Corticosteroid Treatment Induces Chondrocyte Apoptosis in an Experimental Arthritis Model and in Chondrocyte Cultures F

Home , Dexamethasone acefurate

Corticosteroid treatment induces chondrocyte apoptosis in an experimental arthritis model and in chondrocyte cultures F. Nakazawa, H. Matsuno, K. Yudoh,Y. Watanabe1, R. Katayama, T. Kimura

Department of Orthopedic Surgery, Toyama Medical and Pharmaceutical University, Toyama; 1Department of Clinical Biochemistry, Hokkaido Institute of Pharmaceutical Sciences, Katsuraoka, Japan.

Abstract Object In order to examine the mechanisms involved in steroid-induced arthropathy after intra-articular corticosteroid injection, a histological examination was performed in vivo using severe combined immunodeficiency (SCID) mice that were implanted with human articular cartilage into the back (SCID/hu model). In addition, the effect of corticosteroids on chondrocyte apoptosis was evaluated in vitro using cultured human chondrocytes.

Method Human articular cartilage was obtained during knee surgery and implanted subcutaneously into the backs of SCID mice. One month later, weekly injections of corticosteroid (hydrocortisone acatate: 1mg/0.2ml, triamcinolone acetonide: 0.2mg/0.2ml, dexamethasone acetate: 0.1mg/0.2ml) in the subcutaneous cavity around the grafted cartilage in SCID mice were initiated. After six weeks of treatment, the grafted cartilage pieces were removed from the SCID mice and examined histologically. Chondrocyte apoptosis after corticosteroid treatment was also investigated using cultured human chondrocytes.

Result In the corticosteroid treated, grafted articular cartilage, apoptotic chondrocytes were apparent in the superficial and middle layers of cartilage. But a reduced intensity of Safranin O staining was not remarkable. In the cultured chondrocytes, apoptotic changes were also observed after corticosteroid treatment.

Conclusion Corticosteroid treatment induces chondrocyte apoptosis and it may be important to understand the steroid-induced arthropathy.

Key words Corticosteroid, apoptosis, chondrocyte, SCID/hu model.

Clinical and Experimental Rheumatology 2002; 20: 773-781. Steroid-induced chondrocyte apoptosis / F. Nakazawa et al.

