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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 1088 © 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. 1089 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 1091 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%. 1092 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. 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