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Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) distribution in normal and pathological human bone
Bone Vol. 24, No. 3 March 1999:229 –235
Tissue Inhibitor of Matrix Metalloproteinase-1 (TIMP-1) Distribution in Normal and Pathological Human Bone S. BORD,1 A. HORNER,1 C. A. BEETON,1 R. M. HEMBRY,2 and J. E. COMPSTON1 1 2
University of Cambridge School of Clinical Medicine, Addenbrooke’s Hospital, Cambridge, UK Strangeways Research Laboratory, Cambridge, UK
Introduction Degradation of skeletal connective tissue is regulated, at least in part, by the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs), their natural inhibitors. The balance between MMPs and TIMPs may therefore be a determinant of normal bone turnover, and imbalance could thus lead to reduced organization of bone structure. To test this hypothesis, the cellular expression of MMPs and TIMP-1 was investigated by immunohistochemistry in human neonatal rib and osteophytic and heterotopic bone; these differ in their structure, with heterotopic bone showing the least and normal rib the most organized development. In all samples, high levels of MMPs were expressed. Collagenase and stromelysin-2 were detected in chondrocytes, osteoblasts, and osteoclasts, whereas gelatinase-B was confined to osteoclasts and mononuclear cells. Matrix-associated stromelysin-1 was present in fibrous tissue and osteoid. In contrast, the expression of TIMP-1 varied markedly between the three types of bone. In heterotopic bone only occasional low level TIMP-1 expression was detected in chondrocytes and osteoblasts. Osteophytic bone showed varying levels of TIMP-1, which was matrix-bound in fibrous tissue and cell-associated in osteoblasts, chondrocytes, and occasional mononuclear cells. In both types of bone, expression of TIMP-1 by osteoclasts was absent despite large numbers of these cells. Neonatal rib bone showed consistent expression of TIMP-1, particularly in chondrocytes, osteoblasts, and lining cells. In contrast to pathological bone, many osteoclasts were TIMP-1 positive. These results suggest that, in heterotopic and osteophytic bone, the low levels of TIMP-1, and in particular its absence in osteoclasts, may partly explain the more poorly organized bone formation in these pathological bone samples. Furthermore, TIMP-1 may play a role in the regulation of bone modeling and remodeling in normal developing human bone. (Bone 24:229 –235; 1999) © 1999 by Elsevier Science Inc. All rights reserved.
Matrix metalloproteinases (MMPs), a family of proteolytic enzymes, are capable of degrading all major components of the extracellular matrix. Their activity is regulated by natural specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs). A balance between MMPs and TIMPs is necessary for many physiological processes, and it has been suggested that imbalance may lead to the destruction of connective tissue, which occurs in a number of pathological events, including osteoarthritis and rheumatoid arthritis,9,11 tissue metastasis,24 and periodontal disease.21 Although a number of other proteinases are also involved in the degradation of bone matrix, differences in MMP and TIMP production may be important in both physiological regulation of bone turnover and in pathological bone formation. Currently the TIMP family consists of four identified members (TIMP-1, -2, -3, -4). TIMP-1, a 28 kDa glycosylated protein12 has been the most widely studied. TIMP-24 is a smaller 22 kDa nonglycosylated protein and shares 40% sequence homology with TIMP-1. TIMP-338 is bound by connective tissue matrix, and TIMP-4, the most recently identified family member,18,22 is reported to be more closely related to TIMPs-2 and -3 than to TIMP-1. All active MMPs are inhibited by naturally occurring TIMPs. They form noncovalent bimolecular complexes with the active form, and occasionally with the latent form, of individual MMPs in a 1:1 molar ratio, with the inhibitory activity of the molecule residing in the N-terminal domain.10 Several amino acid sequences are thought to be important in this mechanism.37 A mutational study in which single amino acid residues in the N-terminal domain of TIMP-1 were changed demonstrated that no single residue was responsible for inhibition, and concluded that the inhibitor bound mainly to specific surface sites on the MMPs, preventing substrate binding by steric hindrance.31 In addition to their ability to bind to active MMPs, TIMPs can bind to certain latent MMPs. TIMP-1 binds to progelatinase-B,17,35 whereas progelatinase-A binds to TIMP-2 and -3,29 the Cterminal loop of TIMPs being involved in progelatinase-B29 and progelatinase-A binding.36 In this study, the cellular production of TIMP-1 and the MMPs, collagenase, gelatinase-B, stromelysin-1, and stromelysin-2 was examined by immunohistochemistry in three types of human bone. Neonatal rib bone was used as an example of normal developing bone. Heterotopic and osteophytic bone can be regarded as pathological and show less organized development. The aim of this study was to test the hypothesis that the relative expression of MMPs and TIMP-1 contribute to the regulation of human bone formation.
Key Words: Tissue inhibitor of matrix metalloproteinase; Matrix metalloproteinase; Bone; Cartilage; Osteoclasts; Osteoblasts.
Address for correspondence and reprints: Dr. Sharyn Bord, Department Medicine, Level 5, Box 157, Addenbrooke’s Hospital, Cambridge CB2 2QQ, UK. E-mail: [email protected] © 1999 by Elsevier Science Inc. All rights reserved.
