17.
18.
19.
20.
and analgesic activity of Cedrus deodava (Roxb.) Lond.
wood oil. J Ethnopharmacol 1999;65:21–7.
Kim SY, Son KH, Chang HW, Kang SS, Kim HP.
Inhibitory effects of plant extracts in adjuvant-induced
arthritis. Arch Pharm Res 1997;20:313–7.
Simons JM, ’t Hart BA, Ip Vai Ching RAM, Van Dijk H,
Labadie RP. Metabolic activation of natural phenols into
selective oxidative burst agonists by activated human
neutrophils. Free Rad Biol Med 1990;8:251–8.
Beukelman CJ, Van den Berg AJJ, Kroes BH, Labadie
RP, Mattsson EE, Van Dijk H. Plant-derived metabolites
with synergistic antioxidant activity. Immunol Today
1995;16:108.
Stolk J, Hiltermann JTN, Dijkman JH, Verhoeven
AJ. Characteristics of the inhibition of NADPH oxidase
activation in neutrophils by apocynin a methoxy sustituted
catechol. Am J Respir Cell Mol Biol 1994;11:95–102.
Engels F, Renirie BF, ’t Hart BA, Labadie RP, Nijkamp
FP. Effects of apocynin, a drug isolated from the roots of
Picrorhiza kurroa, on arachidonic acid metabolism. FEBS
Lett 1992;305:254–6.
’t Hart BA, Simons JM, Knaan-Shanzer S, Bakker NPM,
Labadie RP. Anti-arthritic activity of the newly developed
neutrophil oxidative burst antagonist apocynin. Free Rad
Biol Med 1990;9:127–31.
Mattsson E, van Dijk H, van Kessel K, Verhoef J, Fleer A,
Rollof J. Intracellular pathways involved in tumor necrosis
factor alpha release by human monocytes on stimulation
with lipopolysaccharide or staphylococcal peptidoglycan
are partly similar. J Infect Dis 1996;173:212–8.
Van Roon JAG, van Eden W, van Roy JLAM, Lafeber
FPJG, Bijlsma JWJ. Stimulation of suppressive T cell
responses by human but not bacterial 60kD heat-shock
protein in synovial fluid of patients with rheumatoid
arthritis. J Clin Invest 1997;100:459–63.
Van Roon JAG, van Roy JLAM, Duits A, Lafeber FPJG,
Bijlsma JWJ. Proinflammatory cytokine production and
cartilage damage due to rheumatoid synovial T-helper-1
21.
22.
23.
24.
25.
26.
27.
28.
1093
activation is inhibited by IL-4. Ann Rheum Dis
1995;54:836–40.
Van Roon JAG, Verhoef CM, van Roy JLAM, GmeligMeyling FHJ, Huber-Bruning O, Lafeber FPJG et al.
Decrease in peripheral type 1 over type 2 T cell cytokine
production in patients with rheumatoid arthritis correlates
with an increase in severity of disease. Ann Rheum Dis
1997;56:600–60.
Lafeber FPJG, van der Kraan, PM, van Roy JLAM,
Huber-Bruning O, Bijlsma JWJ. Articular cartilage explant
culture; an appropriate in vitro system to compare
osteoarthritic and normal human cartilage. Conn Tiss Res
1993;29:287–99.
Lafeber FPJG, van der Kraan PM, Huber-Bruning O,
van der Berg WB, Bijlsma JWJ. Osteoarthritic human
cartilage is more sensitive to transforming growth factor
b than is normal cartilage. Br J Rheumatol 1993;32:281–6.
Lafeber FPJG, Veldhuijzen JP, van Roy JLAM, HuberBruning O, Bijlsma JWJ. Intermittent hydrostatic compressive force stimulates exclusively the proteoglycan synthesis of osteaorthritic human cartilage. Br J Rheumatol
1992;31:437–42.
Johnston SA, Budsberg SC. Nonsteroidal antiinflammatory drugs and corticosteroids for the management of canine osteoarthritis. Vet Clin North Am Small
Anim Pract 1997;27:841–62.
Palmoski MJ, Brandt KD. In vivo effect of aspirin on
canine osteoarthritic cartilage. Arthritis Rheum 1983;
26:994–1001.
Fukaura H, Kent SC, Pietrusewiez MJ, Khury SJ, Weiner
HL, Hafler DA. Induction of circulating myelin basic
protein and proteolipid specific TGFb1 secreting Th3 cells
by oral administration of myelin in multiple sclerosis
patients. J Clin Invest 1996;98:70–7.
Huskisson EC, Berry H, Gishen P, Jubb RW, Whitehead J,
on behalf of the LINK study group. Effects of antiinflammatory drugs on progression of osteoarthritis of the
knee. J Rheumatol 1995;22:1941–6.