ARTICLE |
Correspondence to: Emma Kavanagh, Dept. of Veterinary Basic Sciences, The Royal Veterinary College, University of London, London NW1 0TU, UK. E-mail: e.kavanagh@vcl.ac.uk
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Summary |
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Changes in glycosaminoglycan (GAG) content and distribution are vital for joint development. However, their precise character has not been established. We have used immunohistochemistry (IHC) and "critical electrolyte" Alcian blue staining to assess such changes in developing chick and rabbit joints. IHC showed chondroitin sulfate labeling in chick epiphyseal cartilage but not in interzones. In contrast, prominent labeling for keratan sulfate (KS) was restricted to chick cartilageinterzone interfaces. In rabbit knees, KS labeling was also prominent at presumptive cavity borders, but weak in interzone and cartilage. Selective pre-digestion produced appropriate loss of label and undersulfated KS was undetectable. Quantification of Alcian blue staining by scanning and integrating microdensitometry showed prominent hyaluronan-like (HA-like) interzone staining, with chondroitin sulfate and weaker KS staining restricted to epiphyseal cartilage. Hyaluronidase decreased HA-like staining in the interzone. Surprisingly, keratanases also reduced HA-like but not sulfated GAG (sGAG-like) staining in the interzone. Chondroitinase ABC had little effect on HA-like staining but decreased sGAG staining in all regions. Rabbit joints also showed HA-like but not KS staining in the interzone and strong chondroitin sulfate-like staining in epiphyseal cartilage. Our findings show restricted KS distribution in the region close to the presumptive joint cavity of developing chick and rabbit joints. Alcian blue staining does not detect this moiety. Therefore, it appears that although histochemistry allows relatively insensitive quantitative assessment of GAGs, IHC increases these detection limits. This is particularly evident for KS, which exhibits immunolabeling patterns in joints from different species that is consistent with a conserved functional role in chondrogenesis. (J Histochem Cytochem 50:10391047, 2002)
Key Words: articular cartilage, joint development, hyaluronan, glycosaminoglycans, chondrogenesis, quantitative histochemistry, immunochemistry, embryonic, chick, rabbit
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Introduction |
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THE FORMATION of a synovial joint requires several processes, such as separation of the elements during cavitation, formation and insertion of ligaments and menisci, and elaboration of synovial lining, to occur in unison with continuing growth. For all these processes to occur many signals are required and these must be co-ordinated by mechanisms that we do not yet fully understand. Regardless of the mechanisms involved, changes in the extracellular matrix (ECM) are essential during the elaboration of synovial joint cavities and the articular surfaces.
The putative articular cartilage can be distinguished from the underlying epiphyseal cartilage in the mouse and rabbit at very early stages of joint development. The former contains collagen Type V and fibromodulin and the latter contains matrilin-1 (
It is possible to distinguish among the GAGs HA, chondroitin and dermatan sulfate (CS and DS) and keratan sulfate (KS) using the cationic copper phthalocyanin dye Alcian blue (AB). This is bound electrostatically by carboxyl, phosphate, and sulfate groups in the tissues. Binding is dependent on the charge density of the substrate and on the pH and concentration of other cations in the dye bath. It has been proposed that for steric reasons AB is a very good dye for GAGs (
The sulfated GAGs can also be localized using monoclonal antibodies. Antibodies raised against chondroitin-4- and chondroitin-6-sulfates, 2B6 and 3B3, respectively, recognize epitopes at the GAG linkage region to the core protein (
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Materials and Methods |
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All reagents and labeled antibodies were from Sigma (Poole, UK) unless otherwise stated.
Tissue Preparation
Chick Limbs.
Embryonic White Leghorn chick hind limbs from Hamburger and Hamilton stages 39 and 41 (at least three chicks at each stage) were mounted on cork, covered with 10% PVA (grade GO4/140; Waker Chemicals, Surrey, UK) and immersed in n-hexane (grade low in aromatic hydrocarbons; BDH, Poole, UK) cooled to -70C. Samples were stored at -70C until used (
Rabbit Limbs. Hind limbs distal to the mid-femoral region were removed from New Zealand White rabbit fetuses aged 17, 20, and 25 days (at least two rabbits at each stage). Tissues were fixed in 4% paraformaldehyde in 0.05 M Tris-HCl buffer, pH 7.3, for 18 hr, then washed extensively in buffer. With the exception of 17-day joints, tissues were decalcified in 14.3% EDTA, pH 7.0, until radiographically free of calcium. After washing, the tissue was dehydrated, cleared in methyl salicylate, and embedded in paraffin. Sagittal longitudinal sections were cut at 7 µm. For histological observations, sections of both chick and rabbit joints were stained with 0.1% Toluidine blue in 0.1 M acetate buffer at pH 6.2 or with hematoxylin and eosin.
