Histochemical Journal 23, 503-508 (1991)
Type III collagen in the intervertebral disc S. R O B E R T S 1., J. M E N A G E 1, V. D U A N C E 2 a n d S. F. W O T T O N 2
1Charles Salt Research Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, Shropshire, SYIO 7AG and 2Muscle and Collagen Research Group, Department of Veterinary Medicine, University of Bristol, Langford BS18 7DY, UK Received 28 November 1990 and in revised form 23 April 1991
Summary Several collagen types have now been isolated from the intervertebral disc, although type III collagen has previously only been extracted from human pathological disc. In this study, type III collagen has been isolated from normal human and bovine inter"vertebral disc and immunolocalized in sections of rat, sheep, bovine and 'normal' human intervertebral disc of various ages. Staining with antisera to type III collagen is localized primarily around the cells. Results indicate that cells of the disc sit in 'chondrons', similar to those seen in the deep and mid zones of articular cartilage. We suggest that type III collagen is present in the intervertebral disc and hypothesize that it may be involved in the organization of the pericellular environment, perhaps linking the chondron capsule to the interterritorial matrix.
Introduction The intervertebral disc consists of two regions, the inner nucleus pulposus and outer annulus fibrosus. Collagen accounts for approximately 15% and 50% of the dry weight respectively (Ayad & Weiss, 1986). Eyre and Muir (1974) demonstrated collagen polymorphism in pig intervertebral disc by identifying the presence of two molecular species. They found that the disc contained both types I and II collagen, with type I being predominant in the outer annulus, but the proportion of type II increasing and becoming predominant towards the nucleus pulposus. Since then the picture has become increasingly complex with five further collagen types reported to occur in intervertebral disc. Types II, VI, IX and XI have been isolated from the nucleus pulposus (Eyre & Muir, 1977; Ayad et al., 1981; Ayad et al., 1982; Ayad & Weiss, 1986; Wu et al., 1987) and types I, II, V, VI, IX and XI from the annulus fibrosus (Eyre & Muir, 1977; Ayad & Weiss, 1986; Wu et al., 1987). Type III collagen has been demonstrated by immunofluorescence in both normal and pathological h u m a n intervertebral discs (Roberts et aI., 1982; Stevens et al., 1982) but has only been isolated biochemically from degenerate intervertebral discs (Adam & Deyl, 1984). In view of these findings we have investigated the presence of type III collagen by immunohistochemistry in intervertebral discs and in the adjacent cartilage endplates, from animals of various species and of * To whom all correspondence should be addressed. 0018--2214/91 $03.00 +.12 9 1991 Chapman & Hall
different ages. Particular attention has been paid to the location of the staining in relation to the cells of the tissues. In addition, we have partially purified extracted material from h u m a n and bovine intervertebral discs and examined these extracts for type III collagen using a highly sensitive immunoblotting procedure.
Materials and methods Specimens Intervertebral discs were collected from newborn, seven week and seven month old rats, newborn and seven month old sheep and 18 month old cattle. Human discs (34) were removed from the lumbar spine of 14 individuals as soon as possible post mortem (age range 21-85 years, mean 64 _+ 19). The sagittal slice was removed (see Fig. 1) and columns from both nucleus and annulus, with adjoining cartilage endplate and bone, were frozen in hexane and stored in liquid nitrogen until required. Preparation of antisera Antisera were raised against type III collagen which had been pepsin extracted from human placenta. Antisera were raised in sheep, goat or guinea pig; that from sheep and goat was affinity purified (Stephens et al., 1982). Specificity of the antisera was checked by ELISA for cross reactivity against types I, III, IV and VI collagens. There was minimal cross reaction with any type, other than type III collagen. Sheep anti-type III collagen antibody was used at a dilution of 1:250 for immunofluorescence and 1:1000 for
504 immunoperoxidase and was used routinely. Guinea pig anti-type III and goat anti-type III collagen, when used for immunofluorescence, were diluted 1 in 20 and 1 in 5 respectively. All dilutions were in phosphate buffered saline (PBS) or TRIS buffered saline (TBS) for immunofluorescence or immunoperoxidase respectively.
Commercial antisera Antiserum to human and bovine type III collagen was obtained from Southern Biotechnology Associates Inc., USA (via Bionuclear Services Ltd, UK). It was used at a dilution of 1:100 and 1:1000 for immunofluorescence and immunoperoxidase respectively. Fluorescein- and peroxidase-labelled secondary antibodies were obtained from Nordic Immunologicals Ltd, UK and used at a dilution of 1:50.
