Immunoperoxidase localization of type X collagen ... - Semantic Scholar

Bioscience Reports, Vol. 6, No. 2, 1986

Immunoperoxidase Localization of Type X Collagen in Chick Tibiae Alvin P. L. Kwan, 1 Anthony J. Freemont, 2 and Michael E. Grant 1'3 Received November 14, 1985

KEY WORDS: type X collagen,tibiotarsus, hypertrophicchondrocytes,calcification

Type X collagen was prepared from medium of long-term cultures of embryonic chick tibiotarsal chondrocytes. Antibodies to type X collagen were raised and used in immunoperoxidase localization studies with embryonic and growing chick tibiotarsus. Strong anti-type X collagen reactivity was detected mainly in the region of hypertrophic chondrocytes, and to a lesser extent in the zone of calcified cartilage. No reactivity was detected in the proliferative zone nor the superficial layer of the cartilage growth plate. These results suggest that type X collagen may play a key role in matrix calcification during growth and development of the skeletal system. INTRODUCTION Until the recent discoveries of other collagenous peptides known as the cartilage minor collagens (for review see Mayne and yon der Mark, 1983), type II collagen was thought to be the only collagenous species in cartilage (Miller, 1976). The identification of the lc~2e3~ collagen provided the first evidence for collagen heterogeneity in cartilage (Burgeson and Hollister, 1979). More recently, disulphide-bonded type IX collagen has been demonstrated in biosynthetic studies as well as in pepsin digests of both avian and mammalian cartilages (Shimokomaki et al., 1980, 1981; Ayad et al., 1981, 1982; Reese and Mayne, 1981; vonder Mark et al., 1982; Gibson et al., 1983; Bruckner et al., 1983). 1 Department of Biochemistry,Medical School, Universityof Manchester, Manchester M13 9PT, UK. 2 Department of Pathology, Medical School, Universityof Manchester, Manchester M13 9PT, UK. 3 To whom reprint requests should be sent. 155 0144-8463/86/0200-0155505.00/0 ,~ 1986 Plenum Publishing Corporation

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The structure of type IX collagen has been elucidated by electron microscopy (Reese et al., 1982) and cDNA techniques (Ninomiya et al., 1984; van der Rest et al., 1985) and evidence is emerging that it contains covalently bound glycosaminoglycans (Bruckner et al., 1985; Vaughan et al., 1985). Immunolocalization studies suggest that this cartilage collagen exhibits a pericellular distribution (Duance et al., 1982; Ricard-Blum et al., 1982; Evans et al., 1983; Hartmann eC al., 1983). The low-molecular-weight G collagen with polypeptides of M~ 59000, now designated as type X collagen, was first described by Gibson et aI. (1981, 1982) in chick embryo chondrocyte cultures. This short chain collagen has also been detected by other investigators in cell and organ culture systems (Schmid and Conrad, 1982a, b; Capasso et al., 1982, 1984a; Mayne et al., 1983). Radiolabelling experiments with chondrocytes from histologically distinct zones of the chick embryo tibiae have shown that type X collagen is synthesized by cells undergoing hypertrophy (Capasso et al., 1982, 1984b; Schmid and Linsenmayer, 1983; Mayne et al., 1983; Schmid et al., 1984; Kielty et al., 1985). Analogous studies conducted on the cephalic calcified and caudal cartilaginous regions of embryonic chick sternal tissue have also indicated that type X collagen synthesis is confined to hypertrophic chondrocytes involved in the process of endochondral calcification (Gibson et al., 1984; Gibson and Flint, 1985). In the present report, we describe the production of antibodies against type X collagen, demonstrate that type X collagen deposition is closely associated with endochondral development, and conclude that previous concepts of cartilage calcification and bone formation may need revision. MATERIALS AND METHODS