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Materials and Methods Tissue The present study was conducted on neonatal rib, osteophytes, and heterotopic bone. Neonatal ribs were removed at postmortem (28 –37 weeks gestation). Osteophytes were obtained following routine shoulder and hip-joint replacement surgery. Heterotopic bone was removed from sites of ectopic bone formation at the hip of two and shoulder of one patient (mean age 29 years) who had previously sustained severe head injury. Informed written consent was given and approval obtained from the local ethics committee. Following removal, the tissue was immediately placed on ice and then immersed in 5% polyvinylalcohol (PVA) for 2 min, snap frozen in liquid nitrogen, and stored at 270°C. Frozen sections were obtained using a Bright cryostat (Huntingdon, UK) with a cabinet temperature of 230°C, equipped with a slow-drive high-torque motor with automatic speed control and a highly polished tungsten carbide knife. Histology Undecalcified, unfixed 9 mm thick frozen sections were stained with Diff-Quik (Baxter Dade AG) or 1% toluidine blue for general morphology, and von Kossa with a van Giesen counterstain to assess matrix mineralization. Alkaline phosphatase staining to identify osteoblasts was detected using a coupled reaction with a-naphthyl acid phosphate and fast-red TR.8 Tartrateresistant acid phosphatase (TRAP) staining in osteoclasts and preosteoclasts was demonstrated by reactivity with AS-BI phosphate and sodium tartrate, postcoupled with fast garnet.25 Antibodies Well-characterized specific polyclonal antibodies to gelatinase-B (MMP-9),30 stromelysin-1 (MMP-3),2 stromelysin-2 (MMP10),19 collagenase (MMP-1),19,28 and TIMP-120 were raised in sheep. Immunoglobulins for all antisera were prepared by triple ammonium sulfate precipitation. Pooled normal sheep serum (NSS) was used as a control at the same IgG concentration as the antibodies. The secondary antibody was a biotinylated rabbit antisheep IgG (Vector Laboratories). Immunohistochemistry Sets of serial sections, 9 mm in thickness, were picked off onto glass slides, coated with 2% 3-aminopropyltriethoxysilane (APES), air dried for 10 min, fixed in 4% paraformaldehyde for 22 min at room temperature, and washed in phosphate-buffered saline (PBS). Immunolocalization was carried out using an indirect immunoperoxidase system. Endogenous peroxidase and nonspecific binding were blocked by incubation with ImmunoPure (Pierce) peroxidase suppressor for 22 min and extensive washing followed by incubation in 20% normal rabbit serum (12 min) prior to the primary antibody incubation (10 mg/mL, overnight at 4°C in a humid chamber). Following further washing, the second antibody was applied (5 mg/mL, 40 min at room temperature). The sections were washed and the signal amplified using avidin-biotin complex (ABC, Vector Laboratories) for 30 min at room temperature. Sites of antigen binding were visualized using 3,39-diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC) as chromogens. Some sections were lightly counterstained with hematoxylin to detect nuclei. Sections were mounted in aqueous mount (90% glycerol in PBS) and observed under bright-field microscopy on an Olympus
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BH-2 microscope. Photographs were taken using Ektachrome 64 film. Quantitation Different cell types were identified morphologically and sites of formation and resorption identified by alkaline phosphatase, TRAP, and gelatinase-B. Serial sections were immunolocalized for TIMP-1 and cells within specific areas examined for positive staining. These areas included endochondral bone formation with associated chondrocytes and osteoblasts, and areas of bone modeling and remodeling. For each cell type, the percentage of cells staining was assessed semiquantitatively by counting five fields from each section and the results expressed as the number of cells staining positively as a percentage of the total cell number. These figures were tabulated as follows: no staining scored as “2”; less than 20% of cells staining positively scored “1”; 20%–50% positive cells as “11”; and more than 50% of cells exhibiting positive staining scored “111.” The presence of matrix staining was also noted. Results All three types of bone showed high cellularity with areas of bone modeling, remodeling, and endochondral and intramembranous bone formation (Figures 1 and 2). Neonatal rib bone showed resting chondrocytes, which progressed into columns of proliferating chondrocytes as endochondral ossification proceeded. Chondrocytes underwent progressive hypertrophy, with increasing mineralization of the intercolumnar matrix (Figure 2A). Within the primary spongiosa, osteoid was deposited on the remnants of resorbed cartilage to form spicules of bone, which were then remodeled by osteoclasts and osteoblasts. Alkaline phosphatase staining was particularly apparent on cancellous surfaces in the primary spongiosa and in the more developed regions of cortical bone. TRAP and gelatinase-B staining was evident in multinucleated cells in both marrow spaces and at sites of resorption. Some mononuclear cells were also positive, as were many hypertrophic chondrocytes. A surrounding periosteal collar of mineralized woven bone, containing many osteocytes, was covered by periosteum and contained vascular channels. Heterotopic bone exhibited areas of multilayers of cuboidal alkaline phosphatase-positive osteoblasts associated with thick osteoid seams and numerous osteocytes in the mineralized matrix, suggesting rapid bone formation. Some bone surfaces were smooth and lined with quiescent cells, whereas other areas showed large numbers of gelatinase-B and TRAP-positive osteoclasts along resorbed surfaces of the bone. These features are indicative of high bone turnover. Areas of intramembranous and endochondral ossification were evident, usually surrounded by fibrous tissue or cartilage. Sections viewed under polarized light showed that most of the bone was woven with random distribution of collagen fibrils. Generally, the bone had a disorganized structure, but gave the appearance of being highly active, with large numbers of cells (Figure 1A,B). Osteophytic bone also showed areas of endochondral and intramembranous bone formation with both woven and lamellar bone evident (Figure 1C,D). The chondrocytic differentiation stages through to hypertrophy often appeared truncated, the zone of columnar chondrocytes being shorter and less well organized (Figure 1C) than those seen in the growth plate of normal rib bone. Vascular invasion and extensive resorption of the cartilage by osteoclasts was followed by deposition of woven bone matrix by osteoblasts on the resorbed surfaces; these osteoblasts showed
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Figure 1. (A, B) Undecalcified sections from heterotopic bone demonstrating the high cellularity of the bone. Multilayers of osteoblasts (ob) are seen at sites of bone formation and many osteoclasts (oc) are evident on resorbing surfaces. Numerous osteocytes (o) are apparent in the bone matrix (b). (A) Diff-Quik-stained section viewed under low power. (B) Toluidine blue-stained section viewed under high power showing the resorbed surface of the bone (arrowheads) and adjacent osteoclasts. (C, D) Undecalcified Diff-Quik-stained sections from osteophytic bone. (C) Area of endochondral ossification with calcified cartilage matrix (c) containing vacuolated chondrocytes (arrow). Osteoclasts (arrowhead) are seen at the resorbing surface of the calcified matrix. Mineralized woven bone trabeculae (w) develop from this area of cartilage. (D) Intramembranous ossification with a layer of osteoblasts (arrowhead) apposed to the woven bone matrix (o), which gradually mineralizes (m). Bars: (A) 100 mm; (B–D) 20 mm.
