- Email: [email protected]
Effects of interleukin-1β on chondroblast viability and extracellular matrix changes in bovine articular cartilage explants
Biomedicine & Pharmacotherapy 57 (2003) 314–319 www.elsevier.com/locate/biopha
Original article
Effects of interleukin-1b on chondroblast viability and extracellular matrix changes in bovine articular cartilage explants Giordano Stabellini a,*, Monica De Mattei b, Carla Calastrini b, Nicoletta Gagliano a, Claudia Moscheni a, Michela Pasello b, Agnese Pellati b, Catia Bellucci c, Magda Gioia a b
a Department of Human Anatomy, L.I.T.A. Segrate, University of Milan, Via Mangiagalli 31, 20133 Milan, Italy Section of Histology and Embryology, Department of Morphology and Embryology, University of Ferrara, Ferrara, Italy c Department of Experimental Medicine and Biomedical Science, University of Perugia, Perugia, Italy
Received 23 January 2003; accepted 27 February 2003
Abstract Osteoarthritis is a degenerative disease of joint cartilage, characterized by the progressive and permanent degeneration of cartilage due to an imbalance in normal extracellular matrix turnover. Interleukin-1b is a proinflammatory agent, which is present in an elevated amount in osteoarthritic cartilage, and is thought to play a decisive role in osteoarthritis. Interleukin-1b acts as an important mediator of extracellular matrix changes where its activity is regulated by glycosaminoglycan composition. The aim of this study was to investigate the extracellular matrix changes in bovine cartilage explants following interleukin-1b treatment by morphological, histochemical and biochemical methods. Interleukin-1b stimulated the release of matrix sulfated proteoglycans in the culture medium, and significantly inhibited sulfated proteoglycan synthesis. These events were associated to a strong stimulation of nitric oxide production. Interleukin-1b-treated cartilage showed evident collagen fibers around the chondrocytes, together with diminished glycosaminoglycan sulfate content in the extracellular matrix of the explants. Moreover, the ultrastructure and viability of cells did not change in treated cartilage. Our data show that interleukin-1b modifies the ECM turnover without toxic effect on chondrocytes. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Extracellular matrix; Interleukin-1b; Sulfated proteoglycans
1. Introduction Osteoarthritis is characterized by reduced integrity of joint cartilage, characterized by its progressive and permanent degeneration. It is a major cause of morbidity and is a multifactorial pathology caused by aging, mechanical, genetic, hormonal factors, and is a consequence of an imbalance of normal extracellular matrix turnover, with degradation exceeding synthesis [10,18]. Excessive degeneration of articular cartilage results in the release of several substances into sinovial fluid, inducing inflammatory reaction by sinovial macrophages with the consequent release of cytokines, such as interleukin-1b, into adjacent tissues [15]. Interleukin-1b acts as an important mediator of increased extracellular matrix degradation by stimulating a number of events, such as increase of metalloproteinases, gene expression, nitric oxide production [25] and by suppressing type II * Corresponding author. E-mail address: [email protected] (G. Stabellini). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/S0753-3322(03)00038-6
collagen and proteoglycan synthesis [4,9]. By contrast, other authors have showed that, during articular cartilage degeneration, interleukin-1b levels remain within normal levels [19]. In fibroblast and osteoblast cultures, interleukin-1b stimulates the glycosaminoglycan synthesis in the extracellular matrix [22]; glycosaminoglycan classes such as hyaluronic acid and chondroitin sulfate modulate the cytokine activity [16] and gene expression [3]. Moreover, the hyaluronic acid blocks the decreased proteoglycan concentration in osteoarthritic chondrocyte cultures [23], chondroitin sulfate prevents nitric oxide damage [5] and heparan sulfate regulates gene expression in adult hepatocytes [3]. These data show an important role played by the glycosaminoglycans, relevant components of the extracellular matrix, and suggest that their coordination with cytokine is essential for the differentiation and the maintenance of adult organ functions. In this study we reexamined the changes of extracellular matrix in bovine articular cartilage explants following treat-
G. Stabellini et al. / Biomedicine & Pharmacotherapy 57 (2003) 314–319
ment with interleukin-1b, by morphological, biochemical and histochemical methods. For this purpose, we studied the extracellular matrix components, such as proteoglycans, measuring specific biochemical markers of their metabolism. We analyzed their synthesis and release as well as the production of nitric oxide, an important regulatory molecule involved in matrix degradation [17]. Moreover, glycosaminoglycan classes, such as hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, were distinguished by Alcian blue staining [20], whilst collagen fibers with Sirius red [8]; finally, ultrastructure of chondrocytes was examined by transmission electron microscope.