Preparation and Purification of Type X Collagen Native type X collagen was isolated from the medium of long-term chick embryo tibiotarsal chondrocyte cultures as previously described (Schmid and Linsenmayer, 1983; Kielty et al., 1985). Hypertrophic chondrocytes were maintained in plastic culture flasks for 8 weeks. Collagens were precipitated from the medium by addition of solid (NH4)2SO 4 to 30% saturation:,The iimmonium sulphate precipitate was redissolved in 0.5 M acetic acid, and type II collagen was removed by differential salt precipitation at 0.8 M NaC1. Minor collagens were precipitated by adjusting the NaC1 concentration to 2 M. This precipitate was redissolved in acetic acid and extensively dialysed against 0.02 M Na2HPO4, which precipitated type X collagen. SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) of this precipitate demonstrated that the only Coomassie blue positive bands are bands corresponding to the type X procollagen polypeptides (M r 59000) and type X collagen polypeptides (Mr 49000) (Kielty et al., 1985). This preparation was used in antibody production without further purification.

Preparation of Anti-Type X Collagen Antiserum The immunization regime was similar to that of Evans et al. (1983). Adult rabbits were injected intradermally with type X collagen (0.5 rag) emulsified with complete Freund's adjuvant. After 2 weeks the rabbits were re-injected with 0.25 mg of type X collagen in incomplete Freund's adjuvant. A week after the second booster injection,

Immunolocalization of type X cgllagen

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the rabbits were ear-bled. The freshly collected blood was incubated at 37~ for 1 hr, then at 4~ for 3 hr to allow clotting, Serum was withdrawn from the clot and clarified by centrifugation. Antiserum specificity was assessed by enzyme-linked immunosorbent assay (ELISA) (Rennard et al., 1981). In this assay, 96 well microtiter plates were coated with 200 #1 of either type X collagen or various other types of collagen in 0.02 M Tris/HC1 buffer containing 0.45 M NaC1 at pH 7.4, for 16 hr. The wells were rinsed three times with phosphate buffered saline (PBS) containing 0.05% (w/v) Tween 20. Non-specific binding was blocked by the addition of 0.1% (w/v) bovine serum albumin in PBS. After 4 hr, 200 ~tl of the clarified rabbit serum was added to each coated well and incubated at 4~ for 16 hr. At the end of the incubation, 200 #1 of alkaline phosphatase conjugated anti-rabbit IgG (Miles-Yeda Ltd., Israel) was added and the plates were left at room temperature for 4 hr before the substrate p-nitrophenyl phosphate (Sigma chemical Co. Ltd., UK) was added. The product of the enzyme reaction was measured with a Titertek Multiskan ELISA plate reader (Flow Labs., UK). Immunoiocalization

Blocks of tissues were embedded in Tissue-Tek OCT-embedding compound (Miles Lab. Ltd., UK), frozen in liquid nitrogen and stored at - 70~ before sectioning. Thick sections (5 #m) were cut on a cryostat kept at - 2 5 ~ and were adhered onto microscope slides with polyvinyl acetate. Sections were fixed in acetone for 3 min, air dried and then treated with bovine testicular hyaluronidase (Sigma Chemical Co. Ltd., UK) (186 units/ml in 25 m M NaC1/0.05 M sodium acetate, pH 5) for 60 min in a moist chamber. Treated sections were washed three times with PBS and then incubated with 15/11 of 1:30 diluted rabbit anti-type X collagen antiserum for 30 min followed by incubation with peroxidase-conjugated swine anti-rabbit Ig (Dako, Denmark). Control sections were incubated with either PBS or with normal rabbit serum. After incubation with the second antibody, sections were washed and immersed in developing solutions (0.5 mg/ml 3,Ydiamino-benzidine tetrahydrochloride, containing 4% (v/v) H 2 0 2 in PBS) for 5min. The nuclei were then counterstained with haematoxylin for 30 sec, and the sections then dehydrated in alcohol and mounted on XAM neutral white (BDH Chemicals Ltd., UK) for observation by light microscopy. RESULTS To assess the specificity of the anti-type X collagen antiserum, each well in a row of the ELISA plates was coated with an equal amount of type X collagen and each received one concentration in a series of dilutions ranging from 10,000 ng/well to 312 rig/well. Antiserum was added in serial dilutions across the plates so that each column received the same dilution. As shown in the titration curves in Fig. 1, increasing both the amount of antigen used to coat the wells and the concentration of specific antiserum added resulted in a greater amount of alkaline phosphatase conjugate bound, and therefore of colour produced. From the titration curves, the mid-point concentration of 1,000 ng/well was chosen to coat wells in subsequent assays. Preliminary experiments to check the specificity of the antiserum for type X