intense staining for alkaline phosphatase. Thick osteoid seams were apparent, with large numbers of adjacent osteoblasts. Many osteoclasts were seen on both resorbing bone surfaces and in marrow spaces between developing trabeculae (Figure 1C), their osteoclastic nature confirmed by TRAP and gelatinase-B positivity. The osteophytes were surrounded by a fibrocartilage collar covered with fibrous tissue. Osteophytic bone was highly cellular
and exhibited rapid growth; however, it displayed a more organized structure than that seen in heterotopic bone. Matrix Metalloproteinases Generally, the pattern of localization of MMPs was similar in all three types of bone. Chondrocytes expressed collagenase and
Figure 2. (A–C) Serial sections of neonatal rib. (A) Von Kossa-stained section viewed under low power shows the growth plate of neonatal rib with mineralized bone (black color) of the primary spongiosa, the mineralizing zone (brown color), and columnar chondrocytes (c). (B) TIMP-1 immunolocalized in chondrocytes with positive staining (arrowhead) visualized by the brown reaction product using DAB substrate. TIMP-1 expression gradually decreases throughout the hypertrophic chondrocytes (h) toward the mineralizing zone (m). (C) Control serial section of (B) immunolocalized with normal sheep serum shows absence of staining in chondrocytes (arrowhead). (D, E) Serial sections of osteophytic bone showing columnar chondrocytes (c) adjacent to mineralized bone (m). (D) Section immunolocalized for TIMP-1 shows chondrocyte nuclei stained blue, with no brown color, indicating absence of TIMP-1. (E) Adjacent section stained for collagenase shows many chondrocytes with brown color reaction product in close proximity to blue nuclei, indicating collagenase synthesis (arrowhead). Bars 5 20 mm.
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stromelysin-2 throughout most regions of differentiation, with a slight decrease toward the mineralizing zone. Stromelysin-1 expression was detected in some chondrocytes, but was more predominant in the matrix surrounding proliferating chondrocytes. Osteoblasts on forming surfaces showed intense staining for stromelysin-2 and collagenase, whereas stromelysin-1, occasionally detected in osteoblasts, was more often localized in the underlying osteoid. Some quiescent lining cells were positive for collagenase. Stromelysin-1 was also immunolocalized in osteocytes and in matrix surrounding their lacunae, especially at sites of bone remodeling. Most mononuclear cells in marrow spaces stained positively for gelatinase-B and stromelysin-2. Many osteoclasts were observed in all three types of bone, the majority of which stained positively for gelatinase-B and stromelysin-2. Immunoreactive collagenase was detected in some osteoclasts, but all were negative for stromelysin-1. TIMP-1 TIMP-1 expression showed marked differences in the three types of bone examined. Generally, TIMP-1 was seen in most cell types in normal rib bone, but only low levels were detected in pathological bone. The lowest level of TIMP-1 expression was seen in heterotopic bone (Table 1). The growth plate of rib bone (Figure 2A) showed immunoreactive TIMP-1 in proliferative and mature chondrocytes with a gradual decrease through the hypertrophic region (Figure 2B); TIMP-1 expression was also seen in the mineralizing zone. In samples of pathological bone the greatest number of cells staining positively for TIMP-1 was observed in proliferating chondrocytes at some sites of endochondral ossification (Table 1); however, other areas of endochondral ossification showed absence of TIMP-1 staining, usually associated with high collagenase and stromelysin-2 expression (Figure 2D,E). Osteoblasts in the pathological bone samples showed varying levels of intensity of TIMP-1 expression, the lowest levels usually being associated with areas of rapid remodeling. In areas of heterotopic bone with high MMP expression, little or no TIMP-1 was detected. Similarly, in sections from osteophytic bone, at sites of newly forming osteoid, low levels of TIMP-1 were occasionally seen, often associated with high levels of collagenase and stromelysin (Figure 3A,B). In contrast, many osteoblasts in the rib bone showed distinct TIMP-1 staining (Figure 3C). Those on forming surfaces within the primary spongiosa were often positive as were osteoblasts on cortical bone surfaces. At the same sites many osteoblasts showed positive collagenase expression (Figure 3D). Some lining cells on quiescent surfaces were immunoreactive for TIMP-1 (Figure 3E). Some osteocytes in rib bone were TIMP-1 positive, whereas very few stained positively in the pathological bone samples (Table 1). TIMP-1 was detected in some mononuclear cells in the marrow spaces and primary spongiosa of rib bone. Many osteoclasts were seen in these areas, with almost half staining positively for TIMP-1 (Figure 3F), but the majority showing gelatinase-B immunoreactivity (Figure 3G). These positive cells were seen both apposed to the bone surface at sites of resorption and apparently migrating within marrow spaces (Figure 3F–H). In contrast, despite high numbers of gelatinase-B-positive osteoclasts in both heterotopic and osteophytic bone, no TIMP-1 expression was detected in these cells (Figure 3I,J) (Table 1). On serial sections these osteoclasts stained positively for TRAP.