2. Materials and methods 2.1. Cartilage explant cultures Explants of bovine articular cartilage (Daini, Italy) were aseptically dissected from metacarpophalangeal joints of 12–18-month-old animals. Full thickness cartilage discs were obtained from the weight-bearing region of the articular surface using a 4 mm dermal punch (Stiefel Laboratories, Italy). Explant discs (three per well, approximately 30 mg total) were cultured at 37 °C in an atmosphere of 5% CO2 in multiwells (Nunc, Denmark, 6.6 × 6.6 cm, 1.6 cm the diameter of each well) containing 1 ml DMEM/F12 supplemented with 10% FBS and antibiotics (penicillin 100 U/ml, streptomycin 0.1 mg/ml). Before any treatment, cartilage explants were maintained for 48 h in medium containing 10% FBS and for an additional 48 h in medium without serum. Five cartilage discs were treated with 50 ng/ml interleukin-1b or medium alone (controls). Twenty-four hours after treatment, the release of proteoglycans in the culture medium and the residual proteoglycan content in the tissue were evaluated by dimethylmethylene blue assay; proteoglycan synthesis was determined by incorporation of Na2–35SO4; glycosaminoglycan classes with Alcian blue, and collagen fibers by Sirius red staining were analyzed, and nitrite was assessed. 2.2. Transmission electron microscopy At the end of each treatment, cartilage explants were fixed in 1% glutaraldehyde in sodium cocadylate buffer, pH 7.4. Subsequently they were post fixed in cold 2% OsO4. After dehydration in acetone, the specimens were embedded in Epon-Araldite. Ultrathin sections were contrasted with 2% uranyl acetate and lead citrate and investigated under a transmission electron microscope (Hitachi H800). 2.3. Cell viability assay At the end of the treatment, chondrocytes were isolated from cartilage explants as previously described [14]. Briefly, cartilage explants were sequentially digested in medium containing pronase (0.17 mg/ml) for 40 min at 37 °C under continuous stirring and then with collagenase (1 mg/ml) for
315
2 h, until complete digestion of the tissue. The same amount of tissue (30 mg) was digested from each sample. Cells were then collected by centrifugation and counted under a light microscope using a hemocytometer at ×40 magnification. Chondrocyte viability was assessed by trypan blue exclusion assay [12]. For each experimental condition the percentage of cell viability was normalized to the control. 2.4. Proteoglycan release Proteoglycan release into culture media was determined as total sulfated glycosaminoglycan, using the dimethylmethylene blue assay, with shark chondroitin sulfate as standard [13]. 2.5. Proteoglycan synthesis During the 24 h of treatment, 5 µCi/ml of Na2–35SO4 was added to culture media. After radiolabeling, explants were rinsed and digested in 20 mM phosphate buffer (pH 6.8) containing 4 mg/ml papain at 60 °C for 12 h [7]. The content of 35S-labeled newly synthesized sulfated proteoglycans were measured following precipitation of the 35Sproteolgicans with cetylpyridinium chloride [27] and filtration into glass fiber filters (Whatman GF/C). Filters were dried and radioactivity was quantified by liquid scintillation counting. 2.6. Nitric oxide production The release of nitrite, a stable breakdown product of nitric oxide, by cartilage explants was measured as an indicator of nitric oxide synthesis. At the end of 24 h treatment, nitrite concentration was determined in the conditioned media according to Green et al. [11]. Briefly, 200 µl culture medium was mixed with an equal volume of the Greiss reagent and absorbance was measured at 550 nm. Nitrite concentration was calculated from a standard curve of sodium nitrite. 2.7. Histochemical investigations Cartilage cultures were fixed in 10% buffered formalin at 4 °C overnight and routine histological procedures were followed. Cartilage cultures were cut in 5 µm sections, deparaffined and stained with Sirius red for collagen fibers and Alcian blue for glycosaminoglycan analysis. 2.8. Sirius red staining Deparaffined sections were immersed for 30 min in saturated aqueous picric acid containing 0.1% Sirius Red F3BA (Sigma, Italy), and observed by light microscope. We performed five slides for sample, each one in triplicate. Collagen values were obtained by connecting a Zeiss Axioplane Microscope to a Kontron Electronic Scanner using Vidas Software. The values are expressed as the relative optical density (arrangement: black = 0, white = 1) and were mean ± S.D. of five determinations for slide.
316
G. Stabellini et al. / Biomedicine & Pharmacotherapy 57 (2003) 314–319
Fig. 2. Proteoglycans release in culture medium by bovine cartilage during interleukin-1b treatment. The values (µg PG per well) are expressed as mean ± S.D. for five experiments. C, controls; IL-1b, interleukin-1b.
determine the distribution of individual glycosaminoglycan. Digestion with testicular hyaluronidase, in particular, selectively removed hyaluronic acid and chondroitin sulfate. Glycosaminoglycan values were obtained by connecting a Zeiss Axioplane Microscope to a Kontron Electronic Scanner using Vidas Software. We prepared three slides for samples; the values are expressed as the relative optical density (arrangement: black = 0, white = 1) and were mean ± S.D. of five determinations for each slide. 2.10. Statistical analysis
Fig. 1. Microphotographs showing the ultrastructure of cartilage cultures. (a) Controls; (b) interleukin-1b treated.
All values are expressed as means ± S.D. of for at least five independent experiments, each performed in triplicate. The statistical analysis was made using the Student’s t-test for paired and unpaired data. P ≤ 0.05 were considered significant.
2.9. Alcian blue staining Sections were cut at 4–7 µm and stained for morphological examination with haematoxylin-eosin. For histochemical analysis, in order to distinguish different glycosaminoglycans, we used Alcian blue 8GX (Sigma, Italy) staining (1.5% Alcian blue in 0.1 M acetate buffer, pH 5.8 containing 0.025 or 0.3 or 0.65 M MgCl2, for 6 h at room temperature). For enzymatic digestion, the sections were incubated with testicular hyaluronidase (Merck, Germany) (1 mg/ml in 0.1 M phosphate buffer, pH 7, 20 h at 37 °C). Control sections were incubated in buffer alone. Glycosaminoglycans were identified by critical electrolyte concentrations at which the polyanions change from binding Alcian to binding Mg2+ [21]. Alcian blue stained polyanions with increasing selectivity as the MgCl2 concentration of the staining solution was raised. At 0.025 M MgCl2, not only glycosaminoglycans but also nucleic acids and sulfated glycoproteins were stained; at 0.3 M MgCl2 the only macromolecules, which were positively stained, were glycosaminoglycan (chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate); at 0.65 M MgCl2 the only stained glycosaminoglycan were heparan sulfate and keratan sulfate. The action of specific enzymes on the section, followed by Alcian blue staining, allowed us to
3. Results 3.1. Ultrastructural analysis Cultures of cartilage maintained with medium alone (Fig. 1a) showed oval-round chondrocytes with abundant rough endoplasmic reticulum in the cytoplasm. The nucleus
Fig. 3. Proteoglycan content in bovine cartilage cultures after interleukin-1b treatment. The values (µg PG per mg tissue) are mean ± S.D. of five experiments. C, controls; IL-1b, interleukin-1b.