Fujio Nakazawa, MD; Hiroaki Matsuno, Introduction Japan. triamcinolone acetonide: 0.2mg/ MD; Kazuo Yudoh,MD; Yasuhiro Watan- Corticosteroid injections are frequently 0.2ml Sankyo Co. Tokyo, Japan. dex- abe, MD; Rie Katayama, MD; Tomoatsu used to achieve local anti-inflammatory amethasone acetat e : 0 . 1 m g / 0 . 2 m l , Kimura, MD. activity (1). And it is well known that B a nyu Co. To kyo Japan) injections Please address correspondence and rep e ated intra - a rticular cort i c o s t e ro i d were performed to the rabbit (Japanese reprint requests to: Fujio Nakazawa MD, therapy interferes with cartilage metab- white rabbit 2 mothes old: Japan Clea Department of Orthopedic Surgery, Toya- ma Medical and Pharmaceutical Universi- olism and may lead to rapid destruction Inc., Tokyo, Japan) knee for six weeks. ty, 2630 Sugitani, Toyama 930-0194, of the joint. In one of the most famous Each corticosteroid was injected to 3 Japan. E-mail: [email protected] t ext-book of rheumat o l ogy, c o rt i c o s- rabbit in the right knee, and the left Received on February 25, 2002; accepted t e roid art h ro p at hy was descri b e d. It knee was used for control. Three days in revised form on September 19, 2002. was printed that abuse of intra-articular after the final injection, the art i c u l a r © Copyright CLINICAL AND injections may result in a Charcot’s- cartilage were extirpated and used for EXPERIMENTAL RHEUMATOLOGY 2002. l i ke art h ro p at hy (2). Cort i c o s t e ro i d s the histological investigation of chon- exe rt powerful anti-infl a m m at o ry drocyte apoptosis and matrix degrada- effects through inhibition of the arachi- tion. donic acid cascade. However, the phar- m a c o l ogical mechanism of cort i c o s- Transplantation of human articular t e roid-induced art h ro p at hy re m a i n s cartilage to the back of the SCID unclear (3-5). mouse (SCID/hu model) Recently, corticosteroids were reported The SCID/hu model, which we have to induce the apoptosis of lymphocytes, previously described (14-16), was used e o s i n o p h i l s , bone and cart i l age of for the study of cort i c o s t e roid tre at- growth plate (6-8). These results sug- ment. Bri e fly, male SCID mice gest that similar corticosteroid-induced ( C B.17/lcr; Japan Clea Inc. , To kyo , apoptosis may occur in articular carti- Japan), 6-7 weeks old and bred under l age. Chondro cyte apoptosis has specific pathogen-free conditions at our already been reported to play important u n ive rsity animal center, we re used. roles in cartilage degradation and in the Articular cartilage, collected at the time pathogenesis of joint cartilage degener- of total knee arthroplasty, was used for ation (9-13). Howeve r, if cort i c o s- i m p l a n t ation. Undamaged cart i l age teroids are able to induce chondrocyte samples from non-weight beari n g apoptosis, this may explain, at least in region such as an inter-condylar part part, the pharmacological mechanism we re collected from 7 OA pat i e n t s of steroid-induced arthropathy. There- (ages were 57-68 years: average was fore, in the present study, we investigat- 63.1 years). These cartilages were usu- ed the histological changes in human ally wasted in the process of surgery. articular cartilage after repeated corti- Parts of these samples were examined c o s t e roid injections using the seve re h i s t o l ogi c a l ly and confi rmed to be combined immu n o d e fi c i e n cy (SCID) intact. All patients gave informed con- mouse in which human articular carti- sent to be tissue donors in this study. lage had been grafted (SCID/hu model) The size of the donor articular cartilage (14-16). In this model, the implanted was adjusted to a block of 5 mm across human articular cart i l age maintained prior to its subcutaneous implantation the ch a ra c t e ristics of in vivo h u m a n into SCID mice.The mice were anes- cartilage, and enabled histological ex- thetized with dimethylether, according amination of the grafted cartilage after to the guidelines established by our c o rt i c o s t e roid tre atment without any u n ive rs i t y ’s animal ethics committee, confounding mechanical influences. In and the human articular cart i l age addition we examined the induction of bl o cks we re grafted subcutaneously chondrocyte apoptosis in vitro. into their backs. All surgical pro c e- d u res we re perfo rmed under steri l e Material and methods conditions and the result of culture test Preliminary examination to invest the was negative. effect of corticosteroid on the rabbit articular cartilage Corticosteroid injection protocol Weekly corticosteroids (hydrocortisone The initial injection of corticosteroid a c e t at e : 1 m g / 0 . 2 m l , B a nyu Co. To kyo was administered one month post-