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2-

A405nm

1-

1:100

1:300

1:900

Antiserum

1::)700

1:8100

1!24300

Dilution

Fig. 1. Titration curves of native type X collagen coating and rabbit antitype X collagen antiserum. Concentrations of type X collagen used are: (0) 312ng/well, (A) 625ng/well, (11) 1250ng/well, (O) 2500ng/well, (A) 5000ng/well, (71) 10 000ng/well.

collagen revealed weak cross-reactivity with bovine fibronectin (results not shown) which may be attributable to traces of fibronectin-related peptides from the culture medium remaining in the type X collagen preparation. Accordingly, the antiserum was passed down a column of fibronectin-Sepharose, prepared as described by Axen et aI. (1967), to remove all antifibronectin antibodies. The unbound fractions were pooled and tested against all the collagen types known to occur in cartilaginous tissues. Only type X cross-reactivity was observed and no reactivity was detected with chick collagen types I, II, IX and 1~2~3~ (Fig. 2). Immunohistochemical experiments (results not shown) employing this purified antiserum on sections of 17-day-old chick embryo tibiotarsus demonstrated that type X reactivity occurs only in the zone of hypertrophic chondrocytes, an observation consistent with results obtained from radiolabelling experiments (Schmid and Linsenmayer, 1983; Kielty et al., 1985) and with the immunofluorescence studies of Schmid and Linsenmayer (1985a, b). No type X reactivity was detected in the permanent cartilaginous region of the sternal cartilage taken from the 17-day-old chick embryo, thus confirming by immunochemical techniques the biochemical observation that the embryonic chick sternal cartilage has little detectable type X collagen prior to day 17 of development in ovo (Kielty et al., 1984; Gibson and Flint, 1985). In further studies on the growing chick (10 days old) it has been possible to demonstrate a more definitive relationship of type X collagen to the centre of hypertrophy within the epiphysis of the tibiotarsus. As shown in Fig. 3, anti-type X collagen antibodies reacted strongly with the connective tissue matrix within the hypertrophic zone of the secondary ossification centre. Less intense reactivity is also concentrated within the calcified cartilage and discrete, longitudinally oriented portions of adjacent bone

Immunolocalization of type X collagen

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A405 nm

.5

1=100

1=300

1 : 900

Antiserum

1=2700

1=8100

1"24300

Dilution

Fig. 2. Direct enzyme linked immunosorbent assay with coating of type X collagen (ll), chick type I and II collagen mixture in 1:1 ratio (O), type IX collagen ( ~ ) and 1c~2c~3c~collagens (A) at concentrations of 1000ng/well. The curve ( 9 shows the reactivity of the control rabbit serum against native type X collagen.

trabeculae. Osteoblast-derived and periosteal bone gave little or no reaction with the purified antiserum. DISCUSSION Evidence for the existence of the short chain cartilage collagen type X first came from biosynthetic experiments with embryonic chick chondrocytes in culture (Gibson et al., 1981, 1982; Schmid and Conrad, 1982a, b). This novel collagen shares a number of properties with the interstitial fibrillar collagens (types I, II, and III) in that, although only approximately half their size, type X collagen is susceptible to digestion with vertebrate collagenase (Gibson et al., 1983) and appears to undergo processing in a manner analogous to the conversion of type II procollagen to collagen (Kielty et al., 1985). Whether or not type X collagen is related to the fibrillar pericellular capsule surrounding chondrocytes in the deep layers of articular cartilage (Poole et al., 1984) remains to be determined but the studies presented here (Fig. 3) add to the growing evidence that type X collagen is found in the hypertrophic zones of cartilage where calcification and bone remodelling are occurring. In our present study we have concentrated on the localization of type X collagen in the developing 10-day-chick tibiotarsus, and particularly in the secondary ossification centre of the epiphysis. At this site chondrocytes differentiate radially in a pattern similar to that seen in the growth plate cartilage (Stocum et al., 1979), for the cells pass through a continuum of cell proliferation, elongation and hypertrophy. Degeneration of the aged hypertrophic chondrocytes is then followed by matrix erosion, invasion of