G. Stabellini et al. / Biomedicine & Pharmacotherapy 57 (2003) 314–319
317
pseudopodia were present and rough endoplasmic reticulum was well developed. 3.2. Proteoglycans release and synthesis
Fig. 4. Proteoglycan synthesis in bovine cartilage cultures during interleukin-1b treatment. The values (cpm/mg tissue) are mean ± S.D. of five experiments. C, controls; IL-1b, interleukin-1b.
was round and presented euchromatin and evident nucleolus. The interleukin-1b treated cartilages did not show differences compared to controls (Fig. 1b). Several cytoplasmatic
Fig. 5. Nitric oxide production by bovine cartilage cultures during interleukin-1b treatment. The values (µM NO per mg well) are mean ± S.D. of five experiments. C, controls; IL-1b, interleukin-1b.
In vitro interleukin-1b significantly increased (P < 0.01) the release of matrix proteoglycans in the culture medium when compared to controls (74.01 ± 10.36 and 35.3 ± 5.3 µg PG per well, respectively) (Fig. 2) and decreased (P < 0.05) the proteoglycans content in interleukin-1b treated bovine cartilage cultures compared to controls (58.08 ± 8.78 and 64.23 ± 9.63, respectively) (Fig. 3). Fig. 4 shows that the cytokine inhibited proteoglycan synthesis measured by 3Hglucosamine incorporation (P ≤ 0.01) in treated cartilage (1256 ± 238 cpm/mg tissue) when compared to controls (1966 ± 394 cpm/mg tissue). These events were associated to a strong stimulation of nitric oxide production (P ≤ 0.01) in interleukin-1b treated cartilage (49.56 ± 6.45) compared to untreated cultures (3.18 ± 0.56) (Fig. 5). Sirius red staining showed evident collagen fibers around the chondrocytes of treated cartilage, when compared to controls (Fig. 6a,b). Alcian blue staining did not show differences in the alcianophilia at 0.025 and 0.3 M MgCl2 before jaluronidase treatment (Table 1). Whereas, after testicular jaluronidase digestion, the alcianophilia of cartilages in 0.025 and 0.3 M MgCl2 significantly decreased in interleukin-1b treated cultures compared to control (Table 1 and Fig. 6c,d). Since the enzyme digests hyaluronic acid and chondroitin sulfate, the decrease of alcianophilia can be considered as an indicator of a lower content of hyaluronic acid and chondroitin sulfate in the extracellular matrix of interleukin-1b treated cultured cartilages. Table 2 shows the effects of interleukin-1b on cell viability. The total number of recovered cells did not signifi-
Fig. 6. Sirius red staining of control (a) and interleukin-1b treated (b) bovine cartilages. Collagen fibers around the chondrocytes after interleukin-1b treatment are evident (b). Alcian blue staining in 0.3 M MgCl2 after testicular hyaluronidase digestion of control (c) and interleukin-1b treated cartilages (d). Magnification, 250×.
318
G. Stabellini et al. / Biomedicine & Pharmacotherapy 57 (2003) 314–319
Table 1 Histochemical evaluation of control cartilages and 24 h after interleukin-1b treatment Cartilage Controls Capsula Interter. A. Treated Capsula Interter. A.
AB in 0.025 M MgCl2 Before Jal
After Jal
AB in 0.3 M MgCl2 Before Jal
After Jal
142.52 ± 2.60 166.28 ± 2.15
170.92 ± 2.02 184.05 ± 3.59
159.05 ± 2.40 152.19 ± 2.16
156.50 ± 2.00 178.60 ± 1.86
149.76 ± 4.24 165.60 ± 2.05
185.55 ± 3.00 197.07 ± 2.09
151.40 ± 2.45* 147.40 ± 2.95*
165.04 ± 1.97* 187.21 ± 3.99*
Values were obtained by a Kontron Electronic Scanner and are expressed in optical density (mean ± S.D.). Jal, testicular hyaluronidase; AB, Alcian blue; Interter. A., interterritorial area. * P < 0.01.
cantly change 24 h after interleukin-1b treatment, compared to controls. 4. Discussion and conclusions Our data show that interleukin-1b modifies the cartilage extracellular matrix turnover by inhibiting proteoglycan synthesis and increasing proteoglycan release in medium, leading to a lowered proteoglycan content in the cartilage, without modifying the chondrocyte viability. The cytokine stimulates the production of nitric oxide production that is able to induce apoptosis. Our data did not evidence any morphological change in chondrocytes of interleukin-1b treated cartilages compared to controls. The observation of nitric oxide increase without the evidence of apoptosis processes can be due to the short maintenance (24 h) of cartilage in culture. The decrease of alcianophilia after jaluronidase digestion in interleukin-1b treated cartilage at 0.025 and 0.3 M MgCl2 shows that in treated cartilage non-sulfated (hyaluronic acid) and sulfated glycosaminoglycans (chondrotin sulfate, dermatan sulfate, keratan sulfate and heparan sulfate) decrease. The extracellular matrix changes could modify its physiological functions. In particular, since hyaluronic acid blocks the degradative effects of cytokines on the extracellular matrix [23], hyaluronic acid and chondroitin sulfate content can be related to cytokine activity [16], and since displays a protective effect against nitric oxide damage [5], the extracellular matrix changes can play an important role in maintaining osteoarthritic pathogenesis. In fact, according to Sztrolovics et al. [24], it has been proposed that the alteration in hyaluronate degradation can be an alternative mechanism for the regulation of proteoglycan release from cartilage following interleukin-1b stimulation. Our data, therefore, suggest that interleukin-1b effects on cartilage first modify the activity of the enzymes involved in proteoglycan synthesis and
degradation and, more in general, in the metabolic pathways of glycosaminoglycans. Moreover, interleukin-1b is able to up-regulate metalloproteinases gene expression, inducing increased proenzyme synthesis and secretion of these family enzymes involved in extracellular matrix cartilage degradation [4]. Our results evidenced a decreased glycosaminoglycans and proteoglycans content following interleukin-1b treatment: this may have the effect of “unmasking” collagen fibers, rendering them more susceptible to the digestion by metalloproteinases, whose gene expression is stimulated by interleukin-1b treatment [1,26]. Since a deficit in the production of a natural antagonist of the IL-1 receptor has been demonstrated [1] it is possible that an individual sensibility to IL-1b stimulation occurs. These speculations are in agreement with data showing that cartilage degenerates without IL-1b variations [19], and that glucosamine is not able to reverse proteoglycan decrease in interleukin-1b-treated bovine cartilage [6]. In conclusion, our data suggest that interleukin-1b in bovine metacarpophalangeal cartilage acts directly on the pathways of glycosaminoglycan synthesis and degradation, without modifying the viability of chondrocytes; moreover, suggest that the changes in ECM composition may play an important role in maintaining osteoarthritis damage. Since the susceptibility to IL-1b concentrations varies in cartilage at different anatomical locations [2] and in vitro system, where several stimulation such as hormones and cytokines are missing, this must always be considered when comparing in vivo and in vitro data. Further work, however, is necessary to investigate the relation between interleukin-1b and of cartilage damage. References [1] [2]
Table 2 Cell viability of bovine cartilage cultures 24 h after interleukin-1b treatment C IL-1b
% Chondrocytes viability 100 98 ± 4.8
The percentage of cell viability was normalized to controls (100%). The values are mean ± S.D. of five experiments. C, controls; IL-1b, interleukin-1b.