774 Steroid-induced chondrocyte apoptosis / F. Nakazawa et al. implantation, after the graft had taken 3% H2O2. The specimens we re then 12 hours, the cells in the control wells in the SCID mice. Six cartilage pieces l abeled in TdT bu ffer (30 mM Tri s - received PBS only. The concentration were acquired from 1 donor and a total HCl, pH 7.4, 140 mM sodium cacody- of cort i c o s t e roid (10- 4M) was ch o s e n of 40 mice were used in this experi- late, 1 mM cobalt chloride) and reacted fo l l owing the prior art i cle (8). To ment. Pa i rs of cort i c o s t e ro i d - t re at e d with biotiny l ated dUTP dissolved in analysis membrane changes associated and control mice were implanted with TdT buffer. Finally, the samples were with apoptosis, FITC-Annexin V bind- a rticular cart i l age from the same stained with 3,3’-diaminobenzidine. ing to the cellular membrane, and PI patient. Weekly corticosteroid (hydro- For electron microscopy, the samples staining of cellular DNA we re per- cortisone acetate: 1mg/0.2ml: 1.4 x 10- were prepared as previously described formed as follows (18). After washing 2M; triamcinolone acetonide: 0 . 2 m g / (16). Briefly, they were fixed in 0.1 M twice with PBS, 1 x 105 cells were re- 0 . 2 m l : 2.5 x 10- 3 M; dex a m e t h a s o n e cacodylated buffer containing 3% glu- suspended in binding buffer (10 mM acetate: 0.1mg/0.2ml: 1.1 x 10-3M) in- t a ra l d e hyde at 4ûC for 1 hour. A f t e r Hepes/NaOH, pH 7.4, 140 mM NaCl, jections were given in the subcutaneous subsequent incubation in 1% OsO4 for 2.5 mM CaCl2). FITCÐAnnexin V was cavity around the grafted tissue of 30 1 hour, the samples were dehydrated a dded at a final concentration of 1 SCID mice (hydrocortisone acatate: 10 and embedded in epon, then sliced to µg/ml, and 0.1 volume of PI (10 µg/ml mi c e : tr iamcinolone acetonide: 10 mice: obtain ultra-thin sections 1000Å thick. in binding buffer) was added to the cell dexamethasone acetate: 10 mice). Tri- The sections we re then stained with suspension. The mixture was incubated amcinolone acetonide (TA) is clinically uranyl acetate and lead citrate for elec- for 10 minutes in the dark at room tem- used for intra - a rticular injection in tron microscopic observation. p e rat u re, and the fl u o rescence wa s humans (1). Genera l ly, the optimal measured using a FACScan. dose of TA for treatment of arthritic Human articular chondrocytes culture To analysis the nuclear fragmentation disease has been considered to be 10- Articular cartilages were removed from with apoptosis, the cells were cultured 40 mg/1-4 ml: 2.5 x 10-2 M per single 7 OA patients. Part of each sample was with cort i c o s t e roid (hy d ro c o rt i s o n e injection in human. As a control, phos- engrafted into a SCID mouse, and the acetate, triamcinolone acetonide, dex- phate-buffered saline (PBS) alone was remaining portion of the sample was amethasone acetat e : 1 0- 4M) for 72 injected in the same manner into the used for in vitro experiments. Articular h o u rs and fi xed with 70% ethanol. other 10 SCID mice.The weekly injec- chondrocytes were isolated as reported After washing twice with PBS, 1 x 105 tions we re perfo rmed for six we e k s . previously (17), and analyses were per- cells we re stained with pro p i d i u m Three days after the final injection, the fo rmed on each sample 3 times. iodide (PI) (1 µg/ml) and the ratio of implanted tissues were removed from B ri e fly, c a rt i l age specimens we re f rag m e n t ation was measured using a mice in each treatment group, and used minced under sterile conditions and FACScan (Becton Dikinson Co., for the histological inve s t i gation of t re ated with 1 mg/ml hya l u ro n i d a s e Mountain View, CA). ch o n d ro cyte apoptosis and mat ri x (type-1 from bovine testes, Sigma, St For TUNEL staining, ch o n d ro cy t e s degradation. Louis, MO) in PBS at 37ûC for 15 min- were cultured with different doses of utes. After washing, the samples were the corticosteroid (TA: 10-7 to 10-3 M) Histological examination of implanted d i gested with 0.25% trypsin (type-1 for 72 hours. We used TA to investigate cartilage after corticosteroid treatment from bovine pancreas, Sigma) in PBS the optimal concentration of induction Upon removal, the implanted articular at 37ûC for 30 minutes, followed by chondrocyte apoptosis because TA has cartilage pieces were fixed in 4% neu- digestion in DMEM containing 0.25% been widely used to intra - a rt i c u l a r tral-buffered formaldehyde. Embedded collagenase (type II, Sigma) on a gyrat- injection. The cells in the control wells in paraffin wax, each tissue block was ing shaker, until the tissue fragments received PBS only, and stained in the cut into 6 µm sections and applied to were dissolved. Isolated cells were then same manner as for the articular carti- slides. Sections were dewaxed, hydrat- washed with DMEM and cultured in lage. ed and stained with hematoxylin and DMEM containing 10% FCS and 50 eosin (HE) and Safranin O (SO) at µg/ml of L-ascorbat e, 100 U/ml of Evaluation of the viability of cultured room temperature. Sections were also penicillin and 100 µg/ml of strep t o- chondrocytes after corticosteroid a n a ly zed using terminal deox y nu- mycin. administration (XTT assay) cl e o t i dyl tra n s fe rase (TdT)-mediat e d C h o n d ro cyte viability was measure d dUTP nick end labeling (TUNEL) Corticosteroid-induced apoptosis in using the XTT (2,3-bis [2-Methoxy-4- staining, as previously described (16). cultured chondrocytes ni t ro - 5 - s u l fo p h e ny l ] - 2 H - t e t ra zo l i u m - 5 - B ri e fly, the tissue sections we re C o rt i c o s t e roid-induced ch o n d ro cy t e carboxanilide) assay (19), as follows. dewaxed and hydrated, and the proteins apoptosis was examined using fl ow Chondrocytes (1 x 10 4 cells/well) were we re stripped off using 20 µg/ml of cytometric analysis and TUNEL stain- seeded into 96-well fl at - b o t t o m e d p roteinase K (Boehri n ge r- M a n n h e i m ing. For flow cytometric analysis, the plates, and were incubated with various Co. Ltd. Germany) at room tempera- cells were cultured with corticosteroid c o n c e n t rations of cort i c o s t e roid (TA : ture prior to TUNEL staining. Endoge- (hydrocortisone acetate, triamcinolone 10-7 to 10-3 M) for 72 hours at 37ûC in a -4 nous peroxidase was quenched with acetonide, dexamethasone: 10 M) for 5% CO2 atmosphere. The cells in the