Immunolocalization Frozen sections were cut on a Bright's rotary cryostat at a thickness of 6 ~tm and collected onto polylysine coated slides. Following enzymatic digestion (see below) sections were fixed with 4% paraformaldehyde or 10% formaldehyde for 10rain before being stained by indirect immunofluorescence or immunoperoxidase (Newton, 1984) respectively. Peroxidase staining was demonstrated with 3,4,3,4-tetra-aminobiphenyl hydrochloride (BDH; 0.6 mg m1-1 of TBS containing 4 ~ m1-1 of 10 volume solution hydrogen peroxide). Incubations with all antisera were for 30 min at 37~ in a humid chamber. No blocking agents were used for immunofluorescence, whilst for immunoperoxidase 10% normal serum, of the same species as the section, was included with the peroxidase label. Normal serum and PBS or TBS controls were included in each experiment. Adjacent sections were stained with Haematoxylin and Eosin. Fluorescence was viewed on a Leitz Dialux 20 microscope fitted with incident illumination and Phloempak 2.4 filter unit.
Enzyme pretreatment Before staining, sections were enzymatically digested by one of the following: (a) Hyaluronidase (ovine testicular, Sigma type V, 1250 units mg-1): 2mg m1-1 in 0.025M sodium chloride, 0.05 M sodium acetate buffer adjusted to pH 5.0; incubated for 120 min at 37~ This regime was used routinely. (b) Chondroitinase ABC (Seikagaku Kogyo Co. Ltd from ICN Biochemicals Ltd, UK): 0.25 units ml -I in 0.1 M tris acetate at pH 7.6; incubated for 90min at room temperature. (c) Hyaluronidase + chondroitinase: 2 mg ml 1 hyaluronidase incubated for l h at room temperature at pH 5, washed in tris acetate buffer, and incubated for a
ROBERTS, MENAGE, DUANCE and W O T T O N further 90rain at room temperature with 0.25 units 1TI1-1 chondroitinase ABC at pH 7.6 (d) Collagenase 1 (Type III, Advance Biofactures Corporation): 30 units m1-1 in 0.05 M Tris and 0.01M calcium acetate, pH 7.2; incubated for 30 rain at room temperature. (e) Collagenase 2 (Type III, Advance Biofactures Corporation): 30 units m1-1 in 0.05 M tris and 0.01 M calcium acetate pH 7.2; incubated overnight at room temperature, with fresh solution applied 2x 30 min and l x 60 min at 37~ (f) No enzyme treatment.
Biochemical analysis Nucleus pulposus and annulus fibrosus were separated from both human and bovine discs and were extracted with 4 M guanidine hydrochloride in 0.05 M Tris, pH 7.4, following treatment with pepsin (1:100 weight/wet weight of tissue in 0.5 M acetic acid). Dialysis against water produced a precipitate which was freeze dried and analysed by SDSpolyacrylamide gel electrophoresis (Laemmli, 1970) and Western blotting (Towbin et al., 1979). Detection was carried out using the guinea pig anti serum to human type III collagen with either an alkaline phosphatase or a peroxidase conjugated second antibody. Alkaline phosphatase detection was performed using a mixture of 5-bromo-4chloro-3-indolyl phosphate (Sigma) and Nitro Blue Tetrazolium (NBT) (Sigma) as substrate (Harlow & Lane, 1988). Peroxidase activity was detected using the highly sensitive light generating procedure (ECL Detection System, Amersham).
Results
Distribution pattern in disc Staining with all four p r e p a r a t i o n s of antisera to t y p e III collagen occurred a r o u n d the cells of the rat, s h e e p a n d h u m a n intervertebral disc. This w a s m o r e obvious in the nucleus p u l p o s u s t h a n in the a n n u l u s fibrosus. Typically a b a n d a r o u n d , b u t not b o r d e r i n g the cell (A; Fig. 2), a l w a y s stained positively w h e r e a s the cell itself a n d the area directly adjacent to it varied, s o m e t i m e s b u t not always staining. In s o m e cases the region (A) w h i c h typically stained h a d the a p p e a r a n c e of a series of concentric rings (Fig. 3). In addition to the pericellular pattern, there w a s in s o m e discs a degree of staining of the fibres within the interterritorial matrix. In adjacent sections stained w i t h H a e m a t o x y l i n a n d Eosin these discs often s h o w e d d e g e n e r a t i v e c h a n g e s such as clustering of the cells or disorganization of the matrix. In m a n y
Fig. 1. Schematic representation of sampling of human intervertebral disc. A : anterior, P : posterior. Fig. 2. Peroxidase staining for type III collagen in the nucleus of human intervetebral disc. The capsule and beyond (A) is positive while the cell (C) can be positive (a) or negative (b). (c) Control section incubated with normal sheep serum in place of antiserum, x600. Fig. 3. Immunofluorescent staining for type III collagen sometimes forms a series of concentric rings. Section of human nucleus, stained with antiserum raised in sheep, x600. Fig. 4. Heavy staining for type III collagen was seen in the human intervertebral disc (D) where it bordered the endplate (E). Nucleus pulposus, sheep antiserum with peroxidase label, x600.