[3]
[4]
Abramson SB, Amin A. Blocking the effects of IL-1 in rheumatoid arthritis protects bone and cartilage. Rheumatology 2002;41:972. Barakat AF, Elson CJ, Westacott CI. Susceptibility to physiological concentrations of IL-1b varies in cartilage at different anatomical locations on human osteoarthritic knee joints. Osteoarthritis Cartilage 2002;10:264. Brill S, Zvibel I, Halpern Z, Oren R. The role of fetal and adult hepatocyte extracellular matrix in the regulation of tissue-specific gene expression in fetal and adult hepatocytes. Eur J Cell Biol 2002; 81:43. Chevalier X. Upregulation of enzymatic activity by interleukin-1 in osteoarthritis. Biomed Pharmacother 1997;51:58.
G. Stabellini et al. / Biomedicine & Pharmacotherapy 57 (2003) 314–319 [5] [6]
[7]
[8]
[9]
[10] [11]
[12]
[13] [14]
[15]
[16]
Conrozier T. Death of articular chondrocytes. Mechanisms and protection. Presse Med 1998;27:1859. De Mattei M, Pellati A, Pasello M, de Terlizzi F, Massari L, Gemmati D, et al. High doses of glucosamine-HCl have detrimental effects on bovine articular cartilage explants cultured in vitro. Osteoarthritis Cartilage 2002;10:816. Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 1986;883:173. Gagliano N, Arosio B, Grizzi F, Masson S, Tagliabue J, Dioguardi N, et al. Reduced collagenolytic activity of matrix metalloproteinases and development of liver fibrosis in the aging rat. Mech Ageing Dev 2002;123:413. Gouze JN, Bordji K, Gulberti S, Terlain B, Netter P, Magdalou J, et al. Interleukin-1beta down-regulates the expression of glucuronosyltransferase I, a key enzyme priming glycosaminoglycan biosynthesis: influence of glucosamine on interleukin-1beta-mediated effects in rat chondrocytes. Arthritis Rheum 2001;44:351. Ghosh P, Smith M. Osteoarthritis, genetic molecular mechanisms. Biogerontology 2002;3:85. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 1982;126:131. Grimshaw MJ, Mason RM. Bovine articular chondrocyte function in vitro depends upon oxygen tension. Osteoarthritis Cartilage 2000;8: 386. Handley CJ, Buttle DJ. Assay of proteoglycan degradation. Methods Enzymol 1995;248:47. Kuettner KE, Pauli BU, Gall G, Memoli VA, Schenk RK. Synthesis of cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture characteristics, and morphology. J Cell Biol 1982;93:743. Iniguez MA, Pablos JL, Carreira PE, Cabr F, Gomez-Reino JJ. Detection of COX-1 and COX-2 isoform in synovial fluid cells from inflammatory joint diseases. Br J Rheumatol 1998;37:773. Locci P, Marinucci L, Lilli C, Martinese D, Becchetti E. Transforming growth factor b1-hyaluronic acid interaction. Cell Tissue Res 1995; 281:317.
319
[17] Lotz M. The role of nitric oxide in articular cartilage damage. Rheum Dis Clin North Am 1999;25:269. [18] Malemud CJ. Fundamental pathways in osteoarthritis: an overview. Front Biosci 1999;15:D659. [19] Sakano Y, Terada N, Ueda H, Fujii Y, Hamada Y, Akamatsu N, et al. Histological study of articular cartilage in experimental rat knee arthritis induced by intracapsular injection of cationic polyethyleneimine. Med Electron Microsc 2000;33:246. [20] Scott JE. The molecular biology of histochemical staining by cationic phthalocyanin dyes: the design of replacements for Alcia blue. J Microsc 1980;119:373. [21] Scott JE, Dorling J. Differential staining of acid glycosaminoglycans (mucopolysaccharides) by Alcian blue in salt solutions. Histochimie 1965;5:221. [22] Stabellini G, Calvitti M, Baroni T, Marinucci L, Calastrini C, Carinci P, et al. Glycosidases during chick embryo lung development and their colocalization with proteoglycans and growth factors. Eur J Histochem 2002;46:41. [23] Stove J, Gerlach C, Huch K, Gunther KP, Puhl W, Scharf HP. Effects of hyaluronan on proteoglycan content of osteoarthritic chondrocytes in vitro. J Orthop Res 2002;20:551. [24] Sztrolovics R, Recklies AD, Roughley PJ, Mort JS. Hyaluronate degradation as an alternative mechanism for proteoglycan release from cartilage during interleukin 1b-stimulated catabolism. Biochem J 2002;362:473. [25] van der Kraan PM, van den Berg WB. Anabolic and destructive mediator in osteoarthritis. Curr Opin Clin Nutr Metab Care 2000;3: 205. [26] Vincenti MP, Brinckerhoff CE. Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex signaling pathways for the recruitment gene-specific transcription factors. Arthritis Res 2002;4:157. [27] Wasteson A, Uthne K, Westermark B. A novel assay for the biosynthesis of sulphated polysaccharide and its application to studies on the effects of somatomedin on cultured cells. Bioc or medium alone (controls). Biochem J 1973;136:1069.