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Fig. 3(a). Longitudinal section through the secondary ossification centre in the epiphysis ofa 10d posthatch chick tibiotarsus.Section was treated with bovine testicular hyalurouidase, and stained for type X collagen. Reaction product is most dense in the hypertrophic zone (H), but is also present within calcifyingcartilage (C) and parts of adjacent trabeculae (T). Peroxidase-anti-peroxidase x 100.

vascular elements and finally calcification of the cartilage matrix. With the purified antibodies it was possible to demonstrate that type X collagen reactivity was localized mainly to the zone of cell hypertrophy and cell orientation prior to calcification of the chondroid matrix. Reactivity could be identified within trabeculae but only with certainty in areas of residual calcified cartilage. As yet, the role of type X collagen within the skeletal matrix remains unclear. The possibility that type X collagen production m a y be related to invasion of vascular elements (Capasso et al., 1984b) has been considered unlikely by Schmid and Linsenmayer (1985b), who have suggested that type X collagen may provide a permissive matrix for calcification. Such a conclusion is supported by our immunohistochemical studies. Experiments conducted with chondrocytes in culture demonstrating dramatic influences of matrix components and especially calcium and

Imm~anolocalization of type X collagen

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Fig. 3(b). At higher power the density of reaction product in the hypertrophic zone (H) is apparent and it is particularly noteworthy that type X collagen is also concentrated (see arrows) between the chondrocyte columns close to the calcification front (c0. Peroxidaseanti-peroxidase x 300.

phosphate levels on the regulation of type X collagen synthesis (Bates et al., 1985) add further evidence to the suggestion that this collagen plays a key role in e n d o c h o n d r a l bone formation.

ACKNOWLEDGMENTS We thank Mrs. Patricia Fielding for excellent technical assistance and the financial support of G.D. Searle (Research and Development) Ltd. and the Science and Engineering Research Council is gratefully acknowledged.