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506 cases, cells at the junction between the cartilage endplate and disc showed particularly heavy staining with antisera to type III collagen (Fig. 4). No difference was seen with either location within the lumbar spine, or with age of the specimen. Staining in the cartilage endplate Cells of the cartilage endplate have an elongated, fibroblast-like appearance, lying horizontally in the same direction as the collagen fibres. When stained with antisera to type III collagen, sections of endplate show a varied pattern, some cells and sometimes the area close to them, staining positively (Fig. 5), whilst other cells are negative. Abnormalities can be demonstrated on sections stained with Haematoxylin and Eosin, such as splits in the endplate, or Schmorl's nodes, where the disc protrudes into the vertebral body. In these abnormal regions, there was usually a greater degree of staining with anti type III collagen. Effect of enzyme pretreatments Sections which were incubated initially with hyaluronidase, chondroitinase ABC, both of these sequentially or collagenase 1 (see Materials and methods) stained with similar intensity. In contrast those sections which were subjected to no enzyme pretreatment, or to collagenase 2 incubation (see Materials and methods) showed the same staining pattern but at a greatly reduced intensity. Biochemical analysis Biochemical analysis revealed small amounts of type III collagen in the annulus and nucleus of bovine and human discs after reduction with mercaptoethanol (Fig. 6). The type III collagen, which was not detectable on the gel by Coomassie Blue staining (not shown), only became apparent after immunoblotting, indicating that the amounts extracted were small, less than 2% of the total protein, the approximate detection limits of Coomassie Blue staining. Discussion
Type III collagen occurs in many soft distensible connective tissues such as dermis, uterus and blood vessel walls (Gay & Miller, 1978). For the most part, the fibres appear as a fine reticular network, in contrast to the larger well structured fibres of type I collagen. In the intervertebral disc, type III collagen has been demonstrated by immunofluorescence in both normal and pathological human tissue (Beard et al., 1981; Stevens et al., 1982; Adam & Deyl, 1984). However, until now it has only been isolated biochemically from discs showing degenerate changes (Adam & Deyl, 1984). In the present study we have used four preparations of antisera to type III collagen and obtained positive staining with all of them in normal
ROBERTS, MENAGE, DUANCE and WOTTON discs from various species. Although the response was stronger when sections were first digested with hyaluronidase, staining was present without any enzymatic digestion. This is in contrast to staining with antiserum to type VI collagen, which was absent without prior enzymatic digestion (Roberts et al., in press). Results from this and a previous study indicate that the matrix around the cells of the disc is highly organized. Although the staining for type III collagen was predominantly pericellular it generally occupied a region beyond that which stained positively with antiserum to type VI collagen (Roberts et al., in press). The cells of the mid and deep zones of articular cartilage have been shown to sit in a discrete area of pericellular matrix contained within a capsule, the whole structure being described as a chondron (Poole et al., 1986). Minor collagens, including type VI and type IX have been shown to locate preferentially in these pericellular capsules (Poole et al., 1988a,b). It appears that similar structures are present in both the annulus fibrosus and nucleus pulposus of the intervertebral disc. In the disc the pericellular capsule, and in some cases the pericellular matrix adjoining the cell, were labelled with antiserum to type VI collagen, with staining for type III collagen outside this (Fig. 7). Koller et al. (1989) suggest that the globular domains of type VI collagen may be capable of interacting with collagenous structures. Homologous repeats have been found on all three polypeptide chains, which resemble sequence motifs in von Willebrand factor and cartilage matrix protein, both of which bind to collagen fibres (Chu et aI., 1990). If one of these repeats showed a specificity for type III collagen, it is possible that the microfilaments of type VI collagen could interact with type III fibres as proposed by Koller et al. (1989). The relative distribution of staining with antisera to type III and type VI collagen seen in this and previous studies would support this suggestion. Von der Mark et al. (1984) found type W collagen had a similar distribution to type III collagen, the two being associated, for example, in the aortic intima. The similar distribution seen in bone and dentine has led Becker et al. (1986) to suggest that type III collagen is s o m e h o w linked to type VI collagen, at least in these tissues. Type III collagen is also found in connective tissue apparently attempting to repair an injury, for example in wound healing of the dermis or severe osteoarthritis (Gay & Miller, 1978). The increased fibrous staining seen here in intervertebral discs of degenerate histological appearance, may also be an attempt at repair. Perhaps this explains why type III collagen has, until now, only been isolated biochemically from degenerate discs where the levels may be greater than in normal tissue. Immunolocalization does have limitations as a
Type III collagen in intervertebral disc
507
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Fig. 5. Ceils of the human cartilage endplate also stained for type III collagen. Although some cells were negatively stained (N), they were visible under phase contrast. Sheep antiserum, peroxidase label x600. Fig. 6. Immunoblots of material extracted from (a) human type III standard, (b) bovine nucleus, (c) bovine annulus, (d) human nucleus and (e) human annulus, run with and without mercaptoethanol (Me), and blotted with guinea pig anti human type III antibody. Fig. 7. Staining for type VI collagen is seen in the r e , o n of the pericellular capsule, inside the region typically staining for type III collagen. Section of human nucleus stained with antiserum to pepsinized type VI collagen from bovine uterus, kindly donated by Dr S. Ayad, Manchester. Peroxidase label x600.