Original article
Effects of interleukin-1b on chondroblast viability and extracellular matrix changes in bovine articular cartilage explants Giordano Stabellini a,*, Monica De Mattei b, Carla Calastrini b, Nicoletta Gagliano a, Claudia Moscheni a, Michela Pasello b, Agnese Pellati b, Catia Bellucci c, Magda Gioia a b
a Department of Human Anatomy, L.I.T.A. Segrate, University of Milan, Via Mangiagalli 31, 20133 Milan, Italy Section of Histology and Embryology, Department of Morphology and Embryology, University of Ferrara, Ferrara, Italy c Department of Experimental Medicine and Biomedical Science, University of Perugia, Perugia, Italy
Received 23 January 2003; accepted 27 February 2003
Abstract Osteoarthritis is a degenerative disease of joint cartilage, characterized by the progressive and permanent degeneration of cartilage due to an imbalance in normal extracellular matrix turnover. Interleukin-1b is a proinflammatory agent, which is present in an elevated amount in osteoarthritic cartilage, and is thought to play a decisive role in osteoarthritis. Interleukin-1b acts as an important mediator of extracellular matrix changes where its activity is regulated by glycosaminoglycan composition. The aim of this study was to investigate the extracellular matrix changes in bovine cartilage explants following interleukin-1b treatment by morphological, histochemical and biochemical methods. Interleukin-1b stimulated the release of matrix sulfated proteoglycans in the culture medium, and significantly inhibited sulfated proteoglycan synthesis. These events were associated to a strong stimulation of nitric oxide production. Interleukin-1b-treated cartilage showed evident collagen fibers around the chondrocytes, together with diminished glycosaminoglycan sulfate content in the extracellular matrix of the explants. Moreover, the ultrastructure and viability of cells did not change in treated cartilage. Our data show that interleukin-1b modifies the ECM turnover without toxic effect on chondrocytes. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Extracellular matrix; Interleukin-1b; Sulfated proteoglycans
1. Introduction Osteoarthritis is characterized by reduced integrity of joint cartilage, characterized by its progressive and permanent degeneration. It is a major cause of morbidity and is a multifactorial pathology caused by aging, mechanical, genetic, hormonal factors, and is a consequence of an imbalance of normal extracellular matrix turnover, with degradation exceeding synthesis [10,18]. Excessive degeneration of articular cartilage results in the release of several substances into sinovial fluid, inducing inflammatory reaction by sinovial macrophages with the consequent release of cytokines, such as interleukin-1b, into adjacent tissues [15]. Interleukin-1b acts as an important mediator of increased extracellular matrix degradation by stimulating a number of events, such as increase of metalloproteinases, gene expression, nitric oxide production [25] and by suppressing type II * Corresponding author. E-mail address: [email protected] (G. Stabellini). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/S0753-3322(03)00038-6
collagen and proteoglycan synthesis [4,9]. By contrast, other authors have showed that, during articular cartilage degeneration, interleukin-1b levels remain within normal levels [19]. In fibroblast and osteoblast cultures, interleukin-1b stimulates the glycosaminoglycan synthesis in the extracellular matrix [22]; glycosaminoglycan classes such as hyaluronic acid and chondroitin sulfate modulate the cytokine activity [16] and gene expression [3]. Moreover, the hyaluronic acid blocks the decreased proteoglycan concentration in osteoarthritic chondrocyte cultures [23], chondroitin sulfate prevents nitric oxide damage [5] and heparan sulfate regulates gene expression in adult hepatocytes [3]. These data show an important role played by the glycosaminoglycans, relevant components of the extracellular matrix, and suggest that their coordination with cytokine is essential for the differentiation and the maintenance of adult organ functions. In this study we reexamined the changes of extracellular matrix in bovine articular cartilage explants following treat-
G. Stabellini et al. / Biomedicine & Pharmacotherapy 57 (2003) 314–319
ment with interleukin-1b, by morphological, biochemical and histochemical methods. For this purpose, we studied the extracellular matrix components, such as proteoglycans, measuring specific biochemical markers of their metabolism. We analyzed their synthesis and release as well as the production of nitric oxide, an important regulatory molecule involved in matrix degradation [17]. Moreover, glycosaminoglycan classes, such as hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, were distinguished by Alcian blue staining [20], whilst collagen fibers with Sirius red [8]; finally, ultrastructure of chondrocytes was examined by transmission electron microscope.