775 Steroid-induced chondrocyte apoptosis / F. Nakazawa et al.

Fig. 1. Histological examination of the rabbit knee articular cartila ge after different corticosteroids treatment. Corticosteroid-induced arthropathy was not observed in rabbit knee during this experiment. No remarkable change was observed in these tissues by SO staining (A-D). TUNEL-positive apoptotic cells were not increased in corticosteroid treated rabbit knee (E-H) (A, E: control; B, F: hydrocortisone acetate: 1mg/0.2ml; C, G: triamcinolone acetonide: 0.2mg/0.2ml; D, H: dexamethasone: 0.1mg/0.2ml. Original magnification x100). control wells received PBS only. Next, was observed in these cartilage by SO cytes were increased in the corticos- the serum-containing medium was re- s t a i n i n g. TUNEL posotive ap o p t o t i c t e roid tre ated group compaired with moved completely, and 200 µl/well of cells were not increased in corticos- control group. (control: 0.5 ± 0.1/mm2, PBS was added. XTT (final concentra- teroid treated rabbit knee articular car- hy d ro c o rtisone acetat e : 12.1 ± 1.4/ tion 200 µg/ml) and PMS (phenazine tilage (Fig. 1). mm2, triamcinolone acetonide: 9.4 ± m e t h o s u l fat e, final concentration 5 1.2/mm2, dexamethasone acetate: 14.9 µM) we re added to each well. Th e Histological characteristics of the ± 1.7/mm2 re s p e c t ive ly : p < 0.05) chondrocytes were incubated for a fur- SCID mouse-implanted articular (Table I). The appearance of apoptotic ther 4 hours, and the absorbance at 450 cartilage chondrocytes was further confirmed by nm was measured by plate re a d e r Successful implantation of human electron microscopy (Fig. 4). Changes (BIO-RAD lab o rat o ries. Herc u l e s , articular cartilage was observed at 4 t h at are ch a ra c t e ristic of ap o p t o t i c CA). XTT, in combination with PMS weeks following implantation. As we c e l l s , i n cluding nu clear pigmentat i o n (electron-coupling agent), was metab- have reported previously (14-16), the and fragmentation and tightly packed o l i zed by mitoch o n d rial dehy d roge- implanted tissue was integrated into cytoplasmic organelles, were observed nase enzymes of metabolically active the back of the SCID mice, and the his- after corticosteroid administration. cells. The cell viability was calculated tological characteristics of the implant- as follows: Cell viability (%) = (OD ed tissue were similar to those of nor- Corticosteroid induced apoptosis 450 of test sample Ð OD 450 of back- mal cartilage (Figure 2). The articular in cultured chondrocytes ground) / (OD 450 of control sample Ð matrix was intensely stained by SO, C h o n d ro cyte apoptosis was demon- OD 450 of background) x 100. and chondrocytes were observed in the strated by FITC-Annexin V / PI double lacunae.Apoptotic chondrocytes were s t a i n i n g. Phosphat i dyl seri n e, wh i ch Statistical analysis not observed befo re cort i c o s t e ro i d binds to Annexin V, translocates from All data are expressed as means ± stan- t re atment in TUNEL staining (dat a the inner to the outer leaflet of the plas- dard error of the mean (SEM). Welch’s was not shown). ma membrane during an early event of correction test was used for statistical apoptosis. Both FITC-Annexin V and c o m p a risons between groups. A p- Histological examination of the PI negative viable cells were observed value of less than 0.05 was considered articular cartilage and occurrence in the control groups (lower left quad- to be statistically significant. of chondrocyte apoptosis after rangle of Figure 5A). FITC-Annexin V corticosteroid treatment p o s i t ive and PI negat ive ap o p t o t i c Results SO staining could not reveal proteogly- cells, demonstrating Annexin V bind- Histological examination of the rabbit can depletion at articular cartilage in ing and cytoplasmic membrane integri- knee articular cartilage after different the corticosteroid-treated animals (Fig- ty, were observed 12 hours after corti- corticosteroids treatment ures 3A-3D). c o s t e roid administration (lower ri g h t C o rt i c o s t e roid-induced art h ro p at hy As shown in Figures 3E-3L, TUNEL quadrangle of Fig. 5B-5D). These find- was not observed in rabbit knee during staining of the implanted articular car- ings were observed all of three corti- this experimant. No remarkable change tilage revealed that apoptotic chondro- costeroids (B: hydrocortisone acetate,