technique for identifying the presence and distribution of molecules. For example masking of the epitope by other molecules may occur, or there may be cross reaction of the antiserum with a further epitope. Checks have been made in this study to eliminate these likelihoods as far as possible, tn addition, the presence of type III collagen in the extracted material was clearly demonstrated using the immunoblotting technique. We therefore conclude that type III collagen is present in the intervertebral disc. The pericellular location suggests that it is associated with the
chondron capsule and possibly involved in the integration of this structure with the surrounding interterritorial matrix.
Acknowledgements We acknowledge the technical assistance of J. T. Wardate and H. Evans. S. F. Wotton and J. Menage gratefully acknowledge support from the Arthritis and Rheumatism Council.
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References ADAM, M. & DELL, Z. (1984) Degenerated annulus fibrosus of the intervertebral disc contains collagen type III. Ann. Rheum. Dis. 43, 258--63. A Y A D , S. & W E I S S , J. B. (1986) Biochemistry of the intervertebral disc. In The Lumbar Spine and Back Pain, 3rd edn. (edited by JAYSON, M. I. V.) pp. 100--37. London: Churchill Livingstone. A Y A D , S . , A B E D I N , M. Z . , G R U N D Y , S. & W E I S S , J . B . (1981) Isolation and characterisation of an unusual collagen from hyaline cartilage and intervertebral disc. FEBS Lett. 123, 195-9. A Y A D r S., A B E D I N ,
M. Z . , W E I S S , J. B. & G R U N D Y , S. M.
(1982) Characterisation of another short-chain disulphide-bonded collagen from cartilage, vitreous and intervertebral disc. FEBS Lett. 139, 300-4. BEARD, H.K., R O B E R T S , S. & O ' B R I E N , J. P. (1981) Immunofluorescent staining for collagen and proteoglycan in normal and scoliotic intervertebral discs. J. Bone Joint Surg. 63B, 529-34. BECKER~ J . , S C H U P P A N , D.~ B E N Z I A N t I-I., B A L S , T.~ ECKART~ G. FI., C A N T A L U P P I , C. & R E I C H A R T , P. (1986) I m m u n o histochemical distribution of collagens types IV, V, and VI and of pro-collagens types I and III in human alveolar bone and dentine. J. Histochem. Cytochem. 34, 1417-29. C H U r M.~ P A N T . , C O N W A Y ~ D . , SAITTAp B., S T O K E S , D.~ K U O H . , G L A N V I L L E , R. W. r T I M P L r R . , M A N N , K. & D E U T Z -
MANN, R. (1990) The structure of type VI collagen. Ann. N.Y. Acad. Sci. 580, 55-63. LYRE, D. R. a MUIR, H. (1974) Collagen polymorphism: two molecular species in pig intervertebral disc. FEBS Lett. 42, 192q5. LYRE, D. R. & M U I R , H . (1977) Quantitative analysis of types I and II collagens in human intervertebral discs at various ages. Biochim. Biophys. Acta 492, 29-42. GAY, S. & M I L L E R , E. J. (1978) Collagen in the physiology and pathology of connective tissue, pp. 6-7, 105. Stuttgart, New York: Gustav Fischer Verlag. H A R L O W , E. & L A N E , D. (1988) In Antibodies: A Laboratory Manual, p. 407. Cold Spring Harbor Laboratory. K O L L E R , E . , W I N T E R H A L T E R , K. H . & T R U E B , B. (1989). The globular domains of type VI collagen are related to the
collagen-binding domains of cartilage matrix protein and von Willebrand factor. EMBO J. 8, 1073-7. LAEMMLI, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-5. NEWTON, M. R. (1984) Studies on the role of the MHC and passenger leucocytes in transplantation. D. Phil. Thesis. University of Oxford. P O O L E , C. A . , F L I N T , M . H . & B E A U M O N T , B. W. (1986) Chondrons from articular cartilage. Scand. J. Rheumatol. Suppl. 60, 12. POOLE, C. A., AYAD, S. & SCHOFIELD, J. R. (1988a) Chondrons from articular cartilage: Immunolocalization of type VI collagen in the pericellular capsule of isolated canine tibial chondrons. J. Cell Sci. 90, 635-43. P O O L E , C. A . , W O T T O N , S. F. & D U A N C E , V. C. (1988b) Localization of type IX collagen in chondrons isolated from porcine articular cartilage and rat chondrosarcoma. Histochem. J. 20, 567-74. R O B E R T S , S . , B E A R D , I-I. K. & O ' B R I E N , J. IJ. (1982) Biochemical changes of intervertebral discs in patients with spondylolisthesis or with tears of the posterior annulus fibrosus. Ann. Rheum. Dis. 41, 78-85. R O B E R T S , S . , A Y A D , S. & M E N A G E , P. J. (in press) Immunolocalisation of type VI collagen in the intervertebral disc. Ann. Rheum. Dis. S T E P H E N S , H . R . , D U A N C E , V. C . , D U N N , M. J . , BAILEY~ A . J.