2. Materials and methods 2.1. Cartilage explant cultures Explants of bovine articular cartilage (Daini, Italy) were aseptically dissected from metacarpophalangeal joints of 12–18-month-old animals. Full thickness cartilage discs were obtained from the weight-bearing region of the articular surface using a 4 mm dermal punch (Stiefel Laboratories, Italy). Explant discs (three per well, approximately 30 mg total) were cultured at 37 °C in an atmosphere of 5% CO2 in multiwells (Nunc, Denmark, 6.6 × 6.6 cm, 1.6 cm the diameter of each well) containing 1 ml DMEM/F12 supplemented with 10% FBS and antibiotics (penicillin 100 U/ml, streptomycin 0.1 mg/ml). Before any treatment, cartilage explants were maintained for 48 h in medium containing 10% FBS and for an additional 48 h in medium without serum. Five cartilage discs were treated with 50 ng/ml interleukin-1b or medium alone (controls). Twenty-four hours after treatment, the release of proteoglycans in the culture medium and the residual proteoglycan content in the tissue were evaluated by dimethylmethylene blue assay; proteoglycan synthesis was determined by incorporation of Na2–35SO4; glycosaminoglycan classes with Alcian blue, and collagen fibers by Sirius red staining were analyzed, and nitrite was assessed. 2.2. Transmission electron microscopy At the end of each treatment, cartilage explants were fixed in 1% glutaraldehyde in sodium cocadylate buffer, pH 7.4. Subsequently they were post fixed in cold 2% OsO4. After dehydration in acetone, the specimens were embedded in Epon-Araldite. Ultrathin sections were contrasted with 2% uranyl acetate and lead citrate and investigated under a transmission electron microscope (Hitachi H800). 2.3. Cell viability assay At the end of the treatment, chondrocytes were isolated from cartilage explants as previously described [14]. Briefly, cartilage explants were sequentially digested in medium containing pronase (0.17 mg/ml) for 40 min at 37 °C under continuous stirring and then with collagenase (1 mg/ml) for
315
2 h, until complete digestion of the tissue. The same amount of tissue (30 mg) was digested from each sample. Cells were then collected by centrifugation and counted under a light microscope using a hemocytometer at ×40 magnification. Chondrocyte viability was assessed by trypan blue exclusion assay [12]. For each experimental condition the percentage of cell viability was normalized to the control. 2.4. Proteoglycan release Proteoglycan release into culture media was determined as total sulfated glycosaminoglycan, using the dimethylmethylene blue assay, with shark chondroitin sulfate as standard [13]. 2.5. Proteoglycan synthesis During the 24 h of treatment, 5 µCi/ml of Na2–35SO4 was added to culture media. After radiolabeling, explants were rinsed and digested in 20 mM phosphate buffer (pH 6.8) containing 4 mg/ml papain at 60 °C for 12 h [7]. The content of 35S-labeled newly synthesized sulfated proteoglycans were measured following precipitation of the 35Sproteolgicans with cetylpyridinium chloride [27] and filtration into glass fiber filters (Whatman GF/C). Filters were dried and radioactivity was quantified by liquid scintillation counting. 2.6. Nitric oxide production The release of nitrite, a stable breakdown product of nitric oxide, by cartilage explants was measured as an indicator of nitric oxide synthesis. At the end of 24 h treatment, nitrite concentration was determined in the conditioned media according to Green et al. [11]. Briefly, 200 µl culture medium was mixed with an equal volume of the Greiss reagent and absorbance was measured at 550 nm. Nitrite concentration was calculated from a standard curve of sodium nitrite. 2.7. Histochemical investigations Cartilage cultures were fixed in 10% buffered formalin at 4 °C overnight and routine histological procedures were followed. Cartilage cultures were cut in 5 µm sections, deparaffined and stained with Sirius red for collagen fibers and Alcian blue for glycosaminoglycan analysis. 2.8. Sirius red staining Deparaffined sections were immersed for 30 min in saturated aqueous picric acid containing 0.1% Sirius Red F3BA (Sigma, Italy), and observed by light microscope. We performed five slides for sample, each one in triplicate. Collagen values were obtained by connecting a Zeiss Axioplane Microscope to a Kontron Electronic Scanner using Vidas Software. The values are expressed as the relative optical density (arrangement: black = 0, white = 1) and were mean ± S.D. of five determinations for slide.
316
G. Stabellini et al. / Biomedicine & Pharmacotherapy 57 (2003) 314–319
Fig. 2. Proteoglycans release in culture medium by bovine cartilage during interleukin-1b treatment. The values (µg PG per well) are expressed as mean ± S.D. for five experiments. C, controls; IL-1b, interleukin-1b.
determine the distribution of individual glycosaminoglycan. Digestion with testicular hyaluronidase, in particular, selectively removed hyaluronic acid and chondroitin sulfate. Glycosaminoglycan values were obtained by connecting a Zeiss Axioplane Microscope to a Kontron Electronic Scanner using Vidas Software. We prepared three slides for samples; the values are expressed as the relative optical density (arrangement: black = 0, white = 1) and were mean ± S.D. of five determinations for each slide. 2.10. Statistical analysis
Fig. 1. Microphotographs showing the ultrastructure of cartilage cultures. (a) Controls; (b) interleukin-1b treated.
All values are expressed as means ± S.D. of for at least five independent experiments, each performed in triplicate. The statistical analysis was made using the Student’s t-test for paired and unpaired data. P ≤ 0.05 were considered significant.
2.9. Alcian blue staining Sections were cut at 4–7 µm and stained for morphological examination with haematoxylin-eosin. For histochemical analysis, in order to distinguish different glycosaminoglycans, we used Alcian blue 8GX (Sigma, Italy) staining (1.5% Alcian blue in 0.1 M acetate buffer, pH 5.8 containing 0.025 or 0.3 or 0.65 M MgCl2, for 6 h at room temperature). For enzymatic digestion, the sections were incubated with testicular hyaluronidase (Merck, Germany) (1 mg/ml in 0.1 M phosphate buffer, pH 7, 20 h at 37 °C). Control sections were incubated in buffer alone. Glycosaminoglycans were identified by critical electrolyte concentrations at which the polyanions change from binding Alcian to binding Mg2+ [21]. Alcian blue stained polyanions with increasing selectivity as the MgCl2 concentration of the staining solution was raised. At 0.025 M MgCl2, not only glycosaminoglycans but also nucleic acids and sulfated glycoproteins were stained; at 0.3 M MgCl2 the only macromolecules, which were positively stained, were glycosaminoglycan (chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate); at 0.65 M MgCl2 the only stained glycosaminoglycan were heparan sulfate and keratan sulfate. The action of specific enzymes on the section, followed by Alcian blue staining, allowed us to
3. Results 3.1. Ultrastructural analysis Cultures of cartilage maintained with medium alone (Fig. 1a) showed oval-round chondrocytes with abundant rough endoplasmic reticulum in the cytoplasm. The nucleus
Fig. 3. Proteoglycan content in bovine cartilage cultures after interleukin-1b treatment. The values (µg PG per mg tissue) are mean ± S.D. of five experiments. C, controls; IL-1b, interleukin-1b.
G. Stabellini et al. / Biomedicine & Pharmacotherapy 57 (2003) 314–319
317
pseudopodia were present and rough endoplasmic reticulum was well developed. 3.2. Proteoglycans release and synthesis
Fig. 4. Proteoglycan synthesis in bovine cartilage cultures during interleukin-1b treatment. The values (cpm/mg tissue) are mean ± S.D. of five experiments. C, controls; IL-1b, interleukin-1b.
was round and presented euchromatin and evident nucleolus. The interleukin-1b treated cartilages did not show differences compared to controls (Fig. 1b). Several cytoplasmatic
Fig. 5. Nitric oxide production by bovine cartilage cultures during interleukin-1b treatment. The values (µM NO per mg well) are mean ± S.D. of five experiments. C, controls; IL-1b, interleukin-1b.