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Discussion Our results demonstrate that MMPs are widely and differentially distributed throughout developing human bone. The staining patterns observed in both normal and pathological samples of bone were very similar. In contrast, the expression of TIMP-1 was more varied, with distinct differences between normal and pathological bone. In heterotopic bone only occasional low-level TIMP-1 expression was detected, whereas osteophytic bone showed higher levels. In both types of pathological bone no TIMP-1 could be detected in osteoclasts. The widespread distribution of TIMP-1 in normal bone is consistent with a role in the regulation of MMPs during bone development. The growth of osteophytes and heterotopic bone is characterized by endochondral and intramembranous ossification with areas of vascular invasion, cellular proliferation, differentiation, and elaboration of extracellular matrix and advancing mineralization.14,32 These changes resemble those seen in the epiphyseal growth plate of immature growing long bones, thus making these types of bone useful models for studies of bone development.1,6,7,13,26,27 However, bone formation in heterotopic and osteophytic bone is clearly a pathological event and the factors responsible for its regulation may differ from those that operate in normal bone. The pattern of MMP expression in osteophytic and heterotopic bone is consistent with our previously reported results6,7 and it was notable that MMP expression in normal neonatal rib bone was similar to that observed in pathological bone. This was also demonstrated in a previous study comparing stromelysin-1 and -2 expression in osteophytic and rib bone.5 However, in contrast to the pattern of MMP expression there were clear differences in the distribution and levels of TIMP-1 expression between the three types of bone. Several studies have indicated that an imbalance between MMPs and TIMP is associated with pathological events. Lin et al.23 suggested that widespread expression of collagenase with absence or variable levels of TIMP-1 expression could lead to radicular cyst expansion. Berend et al.3 showed that a high ratio of MMP to TIMP in human chondrosarcoma may be indicative of a more invasive and aggressive tumor. However, similar studies in human bone in vivo have not been reported. In vitro studies have demonstrated the potential for TIMP-1 to inhibit bone resorption. Shimizu et al.34 reported that addition of Table 1. Summary of the distribution of TIMP-1 TIMP-1 expression in:
Cartilage/endochondral Proliferating chondrocytes Mature chondrocytes Hypertrophic chondrocytes Mineralized bone Osteoblasts Modeling/remodeling Osteoclasts Osteoblasts Osteocytes Mononuclear cells Lining cells
Normal rib bone
Osteophytic bone
Heterotopic bone
111 111 11 11 (m) 11
11 1 1 1 (m) 1
1 2 2 1 (m) 2/1
11 111 1 11 11
2 11 2 1 2
2 1 2 2/1 2
KEY: 2, no staining or occasional staining; 1, less than 20% cells staining; 11, 20%–50% cells staining; 111, more than 50% cells staining; m, mainly matrix staining.
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Figure 3. (A, B) Serial cryosections of osteophytic bone showing osteoblasts at a forming surface of bone (b). (A) A section immunolocalized with anti-TIMP-1 antibody shows no positive staining in osteoblasts (arrow). (B) Distinct stromelysin expression is detected in osteoblasts (arrow) close to the bone surface. (C, D) Serial sections of neonatal rib showing osteoblasts on a forming surface of bone (b). (C) TIMP-1 immunolocalized in osteoblasts (arrow). (D) Adjacent section stained for collagenase shows positive expression in some osteoblasts (arrow) but absence in others (open arrow). (E) TIMP-1 expression detected in bone (b) lining cells (arrows) on a quiescent surface of neonatal rib. (F, G) Serial cryosections of neonatal rib show osteoclasts in the primary spongiosa. (F) TIMP-1 staining in one osteoclast (arrow) but not in the second osteoclast (open arrowhead) nor in mononuclear cells (arrowhead). (G) Positive gelatinase-B expression in two osteoclasts (arrow and open arrowhead) and in some mononuclear cells (arrowhead). (H) An area of the primary spongiosa in neonatal rib shows positive TIMP-1 staining immunolocalized in a multinucleate osteoclast (arrow) and in some adjacent mononuclear cells (arrowhead). (I, J) Non-counterstained serial cryosections of heterotopic bone (b). (I) Section immunolocalized for TIMP-1 shows no staining in an osteoclast on the bone surface (arrow) or in the marrow space (open arrow). (J) Adjacent section shows gelatinase-B expression immunolocalized in both osteoclasts (arrow and open arrow). Bars 5 20 mm.
exogenous TIMP to bone cultures inhibited bone resorption. Everts et al.,16 using a selective inhibitor of MMPs (CI-I), showed an increase in the amount of nondigested bone matrix in the resorption area facing the ruffled border of osteoclasts, further indicating a role for TIMP as a regulator of bone resorption. Similar experiments by Ellis et al.15 demonstrated that exogenous TIMP and synthetic MMP inhibitors prevented cytokine-induced matrix degradation by MMPs in bovine nasal
cartilage. Our studies demonstrating the presence of TIMP-1 in osteoclasts in normal bone are in agreement with these in vitro experiments, providing in vivo support for a role for TIMP in regulating bone modeling and remodeling. Our results also suggest that TIMP may play a dual role in controlling bone turnover. Immunoreactive gelatinase-B, stromelysin-2, and occasional collagenase were detected in osteoclasts in all types of bone, both on and adjacent to bone surfaces, suggesting that MMPs may
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facilitate the migration of osteoclasts to sites of bone resorption. The presence of TIMP-1 in osteoclasts in normal bone would be consistent with regulation of this migration prior to resorption. In support of this theory, Sato et al.33 demonstrated that isolated osteoclasts were able to migrate through collagen-coated invasion chambers—this process being prevented by the addition of MMP inhibitors. They found no evidence of any involvement of serine or cysteine proteinases, suggesting a unique role for MMPs in this process. In conclusion, this study demonstrates distinct patterns of localization of TIMP-1 and MMPs in different types of human bone in vivo. The presence of MMPs with only low levels of TIMP-1 in pathological bone could result in excessive degradation of the extracellular matrix, and therefore contribute to the poorly organized bone formation seen in heterotopic and osteophytic bone in several ways: first, low levels of TIMP-1 expression in chondrocytes would allow uncontrolled MMP degradation of proteoglycans and induce premature calcification; second, collagenase present in lining cells may play a role in activation of bone remodeling,7 and the absence of TIMP-1 in cells could contribute to increased turnover; and third, the absence of TIMP-1 in osteoclasts would facilitate increased MMP-assisted migration to sites of resorption. In contrast, the widespread expression of TIMP-1 in normal developing bone suggests that it may play a role in controlling MMP activity and thus contribute to the regulation of bone modeling and remodeling in developing human bone. Acknowledgments: J.E.C. is supported by the Wellcome Trust. R.M.H. is funded by the Medical Research Council. The authors are indebted to Chris Constant, Dr. Nick Coleman, and Dr. Wilf Kelsall for their invaluable supply of human bone.