& DUBOWITZ, V. (1982) Collagen types in neuromuscular diseases. J. NeuroI. Sci. 53, 45~2. S T E V E N S , R. L . , RYVARz R . , R O B E R T S O N , W. R . , O ' B R I E N , J. P.
& BEARD, H. K. (1982) Biological changes in the annulus fibrosus in patients with low back pain. Spine 7, 223-33. T O W B I N , H . , S T A E H L I N , T. & G O R D O N , J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4. VON DER MARK, H. / AUMAILLEY,
M . r W I C K , G.t FLEISCI-I-
(1984) Immunochemistry, genuine size and tissue localisation of collagen VI. Eur. J. Biochem. 142, 493-502. wu, J.-I., LYRE, D. R. & SLAYTERr H. S. (1987) Type VI collagen of the intervertebral disc. Biochem. J. 248, 373-81. MAJER,
R.
& TIMPL,
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Type III collagen in the intervertebral disc S. R O B E R T S 1., J. M E N A G E 1, V. D U A N C E 2 a n d S. F. W O T T O N 2
1Charles Salt Research Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, Shropshire, SYIO 7AG and 2Muscle and Collagen Research Group, Department of Veterinary Medicine, University of Bristol, Langford BS18 7DY, UK Received 28 November 1990 and in revised form 23 April 1991
Summary Several collagen types have now been isolated from the intervertebral disc, although type III collagen has previously only been extracted from human pathological disc. In this study, type III collagen has been isolated from normal human and bovine inter"vertebral disc and immunolocalized in sections of rat, sheep, bovine and 'normal' human intervertebral disc of various ages. Staining with antisera to type III collagen is localized primarily around the cells. Results indicate that cells of the disc sit in 'chondrons', similar to those seen in the deep and mid zones of articular cartilage. We suggest that type III collagen is present in the intervertebral disc and hypothesize that it may be involved in the organization of the pericellular environment, perhaps linking the chondron capsule to the interterritorial matrix.
Introduction The intervertebral disc consists of two regions, the inner nucleus pulposus and outer annulus fibrosus. Collagen accounts for approximately 15% and 50% of the dry weight respectively (Ayad & Weiss, 1986). Eyre and Muir (1974) demonstrated collagen polymorphism in pig intervertebral disc by identifying the presence of two molecular species. They found that the disc contained both types I and II collagen, with type I being predominant in the outer annulus, but the proportion of type II increasing and becoming predominant towards the nucleus pulposus. Since then the picture has become increasingly complex with five further collagen types reported to occur in intervertebral disc. Types II, VI, IX and XI have been isolated from the nucleus pulposus (Eyre & Muir, 1977; Ayad et al., 1981; Ayad et al., 1982; Ayad & Weiss, 1986; Wu et al., 1987) and types I, II, V, VI, IX and XI from the annulus fibrosus (Eyre & Muir, 1977; Ayad & Weiss, 1986; Wu et al., 1987). Type III collagen has been demonstrated by immunofluorescence in both normal and pathological h u m a n intervertebral discs (Roberts et aI., 1982; Stevens et al., 1982) but has only been isolated biochemically from degenerate intervertebral discs (Adam & Deyl, 1984). In view of these findings we have investigated the presence of type III collagen by immunohistochemistry in intervertebral discs and in the adjacent cartilage endplates, from animals of various species and of * To whom all correspondence should be addressed. 0018--2214/91 $03.00 +.12 9 1991 Chapman & Hall
different ages. Particular attention has been paid to the location of the staining in relation to the cells of the tissues. In addition, we have partially purified extracted material from h u m a n and bovine intervertebral discs and examined these extracts for type III collagen using a highly sensitive immunoblotting procedure.