In vitro interleukin-1b significantly increased (P < 0.01) the release of matrix proteoglycans in the culture medium when compared to controls (74.01 ± 10.36 and 35.3 ± 5.3 µg PG per well, respectively) (Fig. 2) and decreased (P < 0.05) the proteoglycans content in interleukin-1b treated bovine cartilage cultures compared to controls (58.08 ± 8.78 and 64.23 ± 9.63, respectively) (Fig. 3). Fig. 4 shows that the cytokine inhibited proteoglycan synthesis measured by 3Hglucosamine incorporation (P ≤ 0.01) in treated cartilage (1256 ± 238 cpm/mg tissue) when compared to controls (1966 ± 394 cpm/mg tissue). These events were associated to a strong stimulation of nitric oxide production (P ≤ 0.01) in interleukin-1b treated cartilage (49.56 ± 6.45) compared to untreated cultures (3.18 ± 0.56) (Fig. 5). Sirius red staining showed evident collagen fibers around the chondrocytes of treated cartilage, when compared to controls (Fig. 6a,b). Alcian blue staining did not show differences in the alcianophilia at 0.025 and 0.3 M MgCl2 before jaluronidase treatment (Table 1). Whereas, after testicular jaluronidase digestion, the alcianophilia of cartilages in 0.025 and 0.3 M MgCl2 significantly decreased in interleukin-1b treated cultures compared to control (Table 1 and Fig. 6c,d). Since the enzyme digests hyaluronic acid and chondroitin sulfate, the decrease of alcianophilia can be considered as an indicator of a lower content of hyaluronic acid and chondroitin sulfate in the extracellular matrix of interleukin-1b treated cultured cartilages. Table 2 shows the effects of interleukin-1b on cell viability. The total number of recovered cells did not signifi-
Fig. 6. Sirius red staining of control (a) and interleukin-1b treated (b) bovine cartilages. Collagen fibers around the chondrocytes after interleukin-1b treatment are evident (b). Alcian blue staining in 0.3 M MgCl2 after testicular hyaluronidase digestion of control (c) and interleukin-1b treated cartilages (d). Magnification, 250×.
318
G. Stabellini et al. / Biomedicine & Pharmacotherapy 57 (2003) 314–319
Table 1 Histochemical evaluation of control cartilages and 24 h after interleukin-1b treatment Cartilage Controls Capsula Interter. A. Treated Capsula Interter. A.
AB in 0.025 M MgCl2 Before Jal
After Jal
AB in 0.3 M MgCl2 Before Jal
After Jal
142.52 ± 2.60 166.28 ± 2.15
170.92 ± 2.02 184.05 ± 3.59
159.05 ± 2.40 152.19 ± 2.16
156.50 ± 2.00 178.60 ± 1.86
149.76 ± 4.24 165.60 ± 2.05
185.55 ± 3.00 197.07 ± 2.09
151.40 ± 2.45* 147.40 ± 2.95*
165.04 ± 1.97* 187.21 ± 3.99*
Values were obtained by a Kontron Electronic Scanner and are expressed in optical density (mean ± S.D.). Jal, testicular hyaluronidase; AB, Alcian blue; Interter. A., interterritorial area. * P < 0.01.
cantly change 24 h after interleukin-1b treatment, compared to controls. 4. Discussion and conclusions Our data show that interleukin-1b modifies the cartilage extracellular matrix turnover by inhibiting proteoglycan synthesis and increasing proteoglycan release in medium, leading to a lowered proteoglycan content in the cartilage, without modifying the chondrocyte viability. The cytokine stimulates the production of nitric oxide production that is able to induce apoptosis. Our data did not evidence any morphological change in chondrocytes of interleukin-1b treated cartilages compared to controls. The observation of nitric oxide increase without the evidence of apoptosis processes can be due to the short maintenance (24 h) of cartilage in culture. The decrease of alcianophilia after jaluronidase digestion in interleukin-1b treated cartilage at 0.025 and 0.3 M MgCl2 shows that in treated cartilage non-sulfated (hyaluronic acid) and sulfated glycosaminoglycans (chondrotin sulfate, dermatan sulfate, keratan sulfate and heparan sulfate) decrease. The extracellular matrix changes could modify its physiological functions. In particular, since hyaluronic acid blocks the degradative effects of cytokines on the extracellular matrix [23], hyaluronic acid and chondroitin sulfate content can be related to cytokine activity [16], and since displays a protective effect against nitric oxide damage [5], the extracellular matrix changes can play an important role in maintaining osteoarthritic pathogenesis. In fact, according to Sztrolovics et al. [24], it has been proposed that the alteration in hyaluronate degradation can be an alternative mechanism for the regulation of proteoglycan release from cartilage following interleukin-1b stimulation. Our data, therefore, suggest that interleukin-1b effects on cartilage first modify the activity of the enzymes involved in proteoglycan synthesis and
degradation and, more in general, in the metabolic pathways of glycosaminoglycans. Moreover, interleukin-1b is able to up-regulate metalloproteinases gene expression, inducing increased proenzyme synthesis and secretion of these family enzymes involved in extracellular matrix cartilage degradation [4]. Our results evidenced a decreased glycosaminoglycans and proteoglycans content following interleukin-1b treatment: this may have the effect of “unmasking” collagen fibers, rendering them more susceptible to the digestion by metalloproteinases, whose gene expression is stimulated by interleukin-1b treatment [1,26]. Since a deficit in the production of a natural antagonist of the IL-1 receptor has been demonstrated [1] it is possible that an individual sensibility to IL-1b stimulation occurs. These speculations are in agreement with data showing that cartilage degenerates without IL-1b variations [19], and that glucosamine is not able to reverse proteoglycan decrease in interleukin-1b-treated bovine cartilage [6]. In conclusion, our data suggest that interleukin-1b in bovine metacarpophalangeal cartilage acts directly on the pathways of glycosaminoglycan synthesis and degradation, without modifying the viability of chondrocytes; moreover, suggest that the changes in ECM composition may play an important role in maintaining osteoarthritis damage. Since the susceptibility to IL-1b concentrations varies in cartilage at different anatomical locations [2] and in vitro system, where several stimulation such as hormones and cytokines are missing, this must always be considered when comparing in vivo and in vitro data. Further work, however, is necessary to investigate the relation between interleukin-1b and of cartilage damage. References [1] [2]
Table 2 Cell viability of bovine cartilage cultures 24 h after interleukin-1b treatment C IL-1b
% Chondrocytes viability 100 98 ± 4.8
The percentage of cell viability was normalized to controls (100%). The values are mean ± S.D. of five experiments. C, controls; IL-1b, interleukin-1b.