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J. F. Evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage. J Clin Invest 84:678 – 685; 1989. Docherty, A. J. P., Lyons, A., Smith, B. J., Wright, E. M., Stephens, P. E., Harris, T. J. R., Murphy, G., and Reynolds, J. J. Sequence of human tissue inhibitor of metalloproteinases and its identity to erythroid-potentiating activity. Nature 318:66 – 69; 1985. Dodds, R. A., Connor, J. R., James, I. E., Rykaczewski, E., Appelbaum, E., Dul, E., and Gowen, M. Human osteoclasts, not osteoblasts, deposit osteopontin onto resorption surfaces: An in vitro and ex vivo study of remodeling bone. J Bone Miner Res 10:1666 –1680; 1995. Dodds, R. A., and Gowen, M. The growing osteophyte: A model system for the study of human bone development and remodelling in situ. J Histotechnol 17:37– 45; 1994. Ellis, A. J., Curry, V. A., Powell, E. K., and Cawston, T. E. The prevention of collagen breakdown in bovine nasal cartilage by TIMP-1, TIMP-2 and low molecular weight synthetic inhibitor. Biochem Biophys Res Commun 201:94 – 101; 1994. Everts, V., Delaisse´, J. M., Korper, W., Niehof, A., Vaes, G., and Beertsen, W. Degradation of collagen in the bone-resorbing compartment underlying the osteoclasts involves both cysteine-proteinases and matrix metalloproteinases. J Cell Physiol 150:221–231; 1992. Goldberg, G. I., Strongin, A., Collier, I. E., Genrich, L. T., and Marmer, B. L. Interaction of 92-kDa type-IV collagenase with the tissue inhibitor of metalloproteinases prevents dimerization, complex-formation with interstitial collagenase, and activation of the proenzyme with stromelysin. J Biol Chem 267:4583– 4591; 1992. Greene, J., Wang, M. S., Liu, Y. L. E., Raymond, L. A., Rosen, C., and Shi, Y. N. E. Molecular cloning and characterization of human tissue inhibitor of metalloproteinase-4. J Biol Chem 271:30375–30380; 1996. Hembry, R. M., Bagga, M. R., Reynolds, J. J., and Hamblen, D. L. Immunolocalisation studies of six matrix metalloproteinases and their inhibitors, TIMP-1 and TIMP-2 in synovia from patients with osteo- and rheumatoid arthritis. Ann Rheum Dis 54:25–32; 1995. Hembry, R. M., Murphy, G., and Reynolds, J. J. Immunolocalisation of tissue inhibitor of metalloproteinase (TIMP) in human cells. Characterisation and use of a specific antiserum. J Cell Sci 73:105–119; 1985. Larivee, J., Sodek, J., and Ferrier, J. M. Collagenase and collagenase inhibitor activities in crevicular fluid of patients receiving treatment for localized juvenile periodontitis. J Peridont Res 21:702–715; 1986. Leco, K. J., Apte, S. S., Taniguchi, G. T., Hawkes, S. P., Khokha, R., Schultz, G. A., and Edwards, D. R. Murine tissue inhibitor of metalloproteinases-4 (TIMP-4): cDNA isolation and expression in adult mouse tissues. FEBS Lett 401:213–217; 1997. Lin, S. K., Chiang, C. P., Hong, C. Y., Lin, C. P., Lan, W. H., Hsieh, C. C., and Kuo, M. Y. P. Immunolocalisation of interstitial collagenase (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in radicular cysts. J Oral Pathol Med 26:458 – 463; 1997. Liotta, L. A. Tumour invasion and metastases—role of the extracellular matrix. Cancer Res 46:1–7; 1986. Loveridge, N., Dean, V., Goltzman, D., and Hendy, G. N. Bioactivity of parathyroid hormone and parathyroid-hormone like peptide: Agonist and antagonist activities of amino-terminal fragments as assessed by the cytochemical bioassay and in situ biochemistry. Endocrinology 128:1938 –1946; 1991. Merry, K., Dodds, R., Littlewood, A., and Gowen, M. Expression of osteopontin mRNA by osteoclasts and osteoblasts in modelling human bone. J Cell Sci 104:1013–1020; 1993. Middleton, J., Arnott, N., Walsh, S., and Beresford, J. Osteoblasts and osteoclasts in adult human osteophyte tissue express the mRNAs for insulin-like growth factors I and II and the type IIGF receptor. Bone 16:287–293; 1995. Murphy, G., Allan, J. A., Willenbrock, F., Cockett, M. I., O’Connell, J. P., and Docherty, A. J. P. The role of C-terminal domain in collagenase and stromelysin specificity. Biol Chem 267:9612–9618; 1992. Murphy, G., Houbrechts, A., Cockett, M. I., Williamson, R. A., O’Shea, M., and Docherty, A. J. P. The N-terminal domain of tissue inhibitor of metalloproteinases retains metalloproteinase inhibitory activity. Biochemistry 30: 8097– 8102; 1991. Murphy, G., Ward, R., Hembry, R. M., Reynolds, J. J., Kuhn, K., and Tryggvason, K. Characterization of gelatinase from pig polymorphonuclear leucocytes. A metalloproteinase resembling tumour type IV collagenase. Biochem J 258:463– 472; 1989. O’Shea, M., Willenbrock, F., Williamson, R. A., Cockett, M. I., Freedman, R. B., Reynolds, J. J., et al. Site-directed mutations that alter the inhibitory activity of the tissue inhibitor of metalloproteinase-1: Importance of the
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N-terminal region between cysteine 3 and cysteine 13. Biochemistry 31: 10146 –10152; 1992. Resnick, D. and Niwayama, G. Degenerative disease of extraspinal locations. In: Resnick, D., Ed. Diagnosis of Bone and Joint Disorders, 3rd ed. Vol. 3. Philadelphia: Saunders; 1995; 1263–1372. Sato, T., Foged, N. T., and Delaisse´, J.-M. The migration of purified osteoclasts through collagen is inhibited by matrix metalloproteinase inhibitors. J Bone Miner Res 13:59 – 66; 1998. Shimizu, H., Sakamoto, M., and Sakamoto, S. Bone resorption by isolated osteoclasts in living versus devitalised bone: Differences in mode and extent and the effects of human recombinant tissue inhibitor of metalloproteinases. J Bone Miner Res 5:411– 418; 1990. Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant, G. A., and Goldberg, G. I. SV-40 transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages. J Biol Chem 264:17213–17221; 1989.