GAG Digestion
Hyaluronidase (HAase).
Sections were incubated in 0.5 U/ml Streptococcus dysgalactiae hyaluronidase (AMS Biotechnology; Oxfordshire, UK) in 50 mM Tris buffer, pH 6.0, for 30 min at 37C. This enzyme also acts on chondroitin, but the rate of degradation for chondroitin is 1/101/20 compared with that for hyaluronic acid. This enzyme does not act on CS A, CS C, DS, heparin, HS and KS.
Chondroitinase (Chase).
Chase ABC (Proteus vulgaris; AMS Biotechnology) cleaves N-acetylhexosaminide linkages in chondroitin 4-sulfate, chondroitin 6-sulfate, DS, chondroitin, and HA, yielding mainly disaccharides with -hexuronate at the non-reducing ends. The initial rates of enzymatic degradation of CS C, DS, chondroitin, and HA are 1.0, 0.4, 0.2, and 0.02, respectively, relative to the rate of CS A degradation. This enzyme does not act on KS, heparin, and HS. Chase AC (II Arthro from Arthrobacter aurescens; AMS Biotechnology) cleaves N-acetylhexosaminide linkages in chondroitin, chondroitin 4-sulfate, and chondroitin 6-sulfate, yielding
-unsaturated disaccharides (DDi-OS, DDi-4S, and DDi-6S). The enzyme exhibits a comparable activity towards HA. DS (chondroitin sulfate B) is not attacked. Both were used at 0.1 U/ml in 50 mM Tris acetate buffer, pH 7.0, for 30 min at 37C.
Keratanase (KSase). For digestion of KS, two keratanases were used. KSase II was from Bacillus species (AMS Biotechnology), which hydrolyzes 1, 3-ß-glucosaminidic linkage to galactose in KS, and requires the sulfate at the 6-0-position of the participating glucosamine but acts independently of the sulfate at the 6-0-position of the galactose residue. KSase II was used at 0.001 U/ml in 10 mM acetate buffer, pH 6.5, for 30 min at 37C and was followed by keratanase I from Pseudomonas (AMS Biotechnology). This hydrolyzes ß-galactosidic linkages in KS, in which non-sulfated galactosyl residues participate. Keratanase I was used at 0.1 U/ml in 50 mm Tris buffer, pH 7.4, for 30 min at 37C.
Immunohistochemistry
Antibodies.
Mouse monoclonal antibodies 2B6, 3B3, 5D4, MZ15, and 2D3 were a kind gift from Prof. B. Caterson. MAbs 2B6 and 3B3 recognize epitopes on CS and DS after treatment with Chases. MAb 2B6 recognizes mainly C-4-S epitopes but can also recognize DS (
Frozen Sections.
Sections were brought to ambient temperature, allowed to dry for at least 1 hr, and washed in TBS (pH 7.6). For labeling with MAb 2B6 or 3B3, sections were pre-treated with Chase AC II or with Chase ABC, respectively, and subsequently washed three times in TBS containing 20% Tween-20 (TBS-T) for 5 min to unveil the epitopes for the antibodies (
Paraffin Sections. Sections were de-waxed and re-hydrated. They were incubated in 0.1% trypsin in TBS containing 0.1% CaCl2, pH 7.8, at 37C for 1 hr and after washing were exposed to 2% L-lysine in PBS for 15 min to block free aldehyde groups. The sections were washed in PBS between each treatment. To block nonspecific binding, the sections were pre-incubated in heat-inactivated normal rabbit serum (Harlem Sera Labs; Loughborough, UK) containing 4% bovine serum albumin (BSA) and 0.3% Triton X-100 for 30 min. This was drained from the slide and the sections were incubated overnight at 4C in 1% BSA in TBS containing primary antibody. These were either MAb 5D4 (1:350) or MAb 2D3 (1:150). After washing the sections were incubated in alkaline phosphatase-labeled goat anti-mouse IgG antibodies at 1:50 in 1% BSA for 90 min at RT. The activity of the alkaline phosphatase label was localized using the following reaction medium: 5 µg naphthol AS-BI phosphate dissolved in 1 drop dimethylformamide was added to 5 µg Fast Red TR in 10 ml veronal acetate buffer, pH 9.2. Levamisole (1 µg/ml) was added to inhibit endogenous alkaline phosphatase activity. Sections were incubated for 20 min, washed, and mounted in glycerine jelly. For controls the primary antibody was replaced with normal mouse serum.