776 Steroid-induced chondrocyte apoptosis / F. Nakazawa et al.

C: triamcinolone acetonide, D: dexamethasone acetate: 10-4M). The induction of chondrocyte apoptosis was further confirmed by nuclear fragmentation. PI staining, with a shift to the left due to corticosteroid (hydro- c o rtisone acetat e, t riamcinolone acetonide, dexamethasone acetate: 10-4 M) a d m i n i s t rat i o n , s u ggests the ap p e a r- ance of DNA fragmentation in the cultured chondrocytes (Figs. 5E-5H). In cultured chondrocytes, the number of TUNEL positive apoptotic cells was m a rke d ly increased by cort i c o s t e ro i d (TA: 10-4M) administration compared with the control group (8.9 ± 1.2% ver- sus 0.3 ± 0.05%, respectively; p< 0.05) (Fig. 6, Table II).

Decreased chondrocyte viability after corticosteroid administration Fig. 2. Articular cartilage grafted subcutaneously into SCID mice. At gross appearance (A: 4 weeks after implantation), neo-vascularization was observed at the grafted site and grafted cartilage was From the results of the XTT assay, it placed in the subcutaneous cavity (B). Histological characteristics of the implanted tissue remained was shown that chondrocyte viability similar to those of natural cartilage (C: HE staining, 11 weeks after implantation, original magnifica- was decreased by cort i c o s t e roid ad- tion x100. D: SO staining, 11 weeks after implantation, original magnification x100). m i n i s t ration (89.6 ± 3.1% at 10- 4M

Fig. 3. Histological examination of implanted cartilage after corticosteroid treatment. Proteoglycan depletion was not remarkable in the corticosteroid-treated groups by SO staining ( A: control; B: hydrocortisone acetate-treated group; C: triamcinolone acetonide-treated group; D: dexamethasone-treated group. Original magnification x40). An increase of TUNEL-positive apoptotic cells can be clearly discerned after corticosteroid treatment (E, I: control; F, J: hydrocortisone acetate-treated group; G, K: triamcinolone acetonide-treated group; H,L:dexamethasone-treated group; E-H: original magnification x40; I- J: original magnification x200).