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36. Willenbrock, F., Crabbe, T., Slocombe, P. M., Sutton, C. W., Docherty, A. J. P., Cockett, M. I., et al. The activity of the inhibitor of metalloproteinases is regulated by C-terminal domain interactions: A kinetic analysis of the inhibition of gelatinase A. Biochemistry 32:4330 – 4337; 1993. 37. Woessner, J. F., Jr. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 5:2145–2154; 1991. 38. Yang, T. and Hawkes, S. P. Role of the 21-kDa protein TIMP-3 in oncogenic transformation of cultured chick embryo fibroblasts. Proc Natl Acad Sci USA 89:10676 –10680; 1992.
Date Received: May 12, 1998 Date Revised: October 27, 1998 Date Accepted: October 27, 1998
Tissue Inhibitor of Matrix Metalloproteinase-1 (TIMP-1) Distribution in Normal and Pathological Human Bone S. BORD,1 A. HORNER,1 C. A. BEETON,1 R. M. HEMBRY,2 and J. E. COMPSTON1 1 2
University of Cambridge School of Clinical Medicine, Addenbrooke’s Hospital, Cambridge, UK Strangeways Research Laboratory, Cambridge, UK
Introduction Degradation of skeletal connective tissue is regulated, at least in part, by the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs), their natural inhibitors. The balance between MMPs and TIMPs may therefore be a determinant of normal bone turnover, and imbalance could thus lead to reduced organization of bone structure. To test this hypothesis, the cellular expression of MMPs and TIMP-1 was investigated by immunohistochemistry in human neonatal rib and osteophytic and heterotopic bone; these differ in their structure, with heterotopic bone showing the least and normal rib the most organized development. In all samples, high levels of MMPs were expressed. Collagenase and stromelysin-2 were detected in chondrocytes, osteoblasts, and osteoclasts, whereas gelatinase-B was confined to osteoclasts and mononuclear cells. Matrix-associated stromelysin-1 was present in fibrous tissue and osteoid. In contrast, the expression of TIMP-1 varied markedly between the three types of bone. In heterotopic bone only occasional low level TIMP-1 expression was detected in chondrocytes and osteoblasts. Osteophytic bone showed varying levels of TIMP-1, which was matrix-bound in fibrous tissue and cell-associated in osteoblasts, chondrocytes, and occasional mononuclear cells. In both types of bone, expression of TIMP-1 by osteoclasts was absent despite large numbers of these cells. Neonatal rib bone showed consistent expression of TIMP-1, particularly in chondrocytes, osteoblasts, and lining cells. In contrast to pathological bone, many osteoclasts were TIMP-1 positive. These results suggest that, in heterotopic and osteophytic bone, the low levels of TIMP-1, and in particular its absence in osteoclasts, may partly explain the more poorly organized bone formation in these pathological bone samples. Furthermore, TIMP-1 may play a role in the regulation of bone modeling and remodeling in normal developing human bone. (Bone 24:229 –235; 1999) © 1999 by Elsevier Science Inc. All rights reserved.
Matrix metalloproteinases (MMPs), a family of proteolytic enzymes, are capable of degrading all major components of the extracellular matrix. Their activity is regulated by natural specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs). A balance between MMPs and TIMPs is necessary for many physiological processes, and it has been suggested that imbalance may lead to the destruction of connective tissue, which occurs in a number of pathological events, including osteoarthritis and rheumatoid arthritis,9,11 tissue metastasis,24 and periodontal disease.21 Although a number of other proteinases are also involved in the degradation of bone matrix, differences in MMP and TIMP production may be important in both physiological regulation of bone turnover and in pathological bone formation. Currently the TIMP family consists of four identified members (TIMP-1, -2, -3, -4). TIMP-1, a 28 kDa glycosylated protein12 has been the most widely studied. TIMP-24 is a smaller 22 kDa nonglycosylated protein and shares 40% sequence homology with TIMP-1. TIMP-338 is bound by connective tissue matrix, and TIMP-4, the most recently identified family member,18,22 is reported to be more closely related to TIMPs-2 and -3 than to TIMP-1. All active MMPs are inhibited by naturally occurring TIMPs. They form noncovalent bimolecular complexes with the active form, and occasionally with the latent form, of individual MMPs in a 1:1 molar ratio, with the inhibitory activity of the molecule residing in the N-terminal domain.10 Several amino acid sequences are thought to be important in this mechanism.37 A mutational study in which single amino acid residues in the N-terminal domain of TIMP-1 were changed demonstrated that no single residue was responsible for inhibition, and concluded that the inhibitor bound mainly to specific surface sites on the MMPs, preventing substrate binding by steric hindrance.31 In addition to their ability to bind to active MMPs, TIMPs can bind to certain latent MMPs. TIMP-1 binds to progelatinase-B,17,35 whereas progelatinase-A binds to TIMP-2 and -3,29 the Cterminal loop of TIMPs being involved in progelatinase-B29 and progelatinase-A binding.36 In this study, the cellular production of TIMP-1 and the MMPs, collagenase, gelatinase-B, stromelysin-1, and stromelysin-2 was examined by immunohistochemistry in three types of human bone. Neonatal rib bone was used as an example of normal developing bone. Heterotopic and osteophytic bone can be regarded as pathological and show less organized development. The aim of this study was to test the hypothesis that the relative expression of MMPs and TIMP-1 contribute to the regulation of human bone formation.