Alcian Blue Staining at Critical Electrolyte Concentrations
Adjacent serial cryostat sections were air-dried and fixed in 4% formaldehyde in phosphate buffer, pH 7.4, for 30 min and then washed. Paraffin sections were de-waxed and re-hydrated. Cryostat sections were immersed in 0.025 M acetate buffer containing 0.05% AB 8GX and either 0.025 M, 0.5 M, 0.8 M, or 1.0 M MgCl2 at a final pH of 5.8 for 18 hr. Paraffin sections were treated similarly except that 0.05 M, 0.4 M, 0.8 M MgCl2 concentrations were used. Sections were washed three times in the corresponding buffer containing MgCl2, dehydrated rapidly, and mounted in DPX. Where stated, sections were also stained in this manner after appropriate enzymatic treatment (see above).
Quantification of Alcian Blue Staining in Developing Chick Joint
AB staining intensity was measured in the intermediate layer, articular surface layer (fibrocartilage), and epiphyseal cartilage of both proximal and distal elements in triplicate longitudinal sections (approximately 1 mm apart lateromedially) of at least three embryonic chick joints using a Vickers M85A scanning and integrating microdensitometer (
The staining intensity that can be attributed to "HA-like" content per unit area was calculated in each of the defined zones as the measured intensity of AB staining at 0.025 M MgCl2 ("total GAGs") less the intensity measured in the same zone at 0.5 M MgCl2. Intensity of staining measured at 0.5 M MgCl2 was attributable to sGAG. In some cases, to provide a relative HA or sGAG content, these calculated values were expressed as a percentage of total GAG staining (i.e., that measured at 0.025 M MgCl2). The decreases in "HA-like" or sGAG staining in response to enzymatic pre-treatment were calculated from the quantitative decline (expressed as a percentage of the level evident in undigested sections) in AB staining in serial sections (triplicate sections from at least two joints) that were pre-digested with S. dysgalactiae HAase, Chase ABC, or KSase I and II before AB staining.
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Results |
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Structural Differences in Joint Organization
To accommodate species differences in joint structure, the following nomenclature will be used. At the presumptive joint, a region named the interzone separates the cartilaginous anlagen of the adjacent bones (Fig 1A1D). Classically, this interzone comprises two chondrogenous layers separated by a thin intermediate layer (
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Quantitative Changes in Alcian Blue Staining During Chick Joint Cavitation
Before the overt cavitation (stage 39), levels of HA-like GAG (AB staining intensity measured at 0.025 M MgCl2 less that measured at 0.5 M MgCl2) exhibit no significant differences across distinct regions of chick tibiotarsal joints (Fig 2A). To provide a measure of relative HA content as a fraction of total GAG content, HA-like staining intensity can be compared with staining intensity for total GAGs. Thus, when expressed as a percentage of total GAG, the major species present in interzone is HA-like, where it constitutes between 8595% of total GAGs (Fig 2B). In epiphyseal cartilage it is only 50% of total GAG content (Fig 2B). In contrast, sGAG content exhibits marked differences across the joint. A significantly lower content is found in the intermediate layer and articular layer than in epiphyseal cartilage (Fig 2A). As a percentage of total GAG, sulfated GAG content was lower (<15%) in the articular layers and intermediate layer than in epiphyseal cartilage, where sGAGs constitute approximately 50% of total GAGs (Fig 2A). Further increases in MgCl2 concentration from 0.5 to 0.7 M resulted in the retention of very weak staining in epiphyseal cartilage but none in either the articular layer or intermediate layer (not shown). At 1.0 M MgCl2, sections showed no staining at all (not shown). It is therefore apparent that there is an absence of KS-like Alcian blue staining in the developing articular layer or intermediate layer. This distribution of GAGs did not change significantly at later stages.