Materials and methods Specimens Intervertebral discs were collected from newborn, seven week and seven month old rats, newborn and seven month old sheep and 18 month old cattle. Human discs (34) were removed from the lumbar spine of 14 individuals as soon as possible post mortem (age range 21-85 years, mean 64 _+ 19). The sagittal slice was removed (see Fig. 1) and columns from both nucleus and annulus, with adjoining cartilage endplate and bone, were frozen in hexane and stored in liquid nitrogen until required. Preparation of antisera Antisera were raised against type III collagen which had been pepsin extracted from human placenta. Antisera were raised in sheep, goat or guinea pig; that from sheep and goat was affinity purified (Stephens et al., 1982). Specificity of the antisera was checked by ELISA for cross reactivity against types I, III, IV and VI collagens. There was minimal cross reaction with any type, other than type III collagen. Sheep anti-type III collagen antibody was used at a dilution of 1:250 for immunofluorescence and 1:1000 for
504 immunoperoxidase and was used routinely. Guinea pig anti-type III and goat anti-type III collagen, when used for immunofluorescence, were diluted 1 in 20 and 1 in 5 respectively. All dilutions were in phosphate buffered saline (PBS) or TRIS buffered saline (TBS) for immunofluorescence or immunoperoxidase respectively.
Commercial antisera Antiserum to human and bovine type III collagen was obtained from Southern Biotechnology Associates Inc., USA (via Bionuclear Services Ltd, UK). It was used at a dilution of 1:100 and 1:1000 for immunofluorescence and immunoperoxidase respectively. Fluorescein- and peroxidase-labelled secondary antibodies were obtained from Nordic Immunologicals Ltd, UK and used at a dilution of 1:50.
Immunolocalization Frozen sections were cut on a Bright's rotary cryostat at a thickness of 6 ~tm and collected onto polylysine coated slides. Following enzymatic digestion (see below) sections were fixed with 4% paraformaldehyde or 10% formaldehyde for 10rain before being stained by indirect immunofluorescence or immunoperoxidase (Newton, 1984) respectively. Peroxidase staining was demonstrated with 3,4,3,4-tetra-aminobiphenyl hydrochloride (BDH; 0.6 mg m1-1 of TBS containing 4 ~ m1-1 of 10 volume solution hydrogen peroxide). Incubations with all antisera were for 30 min at 37~ in a humid chamber. No blocking agents were used for immunofluorescence, whilst for immunoperoxidase 10% normal serum, of the same species as the section, was included with the peroxidase label. Normal serum and PBS or TBS controls were included in each experiment. Adjacent sections were stained with Haematoxylin and Eosin. Fluorescence was viewed on a Leitz Dialux 20 microscope fitted with incident illumination and Phloempak 2.4 filter unit.
Enzyme pretreatment Before staining, sections were enzymatically digested by one of the following: (a) Hyaluronidase (ovine testicular, Sigma type V, 1250 units mg-1): 2mg m1-1 in 0.025M sodium chloride, 0.05 M sodium acetate buffer adjusted to pH 5.0; incubated for 120 min at 37~ This regime was used routinely. (b) Chondroitinase ABC (Seikagaku Kogyo Co. Ltd from ICN Biochemicals Ltd, UK): 0.25 units ml -I in 0.1 M tris acetate at pH 7.6; incubated for 90min at room temperature. (c) Hyaluronidase + chondroitinase: 2 mg ml 1 hyaluronidase incubated for l h at room temperature at pH 5, washed in tris acetate buffer, and incubated for a
ROBERTS, MENAGE, DUANCE and W O T T O N further 90rain at room temperature with 0.25 units 1TI1-1 chondroitinase ABC at pH 7.6 (d) Collagenase 1 (Type III, Advance Biofactures Corporation): 30 units m1-1 in 0.05 M Tris and 0.01M calcium acetate, pH 7.2; incubated for 30 rain at room temperature. (e) Collagenase 2 (Type III, Advance Biofactures Corporation): 30 units m1-1 in 0.05 M tris and 0.01 M calcium acetate pH 7.2; incubated overnight at room temperature, with fresh solution applied 2x 30 min and l x 60 min at 37~ (f) No enzyme treatment.