777 Steroid-induced chondrocyte apoptosis / F. Nakazawa et al.

Table I. Number of TUNEL positive chondrocytes in the grafted human articular cartilage metabolic rate in cultured chondrocyte on the back of SCID mouse after different corticosteroids treatment. so that this reduction value does not mean the rate of induction of apopto- ABCD sis. But this result is consistent with the TUNEL positive chondrocytes 0.5±0.1 12.1±1.4* 9.4±1.2* 14.9±1.7* other in vivo and in vitro findings of (/mm2) this study, and confirms that corticosteroid treatment reduces the viability A: control (PBS: 0.2ml) B: hydrocortisoneacatate: 1mg/0.2ml of chondrocytes. C: triamcinolone acetonide: 0.2mg/0.2ml D/ dexamethasone acetate: 0.1mg/0.2ml Discussion Values are mean ± SEM. In this study, we observed chondrocyte *: p<0.05 compared to control (PBS) group. apoptosis after corticosteroid treatment in vivo and in vitro. Repeated intra- a rticular cort i c o s t e roid injections are known to result in a progressive degradation of articular cartilage and joint d e s t ruction (2). This stero i d - i n d u c e d arthropathy may result from the down regulation of matrix synthesis in cartilage after corticosteroid treatment (20). But steroid-induced arthropathy brings i rreve rs i ble ch a n ges to the art i c u l a r c a rt i l age even though intra - a rt i c u l a r s t e roid injection has been cancelled. Fig. 4. Electron microscopic examination of the chondrocyte after corticosteroid treatment. Nuclear So we hypothesized cartilage degrada- pigmentation, fragmentation, and tightly packed cytoplasmic organelles were observed by electron microscopic examination in the corticosteroid-treated group (A: control; B: triamcinolone acetonide- tion was associated with chondrocyte treated group, original magnification x10000). apoptosis. And chondrocyte apoptosis may play an important role in steroid- induced arthropathy. In fact, chondro- TA). A statistically significant decrease 4.2%, p< 0.05) (Table III). The data in- cyte apoptosis has been implicated pre- was observed at 10-3 M TA (74.3 ± dicates the decrease of mitochondrial viously in other diseases,including RA

Fig. 5. Flow cytometric analysis of the cultured chondrocytes. By double staining of FITC-Annexin V / PI, FITC-Annexin V-positive PI-negative apoptotic cells was observed in the lower right quadrangle (B-D) after corticosteroid administration at 12 hours (A: control; B: hydrocortisone acetate 10-4M; C: triamcinolone acetonide 10-4M; D: dexamethasone 10-4M). After 72 hours of corticosteroid administration exhibit fragmented nuclei, as indicated by the presence of a left-shifted peak (A: control; B: hydrocortisone acetate 10-4M; C: triamcinolone acetonide 10-4M; D: dexamethasone 10-4M).

778 Steroid-induced chondrocyte apoptosis / F. Nakazawa et al.

Fig. 6. TUNEL staining of cultured chondrocytes. At low dose of corticosteroid, it was not observed the remarkable increasing of TUNEL positive apoptotic cells. At high dose (10-4M,10-3M) statistically significant increasing was observed compared with control (A: control PBS; B: after 72 hours 10-7M triamcinolone acetonide; C; 10-6 M; D: 10-5 M; E: 10-4 M; F: 10-3M. Original magnification x100 ).