Qualitative Changes in Alcian Blue Staining During Rabbit Joint Cavitation
Before cavitation (days 17 and 20), the epiphyseal cartilage in rabbit knee joints showed strong AB staining at 0.05 M MgCl2, whereas staining in the intermediate layer and articular layers was weaker (Fig 3A). At an MgCl2 concentration of 0.4 M, the staining intensity in epiphyseal cartilage was only slightly reduced, whereas that in the intermediate layer and articular layers was markedly diminished (Fig 3B). At 0.8 M MgCl2, the epiphyseal cartilage retained weak staining but staining in the intermediate layer and developing articular layers was not evident. After cavitation (25 days), AB staining of epiphyseal cartilage and the articular layer was strong at 0.05 M MgCl2. It was reduced in the articular layer, but not in the epiphyseal cartilage, at 0.4 M MgCl2, and all regions were negative at 0.8 M MgCl2 (not shown).
Effect of Enzyme Digestions on GAG Content Determined by Alcian Blue Staining
Effect on HA Content.
In cavitating chick tibiotarsal joints (stage 39), S. dysgalactiae hyaluronidase resulted in a significant reduction of approximately 60% in AB staining for HA-like GAGs in the intermediate layer and articular layers, whereas staining was reduced by only 20% in epiphyseal cartilage (Fig 4A). Serial sections pre-treated with Chase ABC did not exhibit a significant change in staining for HA-like GAGs in any region, and the very small decreases seen in the articular layers are consistent with a restricted action of this enzyme on HA (Fig 4A). In contrast, KSase I and II pre-treatment did produce a decrease of approximately 35% in AB staining for HA-like GAGs in the intermediate layer and articular layers, whereas HA-like staining in the epiphyseal cartilage was not significantly altered (Fig 4A).
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Effect on sGAG Content. S. dysgalactiae HAase treatment of tibiotarsal joints (stage 39) resulted in small decreases of less than 30% in sGAG staining in epiphyseal cartilage and articular layer matrix (Fig 4B). By comparison, Chase ABC pre-treatment produced considerable decreases in AB staining for sGAGs in all regions, by approximately 90% in the epiphyseal cartilage matrix, 75% in articular layers, and 60% in intermediate layer (Fig 4B). KSase I and II digestion failed to modify sGAG staining in articular layers and intermediate layer and produced only a modest decrease of <30% in sGAG staining in epiphyseal cartilage (Fig 4B).
IHC Patterns of GAG Epitope Expression in Embryonic Chick Joints
Immunolabeling for C4S or C6S, using either MAb 2B6 (not shown) or MAb 3B3, respectively, was evident in the ECM of epiphyseal cartilage in cavitating metatarsalphalangeal chick joints (stage 39) but there was no CS labeling in developing articular layers or the intermediate layer (Fig 5A). In serial chick joint sections, both MAb MZ15 and MAb 5D4 exhibited prominent labeling for "sulfated" KS in the ECM of the articular layer, and this was most prominent at the interface between epiphyseal cartilage and the fibrocartilage of the articular layer (Fig 5B). Little if any KS labeling was evident in the intermediate layer, and none was observed in the deeper epiphyseal cartilage (Fig 5B). Using the antibody selective for undersulfated KS (2D3) no labeling was detected in any region of the developing joint (not shown). Immunolabeling for CS and KS epitopes was lost from sections pre-treated with Chase ABC and KSase I and II, respectively (not shown). In all cases, substitution of the primary antibodies by normal mouse serum resulted in loss of labeling (not shown).
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IHC Patterns of KS Epitope Expression in Fetal Rabbit Joints
Immunolabeling for sulfated KS (MAb 5D4) was prominent in the matrix at the junction between the chondrogenic and intermediate layers of the interzone (Fig 6A). Labeling was also evident in the chondrocytes of the epiphyseal cartilage, but little labeling of the matrix was observed. No labeling for undersulfated KS (MAb 2D3) was observed in any regions (not shown). There was no binding of MAb 5D4 after KSase II pre-treatment (Fig 6B), and substitution of primary antibodies by normal mouse serum resulted in complete loss of labeling (not shown).