Biochemical analysis Nucleus pulposus and annulus fibrosus were separated from both human and bovine discs and were extracted with 4 M guanidine hydrochloride in 0.05 M Tris, pH 7.4, following treatment with pepsin (1:100 weight/wet weight of tissue in 0.5 M acetic acid). Dialysis against water produced a precipitate which was freeze dried and analysed by SDSpolyacrylamide gel electrophoresis (Laemmli, 1970) and Western blotting (Towbin et al., 1979). Detection was carried out using the guinea pig anti serum to human type III collagen with either an alkaline phosphatase or a peroxidase conjugated second antibody. Alkaline phosphatase detection was performed using a mixture of 5-bromo-4chloro-3-indolyl phosphate (Sigma) and Nitro Blue Tetrazolium (NBT) (Sigma) as substrate (Harlow & Lane, 1988). Peroxidase activity was detected using the highly sensitive light generating procedure (ECL Detection System, Amersham).
Results
Distribution pattern in disc Staining with all four p r e p a r a t i o n s of antisera to t y p e III collagen occurred a r o u n d the cells of the rat, s h e e p a n d h u m a n intervertebral disc. This w a s m o r e obvious in the nucleus p u l p o s u s t h a n in the a n n u l u s fibrosus. Typically a b a n d a r o u n d , b u t not b o r d e r i n g the cell (A; Fig. 2), a l w a y s stained positively w h e r e a s the cell itself a n d the area directly adjacent to it varied, s o m e t i m e s b u t not always staining. In s o m e cases the region (A) w h i c h typically stained h a d the a p p e a r a n c e of a series of concentric rings (Fig. 3). In addition to the pericellular pattern, there w a s in s o m e discs a degree of staining of the fibres within the interterritorial matrix. In adjacent sections stained w i t h H a e m a t o x y l i n a n d Eosin these discs often s h o w e d d e g e n e r a t i v e c h a n g e s such as clustering of the cells or disorganization of the matrix. In m a n y
Fig. 1. Schematic representation of sampling of human intervertebral disc. A : anterior, P : posterior. Fig. 2. Peroxidase staining for type III collagen in the nucleus of human intervetebral disc. The capsule and beyond (A) is positive while the cell (C) can be positive (a) or negative (b). (c) Control section incubated with normal sheep serum in place of antiserum, x600. Fig. 3. Immunofluorescent staining for type III collagen sometimes forms a series of concentric rings. Section of human nucleus, stained with antiserum raised in sheep, x600. Fig. 4. Heavy staining for type III collagen was seen in the human intervertebral disc (D) where it bordered the endplate (E). Nucleus pulposus, sheep antiserum with peroxidase label, x600.
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506 cases, cells at the junction between the cartilage endplate and disc showed particularly heavy staining with antisera to type III collagen (Fig. 4). No difference was seen with either location within the lumbar spine, or with age of the specimen. Staining in the cartilage endplate Cells of the cartilage endplate have an elongated, fibroblast-like appearance, lying horizontally in the same direction as the collagen fibres. When stained with antisera to type III collagen, sections of endplate show a varied pattern, some cells and sometimes the area close to them, staining positively (Fig. 5), whilst other cells are negative. Abnormalities can be demonstrated on sections stained with Haematoxylin and Eosin, such as splits in the endplate, or Schmorl's nodes, where the disc protrudes into the vertebral body. In these abnormal regions, there was usually a greater degree of staining with anti type III collagen. Effect of enzyme pretreatments Sections which were incubated initially with hyaluronidase, chondroitinase ABC, both of these sequentially or collagenase 1 (see Materials and methods) stained with similar intensity. In contrast those sections which were subjected to no enzyme pretreatment, or to collagenase 2 incubation (see Materials and methods) showed the same staining pattern but at a greatly reduced intensity. Biochemical analysis Biochemical analysis revealed small amounts of type III collagen in the annulus and nucleus of bovine and human discs after reduction with mercaptoethanol (Fig. 6). The type III collagen, which was not detectable on the gel by Coomassie Blue staining (not shown), only became apparent after immunoblotting, indicating that the amounts extracted were small, less than 2% of the total protein, the approximate detection limits of Coomassie Blue staining. Discussion
Type III collagen occurs in many soft distensible connective tissues such as dermis, uterus and blood vessel walls (Gay & Miller, 1978). For the most part, the fibres appear as a fine reticular network, in contrast to the larger well structured fibres of type I collagen. In the intervertebral disc, type III collagen has been demonstrated by immunofluorescence in both normal and pathological human tissue (Beard et al., 1981; Stevens et al., 1982; Adam & Deyl, 1984). However, until now it has only been isolated biochemically from discs showing degenerate changes (Adam & Deyl, 1984). In the present study we have used four preparations of antisera to type III collagen and obtained positive staining with all of them in normal