Key Words: Tissue inhibitor of matrix metalloproteinase; Matrix metalloproteinase; Bone; Cartilage; Osteoclasts; Osteoblasts.
Address for correspondence and reprints: Dr. Sharyn Bord, Department Medicine, Level 5, Box 157, Addenbrooke’s Hospital, Cambridge CB2 2QQ, UK. E-mail: [email protected] © 1999 by Elsevier Science Inc. All rights reserved.
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Materials and Methods Tissue The present study was conducted on neonatal rib, osteophytes, and heterotopic bone. Neonatal ribs were removed at postmortem (28 –37 weeks gestation). Osteophytes were obtained following routine shoulder and hip-joint replacement surgery. Heterotopic bone was removed from sites of ectopic bone formation at the hip of two and shoulder of one patient (mean age 29 years) who had previously sustained severe head injury. Informed written consent was given and approval obtained from the local ethics committee. Following removal, the tissue was immediately placed on ice and then immersed in 5% polyvinylalcohol (PVA) for 2 min, snap frozen in liquid nitrogen, and stored at 270°C. Frozen sections were obtained using a Bright cryostat (Huntingdon, UK) with a cabinet temperature of 230°C, equipped with a slow-drive high-torque motor with automatic speed control and a highly polished tungsten carbide knife. Histology Undecalcified, unfixed 9 mm thick frozen sections were stained with Diff-Quik (Baxter Dade AG) or 1% toluidine blue for general morphology, and von Kossa with a van Giesen counterstain to assess matrix mineralization. Alkaline phosphatase staining to identify osteoblasts was detected using a coupled reaction with a-naphthyl acid phosphate and fast-red TR.8 Tartrateresistant acid phosphatase (TRAP) staining in osteoclasts and preosteoclasts was demonstrated by reactivity with AS-BI phosphate and sodium tartrate, postcoupled with fast garnet.25 Antibodies Well-characterized specific polyclonal antibodies to gelatinase-B (MMP-9),30 stromelysin-1 (MMP-3),2 stromelysin-2 (MMP10),19 collagenase (MMP-1),19,28 and TIMP-120 were raised in sheep. Immunoglobulins for all antisera were prepared by triple ammonium sulfate precipitation. Pooled normal sheep serum (NSS) was used as a control at the same IgG concentration as the antibodies. The secondary antibody was a biotinylated rabbit antisheep IgG (Vector Laboratories). Immunohistochemistry Sets of serial sections, 9 mm in thickness, were picked off onto glass slides, coated with 2% 3-aminopropyltriethoxysilane (APES), air dried for 10 min, fixed in 4% paraformaldehyde for 22 min at room temperature, and washed in phosphate-buffered saline (PBS). Immunolocalization was carried out using an indirect immunoperoxidase system. Endogenous peroxidase and nonspecific binding were blocked by incubation with ImmunoPure (Pierce) peroxidase suppressor for 22 min and extensive washing followed by incubation in 20% normal rabbit serum (12 min) prior to the primary antibody incubation (10 mg/mL, overnight at 4°C in a humid chamber). Following further washing, the second antibody was applied (5 mg/mL, 40 min at room temperature). The sections were washed and the signal amplified using avidin-biotin complex (ABC, Vector Laboratories) for 30 min at room temperature. Sites of antigen binding were visualized using 3,39-diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC) as chromogens. Some sections were lightly counterstained with hematoxylin to detect nuclei. Sections were mounted in aqueous mount (90% glycerol in PBS) and observed under bright-field microscopy on an Olympus
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BH-2 microscope. Photographs were taken using Ektachrome 64 film. Quantitation Different cell types were identified morphologically and sites of formation and resorption identified by alkaline phosphatase, TRAP, and gelatinase-B. Serial sections were immunolocalized for TIMP-1 and cells within specific areas examined for positive staining. These areas included endochondral bone formation with associated chondrocytes and osteoblasts, and areas of bone modeling and remodeling. For each cell type, the percentage of cells staining was assessed semiquantitatively by counting five fields from each section and the results expressed as the number of cells staining positively as a percentage of the total cell number. These figures were tabulated as follows: no staining scored as “2”; less than 20% of cells staining positively scored “1”; 20%–50% positive cells as “11”; and more than 50% of cells exhibiting positive staining scored “111.” The presence of matrix staining was also noted. Results All three types of bone showed high cellularity with areas of bone modeling, remodeling, and endochondral and intramembranous bone formation (Figures 1 and 2). Neonatal rib bone showed resting chondrocytes, which progressed into columns of proliferating chondrocytes as endochondral ossification proceeded. Chondrocytes underwent progressive hypertrophy, with increasing mineralization of the intercolumnar matrix (Figure 2A). Within the primary spongiosa, osteoid was deposited on the remnants of resorbed cartilage to form spicules of bone, which were then remodeled by osteoclasts and osteoblasts. Alkaline phosphatase staining was particularly apparent on cancellous surfaces in the primary spongiosa and in the more developed regions of cortical bone. TRAP and gelatinase-B staining was evident in multinucleated cells in both marrow spaces and at sites of resorption. Some mononuclear cells were also positive, as were many hypertrophic chondrocytes. A surrounding periosteal collar of mineralized woven bone, containing many osteocytes, was covered by periosteum and contained vascular channels. Heterotopic bone exhibited areas of multilayers of cuboidal alkaline phosphatase-positive osteoblasts associated with thick osteoid seams and numerous osteocytes in the mineralized matrix, suggesting rapid bone formation. Some bone surfaces were smooth and lined with quiescent cells, whereas other areas showed large numbers of gelatinase-B and TRAP-positive osteoclasts along resorbed surfaces of the bone. These features are indicative of high bone turnover. Areas of intramembranous and endochondral ossification were evident, usually surrounded by fibrous tissue or cartilage. Sections viewed under polarized light showed that most of the bone was woven with random distribution of collagen fibrils. Generally, the bone had a disorganized structure, but gave the appearance of being highly active, with large numbers of cells (Figure 1A,B). Osteophytic bone also showed areas of endochondral and intramembranous bone formation with both woven and lamellar bone evident (Figure 1C,D). The chondrocytic differentiation stages through to hypertrophy often appeared truncated, the zone of columnar chondrocytes being shorter and less well organized (Figure 1C) than those seen in the growth plate of normal rib bone. Vascular invasion and extensive resorption of the cartilage by osteoclasts was followed by deposition of woven bone matrix by osteoblasts on the resorbed surfaces; these osteoblasts showed