[3]
[4]
Abramson SB, Amin A. Blocking the effects of IL-1 in rheumatoid arthritis protects bone and cartilage. Rheumatology 2002;41:972. Barakat AF, Elson CJ, Westacott CI. Susceptibility to physiological concentrations of IL-1b varies in cartilage at different anatomical locations on human osteoarthritic knee joints. Osteoarthritis Cartilage 2002;10:264. Brill S, Zvibel I, Halpern Z, Oren R. The role of fetal and adult hepatocyte extracellular matrix in the regulation of tissue-specific gene expression in fetal and adult hepatocytes. Eur J Cell Biol 2002; 81:43. Chevalier X. Upregulation of enzymatic activity by interleukin-1 in osteoarthritis. Biomed Pharmacother 1997;51:58.
G. Stabellini et al. / Biomedicine & Pharmacotherapy 57 (2003) 314–319 [5] [6]
[7]
[8]
[9]
[10] [11]
[12]
[13] [14]
[15]
[16]
Conrozier T. Death of articular chondrocytes. Mechanisms and protection. Presse Med 1998;27:1859. De Mattei M, Pellati A, Pasello M, de Terlizzi F, Massari L, Gemmati D, et al. High doses of glucosamine-HCl have detrimental effects on bovine articular cartilage explants cultured in vitro. Osteoarthritis Cartilage 2002;10:816. Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 1986;883:173. Gagliano N, Arosio B, Grizzi F, Masson S, Tagliabue J, Dioguardi N, et al. Reduced collagenolytic activity of matrix metalloproteinases and development of liver fibrosis in the aging rat. Mech Ageing Dev 2002;123:413. Gouze JN, Bordji K, Gulberti S, Terlain B, Netter P, Magdalou J, et al. Interleukin-1beta down-regulates the expression of glucuronosyltransferase I, a key enzyme priming glycosaminoglycan biosynthesis: influence of glucosamine on interleukin-1beta-mediated effects in rat chondrocytes. Arthritis Rheum 2001;44:351. Ghosh P, Smith M. Osteoarthritis, genetic molecular mechanisms. Biogerontology 2002;3:85. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 1982;126:131. Grimshaw MJ, Mason RM. Bovine articular chondrocyte function in vitro depends upon oxygen tension. Osteoarthritis Cartilage 2000;8: 386. Handley CJ, Buttle DJ. Assay of proteoglycan degradation. Methods Enzymol 1995;248:47. Kuettner KE, Pauli BU, Gall G, Memoli VA, Schenk RK. Synthesis of cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture characteristics, and morphology. J Cell Biol 1982;93:743. Iniguez MA, Pablos JL, Carreira PE, Cabr F, Gomez-Reino JJ. Detection of COX-1 and COX-2 isoform in synovial fluid cells from inflammatory joint diseases. Br J Rheumatol 1998;37:773. Locci P, Marinucci L, Lilli C, Martinese D, Becchetti E. Transforming growth factor b1-hyaluronic acid interaction. Cell Tissue Res 1995; 281:317.
319
[17] Lotz M. The role of nitric oxide in articular cartilage damage. Rheum Dis Clin North Am 1999;25:269. [18] Malemud CJ. Fundamental pathways in osteoarthritis: an overview. Front Biosci 1999;15:D659. [19] Sakano Y, Terada N, Ueda H, Fujii Y, Hamada Y, Akamatsu N, et al. Histological study of articular cartilage in experimental rat knee arthritis induced by intracapsular injection of cationic polyethyleneimine. Med Electron Microsc 2000;33:246. [20] Scott JE. The molecular biology of histochemical staining by cationic phthalocyanin dyes: the design of replacements for Alcia blue. J Microsc 1980;119:373. [21] Scott JE, Dorling J. Differential staining of acid glycosaminoglycans (mucopolysaccharides) by Alcian blue in salt solutions. Histochimie 1965;5:221. [22] Stabellini G, Calvitti M, Baroni T, Marinucci L, Calastrini C, Carinci P, et al. Glycosidases during chick embryo lung development and their colocalization with proteoglycans and growth factors. Eur J Histochem 2002;46:41. [23] Stove J, Gerlach C, Huch K, Gunther KP, Puhl W, Scharf HP. Effects of hyaluronan on proteoglycan content of osteoarthritic chondrocytes in vitro. J Orthop Res 2002;20:551. [24] Sztrolovics R, Recklies AD, Roughley PJ, Mort JS. Hyaluronate degradation as an alternative mechanism for proteoglycan release from cartilage during interleukin 1b-stimulated catabolism. Biochem J 2002;362:473. [25] van der Kraan PM, van den Berg WB. Anabolic and destructive mediator in osteoarthritis. Curr Opin Clin Nutr Metab Care 2000;3: 205. [26] Vincenti MP, Brinckerhoff CE. Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex signaling pathways for the recruitment gene-specific transcription factors. Arthritis Res 2002;4:157. [27] Wasteson A, Uthne K, Westermark B. A novel assay for the biosynthesis of sulphated polysaccharide and its application to studies on the effects of somatomedin on cultured cells. Bioc or medium alone (controls). Biochem J 1973;136:1069.