Table II. Percentage of TUNEL positive cells in the cultured chondrocyte with the indicat- ( 2 2 , 23). These rep o rts demonstrat e d ed dose of Triamcinolone acetonide (TA) (0, 10-7, 10-6, 10-5, 10-4, 10-3M). p ro t e og lycan loss and fi s s u res and cysts in articular cartilage. But they did -7 -6 -5 -4 -3 Concentration of TA (M) 0 10 10 10 10 10 not mention empty lacunae in the artic- TUNEL positive cells (%) 0.3±0.05 0.5±0.1 2.2±0.5 4.0±0.7 8.9±1.2* 14.3±1.1* ular cartilage in those animal models. It might be important to invest the Values are mean ± SEM. chondrocyte viability before the break- *: p<0.05 compared tocontrol (PBS) group. down of articular matrix. Because we could not make corticosteroid-induced arthropathy in healthy rabbit knee by Table III. Viability of the cultured chondrocytes with the indicated dose of Triamcinolone acetonide (TA) (0, 10-7, 10-6, 10-5, 10-4, 10-3M). corticosteroid injection, in this experiment we used SCID/hu model. It was Concentration of TA (M) 0 10-7 10-6 10-5 10-4 10-3 obscure why the corticosteroid-induced arthropathy was not observed in the Viability (%) 100 102.2±1.7 94.8±2.3 91.6±2.9 89.6±3.1 74.3±4.2* rabbit knee in our experiment. It might Values are mean ± SEM. cause of the difference of experimental *: p<0.05 compared tocontrol (PBS) group. condition and period. If we had used mu ch volume of cort i c o s t e roid and injected more often times and more and OA (10-13), and this process may o s t e o a rt h ritic human articular knee long term, it might have been observed be linked to arthropathy (13). cartilage (21). We also observed that co rt i c o s t e r oid-induced arth ro p at h y. But Chondrocyte apoptosis is observed in chondrocyte apoptosis is not seen in if so, it would include mechanical in- the corticosteroid-induced arthropathy the comparat ive ly normal cart i l age jury among the result of experiment. knee in human (Fig. 7). But because after intra - a rticular cort i c o s t e roid in- We believe that our experimental con- these cartilages were worn out, it was jection in human knee. So that we used dition was appropriate to evaluate the remained unclear whether chondrocyte animal model to examine the mecha- articular chondrocyte apoptosis induc- apoptosis had been induced by corti- nism of steroid-induced arthropathy. ed by corticosteroid. In SCID/hu mo- c o s t e roid injection or by mech a n i c a l S eve ral animal ex p e riments we re del human articular carti l a ge was main- s t ress. Recently it was rep o rted that undertaken to study the effects of carti- tained the ch a ra c t e ristics of in vivo apoptotic cell death is not a widespread lage damages caused by repeated intra- human cartilage and injected corticos- phenomenon in normal aging and a rticular injection of cort i c o s t e ro i d s t e roid remained pro l o n ged period in