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REFERENCES Ayad, S., Abedin, M. Z., Grundy, S. M., and Weiss, J. B. (1981). FEBS Lett. 123:195 199. Ayda, S., Abedin, M. Z., Weiss, J. B., and Grundy, S. M. (1982). FEBS Lett. 139:300-304. Axen, R., Brath, J., and Ernback, S. (1967). Nature 214:1302-1304. Bates, G. P., Thomas, J. T., Kielty, C. M., Schor, S. L., and Grant, M. E. (1985). J. Cell Biol. (submitted for publication). Bruckner, P., Vaughan, L., and Winterhalter, K. H. (1985). Proc. Natl. Acad. Sci. USA 82:2608-2612. Burgeson, R. E., and Hollister, D. W. (1979). Biochem. Biophys. Res. Commun. 87:1124 1231. Capasso, O., Gionti, E., Pontarelli, G., Ambesi-Impiombato, F. S., Nitsch, L., Tajana, G., and Cancedda, R. (1982). Exp. Cell Res. 142:197-206. Capasso, O,, Quarto, N., Descalzi-Cancedda, F., and Cancedda, R. (1984a). EMBO J. 3:823-227. Capasso, O., Tajana, G., and Cancedda, R. (1984b). Mol. Cell. Biol. 4:1163-1168. Duance, V. C., Shimokomaki, M., and Bailey, A. J. (1982). Biosci. Rep. 2:223-227. Evans, H. B., Ayad, S., Abedin, M. Z., Hopkins, S., Morgan, K., Walton, K. W., Weiss, J. B., and Holt, P. J. L. (1983). Ann. Rheum. Dis. 42:575-581. Gibson, G. J., Schor, S. L., and Grant, M. E. (1981). Biochem. Soc. Trans, 9:550-551. Gibson, G. J., Schor, S. L., and Grant, M. E. (1982). J. Cell. Biol. 93:767-774. Gibson, G. J., Kielty, C. M., Garner, C., Schor, S. L., and Grant, M. E. (1983). Biochem. J. 211:417-426. Gibson, G. J., Beaumont, B. W., and Flint, M. H. (1984). J. Cell Biol. 99:208-216. Gibson, G. J., and Flint, M. H. (1985). J. Cell Biol. 101:277-284. Hartmann, D. J., Magloire, H., Ricard-Blum, S., Jo fire, A., Couble, M., Wille, G., and Herbage, D. (1983). Coll. Rel. Res. 3:349-357. Kielty, C. M., Hulmes, D. J. S., Schor, S. L., and Grant, M. E. (1984). FEBS Lett. 169:I79-184. Kielty, C, M., Kwan, A. P. L., Holmes, D. F,, Schor, S. L., and Grant, M. E. (1985). Biochem. J. 27:545-554. Laemmli, U. K. (1970). Nature (London) 227:680-685. Mayne, R., Reese, C. A., Williams, C. C., and Mayne, P. (1983). In Limb Development and Regeneration (R. O. Kelley, P.F. Goetinck, and J. A. MacCabe, Eds.), Alan R. Liss, New York, pp. 125 135. Mayne, R., and yon der Mark, K. (1983). In Cartilage (B. K. Hall, Ed.), Academic Press, New York, vol. 1, pp. 181-214. Miller, E. J. (1976). Mol. Cell. Biochem. 13:165 192. Ninomiya, Y., and Olsen, B. R. (1984). Proc. Natl. Acad. Sci. USA 81:3014-3018. Poole, C. A., Flint, M. H., and Beaumont, B. W. (1984). J. Anat. 138:113-138. Reese, C. A., and Mayne, R. (1981). Biochemistry 20:5443-5448. Rennard, S. I., Martin, G. R., and Crystal, R. G. (1981). In Immunochemistry of The Extracellular Matrix (H. Furthmayr, Ed.), CRC Press, Boca Raton, Fla, vol. 1, pp. 237-253. Ricard-Blum, S., Hartman, D. J., Herbage, D., Payen-Meyran, C., and Ville, G. (1982). FEBS Lett. 146, 343347. Schmid, T. M., and Conrad, H. E. (1982a). J. Biol. Chem. 257:12444-12450. Schmid, T. M., and Conrad, H. E. (1982b). J. Biol. Chem. 257:12451-12457. Schmid, T. M., and Linsenmayer, T. F. (1983). J. Biol. Chem. 258:9504-9509. Schmid, T. M., and Linsenmayer, T. F. (1985a). J. Cell Biol. 100:59~605. Schmid, T. M., and Linsenmayer, T. F. (1985b). Dev. Biol. 107:373 381. Schmid, T. M., Mayne, R., Bruns, R. R,, and Linsenmayer, T. F. (1984). J. Ultrastruct. Res. 86:186-191. Shimokomaki, M., Duance, V. C., and Bailey, A. J. (1980). FEBS Lett. 121:51-54. Shimokomaki, M., Duance, V. C., and Bailey, A. J. (1981). Biosci. Rep. 1:561-570. Stocum, D. L., Davis, R. M., Leger, M., and Conrad, H. E. (1979). J. Embryol. Exp. Morphol. 54:155-170. van der Rest, M., Mayne, R., Ninomiya, Y., Seidah, N. G., Chretien, M., and Olsen, B. R. (1985). J. Biol. Chem. 260: 220-225. Vaughan, L., Winterhalter, K. H., and Bruckner, P. (1985). J. Biol. Chem. 260:4758-4763. vonder Mark, K., van Menxel, M., and Wiedemann, H. (1982). Eur. J. Biochem. 124:57-62.