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Discussion |
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An overall picture of distribution patterns of different GAGs in developing joints may be deduced from our results. They indicate a distribution in embryonic joints where (a) the epiphyseal cartilage is rich in CS proteoglycans, (b) the articular layers exhibit a selective distribution of KS-rich proteoglycan, and (c) the joint line is HA-rich. Similarity in the GAG distribution in embryonic rabbit joints, with their tri-laminate organization, and chick joints, with the pronounced fibrocartilage articular layer, support a conserved role for these moieties.
Our results using Alcian blue and IHC both indicate that there is very little CS in the developing articular layers of cavitating joints. However, it is present in the underlying epiphyseal cartilage in both species. The results of our enzymatic digestion studies support this, with Chase ABC decreasing sGAG-like AB staining in epiphyseal cartilage by approximately 80%. This is also in agreement with IHC studies in Monodelphis, which show that C6S, but not C4S, stubs on aggrecan are localized in epiphyseal cartilage, but not in the interzone, before cavitation (
The GAG content of the epiphyseal cartilage therefore appears to be predominately CS. However, streptococcal HAase produced a 30% decrease in sGAG AB staining in the epiphyseal cartilage. This could reflect a residual HAase activity towards chondroitin moieties present at this site, or may be caused by degradation of the HA backbone of PG aggregates and subsequent release of sGAGs from the matrix. This effect of HAase is supported by the streptococcal HAase-induced decreases of approximately 20% in AB staining for sGAG components in both the intermediate and developing articular layers (including chick fibrocartilage). Moreover, KSase I and II digestion failed to modify such sGAG AB staining in the articular and intermediate layers. The lack of immunolabeling for CS suggests either that an undersulfated form of KS, not detectable by AB (with greater than 0.5 M MgCl2), is present in these tissues, or that this KS is of small size or at too low a concentration to be detected by AB. However, the IHC labeling indicates unequivocally that KS epitopes are present in interzones of both the chick and the rabbit. The fact that KSase I and II treatment markedly diminishes HA-like AB staining in the intermediate layer and developing articular layers supports this and suggests that although KS is not disclosed on the basis of its charge density, it is indeed present in these regions.
IHC labeling of KS eiptopes using both MAb 5D4 and MAb MZ15 revealed the presence of KS in the matrix of presumptive joint surfaces of the rabbit and at the articular/epiphyseal cartilage interface in the chick. However, the AB staining did not detect this KS-rich band of matrix. Although the degree of sulfation and hence the charge density of KS is variable, this anomaly does not appear to be due to the presence of an undersulfated form of KS at these sites, as none could be detected using MAb 2D3 in the chick or rabbit. Immunolabeling has previously shown that KS is detectable in a similar region of developing Monodelphis joints (
From our studies it is clear that small quantities of KS exhibit a distribution that is restricted to chondrogenous layers of developing joints and that this distribution is conserved across avian, mammalian, and marsupial species. If this distribution were evident only in mammalian and marsupial joints, one might assume that this KS plays a role in either the elaboration of an articular surface or in maintaining chondrogenesis. However, its presence in avian joints, with their distinct fibrocartilaginous articulating surfaces, appears to support the notion that this KS-rich region is intimately involved in establishing a site for continued chondrogenesis.
Examination of changes in sGAG distribution during neural crest development has shown that chondroitin-6-sulfate and certain KS PGs are absent from regions containing actively migrating neural crest cells but are present along their prospective pathways for migration (
Another observation in chick joints is the presence of relatively large quantities of HA in the interzone. This is not surprising, considering the major role HA is believed to play in tissue separation processes (
The immunochemical detection of KS at these sites suggests an enrichment of KS-rich PGs in the chondrogenic regions of developing rabbit and chick joints. This is closely associated with those sites at which HA content is elevated during the cavity-forming process. It is tempting to speculate that these KS-rich regions play a role in limiting local HA-driven tissue disruption and that such conserved distribution of KS plays an essential role within these chondrogenic zones of articular cartilage.
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Acknowledgments |
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We are very grateful to the Arthritis Research Campaign for supporting Dr E. Kavanagh and for the Departmental support for Anne Osborne.
We wish to thank Yvette Bland and Helen Hunt for technical assistance, Prof Bruce Caterson for generously providing the antibodies, and Prof Tim Hardingham and Dr Claire Hughes for helpful discussions and thoughtful consideration of this work.
Received for publication August 20, 2001; accepted February 20, 2002.
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