779 Steroid-induced chondrocyte apoptosis / F. Nakazawa et al.

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And more in ed that cartilage in the growth plate is 10. B L A N C O F J, G U I T I A N R , M A RTUL EV, TORO FJ, GALDO F: Osteoarthritis chondro- this model, without any confounding induced apoptosis by glucocort i c o i d cytes die by apoptosis. A rt h ritis Rheum m e chanical infl u e n c e s , d e s t ru c t e d via glucocorticoid re c eptor (8). A n d 1998; 41: 284-9. grafted cartilage was not observed and chondrocyte apoptosis might be regu- 11. KIM HA, SONG YW :Apoptotic chondrocyte lated by corticosteroid by decreasing d e ath in rheumatoid art h ritis. A rt h ri t i s proteoglycan loss was not remarkable. Rheum 1999; 42: 1528-37. These findings also indicate that chon- bcl-2 and increasing Bax (24). Articu- 12. TEW SR, KWAN APL, HANN A, THOMSON drocyte apoptosis was not induced by lar ch o n d ro cyte apoptosis might be BM,ARCHER CW:The reactions of articular wound or mechanical stress but by cor- induced by same mechanism that in- cartilage to experimental wounding. Arthri - duced via glucocorticoid receptor and tis Rheum 2000; 43: 215-25. ticosteroid treatment. 13. HASHIMOTO S, OCHI RL, KOMIYA S, LOTZ In the cultured ch o n d ro cy t e s , few regulated by decreasing bcl-2 and in- M: L i n k age of ch o n d ro cyte apoptosis and apoptotic cells were observed at low creasing Bax. cartilage degradation in human osteoarthri- doses of corticosteroid. However chon- In concl u s i o n , we have examined a tis. Arthritis Rheum 1998; 41: 1632-8. 14. SAKAI K, M ATSUNO H, M O R I TA I et al. : drocytes apoptosis was observed at the possible mechanism of steroid-induced Potential withdrawal of rheumatoid synovi- high dose of corticosteroid (1 x 10-4 M) arthropathy using SCID/hu model that um by the induction of apoptosis using a wh i ch can be ach i eved cl i n i c a l ly by was engrafted with human art i c u l a r novel in vivo model of rheumatoid arthritis. intra-articular injection. For the above cartilage, and using an in vitro human Arthritis Rheum 1998; 41: 1251-7. 15. M ATSUNO H, S AWAI T, NEZUKA T et al. : reasons, we conclude that weekly high chondrocyte culture. Our results indi- Treatment of rheumatoid synovitis with anti- dose cort i c o s t e roid injection may cate that corticosteroid administration re s h aping human interleukin-6 re c ep t o r induce chondrocyte apoptosis. In this induces the apoptosis of chondrocytes. monoclonal antibody. Arthritis Rheum 1998; a rt i cle three diffe rent cort i c o s t e ro i d s These findings are important for our 41: 2014-21. 16. NA K A Z AWA F, M ATSUNO H, YUDOH K e t (hydrocortisone acetate, triamcinolone understanding of the articular cartilage al.: Methotrexate inhibits rheumatoid syn- a c e t o n i d e, d examethasone acetat e ) degradation process following cortico- ovitis by inducing apoptosis. J Rheumatol were used.Though statistical signifi- steroid administration. 2001; 28: 1800-8. 17. WA K I TA N I S, K I M U R A T, H I RO O K A A e t cance change was not observed among al.: Repair of rabbit articular surfaces with these cori c o s t e ro i d s , d ex a m e t h a s o n e References allograft chondrocytes embedded in collagen acetate was most effective to induce 1. GA F F N Y K , L E D I N G H A M J, P E R RY J D: gel. J Bone Joint Surg [Br] 1989; 71: 74-80. the chondrocyte apoptosis. The differ- Intra-articular triamcinolone hexacetonide in 18. ENGLAND M, RAMAEKERS FCS, SCHUTTE knee osteoarth ri t i s : fa c t o rs influencing the B, REUTELINGSPERGER PM: A novel assay ence of effect of apoptosis induction clinical response. Ann Rheum Dis 1995; 54: to measure loss of plasma membrane asym- might be caused by binding affinity to 379-81. metry during apoptosis of adherent cells in glucocorticoid receptor. It was report- 2. KLIPPEL JH, DIEPPE PA: R h e u m at o l ogy. culture. Cytometry 1996; 24: 131-9.

780 Steroid-induced chondrocyte apoptosis / F. Nakazawa et al.

19. S C U D I E RODA , S H O E M A K E R R H , PAU L L s t e ro i d. A rt h ritis Rheum 1985; 28: 1 3 9 3 - art h ro p at h y. Art h r itis Rheum 1970; 13: 23 6 - 4 3 . KD et al.: Evaluation of a soluble tetrazoli- 1401. 23. BEHRENS F, SHEPARD N, MITCHELL N: Al- um/ formazan assay for cell growth and drug 21. A I G N E R T, H E M M E L M , NEUREITER D e t teration of rabbit articular cartilage by intra- sensitivity in culture using human and other al.: Apoptotic cell death is not a widespread articular injections of glucocorticoids. J Bone tumor cell lines. Cancer Res 1988; 48; 4827- phenomenon in normal aging and osteoarth- Joint Surg 1975; 57-A: 70-6. 33. ritic human articular knee cartilage. Arthritis 24. MOCETTI P, SILVESTRINI G,BALLANTI P et 20. PELLETIER JP, P E L L E T I E R J M: C a rt i l age Rheum 2001; 44: 1304-12. al.:Bcl-2 and Bax expression in cartilage and degradation by neutral proteoglycanases in 22. M O S KOW I T Z RW, DAV I S W, S A M M A R C O J bone cells after high-dose cort i c o s t e ro n e experimental osteoarthritis. Suppression by et al.: Experimentally induced corticosteroid treatment in rats. Tissue Cell 2001; 33: 1-7.

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