ROBERTS, MENAGE, DUANCE and WOTTON discs from various species. Although the response was stronger when sections were first digested with hyaluronidase, staining was present without any enzymatic digestion. This is in contrast to staining with antiserum to type VI collagen, which was absent without prior enzymatic digestion (Roberts et al., in press). Results from this and a previous study indicate that the matrix around the cells of the disc is highly organized. Although the staining for type III collagen was predominantly pericellular it generally occupied a region beyond that which stained positively with antiserum to type VI collagen (Roberts et al., in press). The cells of the mid and deep zones of articular cartilage have been shown to sit in a discrete area of pericellular matrix contained within a capsule, the whole structure being described as a chondron (Poole et al., 1986). Minor collagens, including type VI and type IX have been shown to locate preferentially in these pericellular capsules (Poole et al., 1988a,b). It appears that similar structures are present in both the annulus fibrosus and nucleus pulposus of the intervertebral disc. In the disc the pericellular capsule, and in some cases the pericellular matrix adjoining the cell, were labelled with antiserum to type VI collagen, with staining for type III collagen outside this (Fig. 7). Koller et al. (1989) suggest that the globular domains of type VI collagen may be capable of interacting with collagenous structures. Homologous repeats have been found on all three polypeptide chains, which resemble sequence motifs in von Willebrand factor and cartilage matrix protein, both of which bind to collagen fibres (Chu et aI., 1990). If one of these repeats showed a specificity for type III collagen, it is possible that the microfilaments of type VI collagen could interact with type III fibres as proposed by Koller et al. (1989). The relative distribution of staining with antisera to type III and type VI collagen seen in this and previous studies would support this suggestion. Von der Mark et al. (1984) found type W collagen had a similar distribution to type III collagen, the two being associated, for example, in the aortic intima. The similar distribution seen in bone and dentine has led Becker et al. (1986) to suggest that type III collagen is s o m e h o w linked to type VI collagen, at least in these tissues. Type III collagen is also found in connective tissue apparently attempting to repair an injury, for example in wound healing of the dermis or severe osteoarthritis (Gay & Miller, 1978). The increased fibrous staining seen here in intervertebral discs of degenerate histological appearance, may also be an attempt at repair. Perhaps this explains why type III collagen has, until now, only been isolated biochemically from degenerate discs where the levels may be greater than in normal tissue. Immunolocalization does have limitations as a
Type III collagen in intervertebral disc
507
!,
Fig. 5. Ceils of the human cartilage endplate also stained for type III collagen. Although some cells were negatively stained (N), they were visible under phase contrast. Sheep antiserum, peroxidase label x600. Fig. 6. Immunoblots of material extracted from (a) human type III standard, (b) bovine nucleus, (c) bovine annulus, (d) human nucleus and (e) human annulus, run with and without mercaptoethanol (Me), and blotted with guinea pig anti human type III antibody. Fig. 7. Staining for type VI collagen is seen in the r e , o n of the pericellular capsule, inside the region typically staining for type III collagen. Section of human nucleus stained with antiserum to pepsinized type VI collagen from bovine uterus, kindly donated by Dr S. Ayad, Manchester. Peroxidase label x600.
technique for identifying the presence and distribution of molecules. For example masking of the epitope by other molecules may occur, or there may be cross reaction of the antiserum with a further epitope. Checks have been made in this study to eliminate these likelihoods as far as possible, tn addition, the presence of type III collagen in the extracted material was clearly demonstrated using the immunoblotting technique. We therefore conclude that type III collagen is present in the intervertebral disc. The pericellular location suggests that it is associated with the
chondron capsule and possibly involved in the integration of this structure with the surrounding interterritorial matrix.
Acknowledgements We acknowledge the technical assistance of J. T. Wardate and H. Evans. S. F. Wotton and J. Menage gratefully acknowledge support from the Arthritis and Rheumatism Council.
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