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Figure 1. (A, B) Undecalcified sections from heterotopic bone demonstrating the high cellularity of the bone. Multilayers of osteoblasts (ob) are seen at sites of bone formation and many osteoclasts (oc) are evident on resorbing surfaces. Numerous osteocytes (o) are apparent in the bone matrix (b). (A) Diff-Quik-stained section viewed under low power. (B) Toluidine blue-stained section viewed under high power showing the resorbed surface of the bone (arrowheads) and adjacent osteoclasts. (C, D) Undecalcified Diff-Quik-stained sections from osteophytic bone. (C) Area of endochondral ossification with calcified cartilage matrix (c) containing vacuolated chondrocytes (arrow). Osteoclasts (arrowhead) are seen at the resorbing surface of the calcified matrix. Mineralized woven bone trabeculae (w) develop from this area of cartilage. (D) Intramembranous ossification with a layer of osteoblasts (arrowhead) apposed to the woven bone matrix (o), which gradually mineralizes (m). Bars: (A) 100 mm; (B–D) 20 mm.
intense staining for alkaline phosphatase. Thick osteoid seams were apparent, with large numbers of adjacent osteoblasts. Many osteoclasts were seen on both resorbing bone surfaces and in marrow spaces between developing trabeculae (Figure 1C), their osteoclastic nature confirmed by TRAP and gelatinase-B positivity. The osteophytes were surrounded by a fibrocartilage collar covered with fibrous tissue. Osteophytic bone was highly cellular
and exhibited rapid growth; however, it displayed a more organized structure than that seen in heterotopic bone. Matrix Metalloproteinases Generally, the pattern of localization of MMPs was similar in all three types of bone. Chondrocytes expressed collagenase and
Figure 2. (A–C) Serial sections of neonatal rib. (A) Von Kossa-stained section viewed under low power shows the growth plate of neonatal rib with mineralized bone (black color) of the primary spongiosa, the mineralizing zone (brown color), and columnar chondrocytes (c). (B) TIMP-1 immunolocalized in chondrocytes with positive staining (arrowhead) visualized by the brown reaction product using DAB substrate. TIMP-1 expression gradually decreases throughout the hypertrophic chondrocytes (h) toward the mineralizing zone (m). (C) Control serial section of (B) immunolocalized with normal sheep serum shows absence of staining in chondrocytes (arrowhead). (D, E) Serial sections of osteophytic bone showing columnar chondrocytes (c) adjacent to mineralized bone (m). (D) Section immunolocalized for TIMP-1 shows chondrocyte nuclei stained blue, with no brown color, indicating absence of TIMP-1. (E) Adjacent section stained for collagenase shows many chondrocytes with brown color reaction product in close proximity to blue nuclei, indicating collagenase synthesis (arrowhead). Bars 5 20 mm.
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stromelysin-2 throughout most regions of differentiation, with a slight decrease toward the mineralizing zone. Stromelysin-1 expression was detected in some chondrocytes, but was more predominant in the matrix surrounding proliferating chondrocytes. Osteoblasts on forming surfaces showed intense staining for stromelysin-2 and collagenase, whereas stromelysin-1, occasionally detected in osteoblasts, was more often localized in the underlying osteoid. Some quiescent lining cells were positive for collagenase. Stromelysin-1 was also immunolocalized in osteocytes and in matrix surrounding their lacunae, especially at sites of bone remodeling. Most mononuclear cells in marrow spaces stained positively for gelatinase-B and stromelysin-2. Many osteoclasts were observed in all three types of bone, the majority of which stained positively for gelatinase-B and stromelysin-2. Immunoreactive collagenase was detected in some osteoclasts, but all were negative for stromelysin-1. TIMP-1 TIMP-1 expression showed marked differences in the three types of bone examined. Generally, TIMP-1 was seen in most cell types in normal rib bone, but only low levels were detected in pathological bone. The lowest level of TIMP-1 expression was seen in heterotopic bone (Table 1). The growth plate of rib bone (Figure 2A) showed immunoreactive TIMP-1 in proliferative and mature chondrocytes with a gradual decrease through the hypertrophic region (Figure 2B); TIMP-1 expression was also seen in the mineralizing zone. In samples of pathological bone the greatest number of cells staining positively for TIMP-1 was observed in proliferating chondrocytes at some sites of endochondral ossification (Table 1); however, other areas of endochondral ossification showed absence of TIMP-1 staining, usually associated with high collagenase and stromelysin-2 expression (Figure 2D,E). Osteoblasts in the pathological bone samples showed varying levels of intensity of TIMP-1 expression, the lowest levels usually being associated with areas of rapid remodeling. In areas of heterotopic bone with high MMP expression, little or no TIMP-1 was detected. Similarly, in sections from osteophytic bone, at sites of newly forming osteoid, low levels of TIMP-1 were occasionally seen, often associated with high levels of collagenase and stromelysin (Figure 3A,B). In contrast, many osteoblasts in the rib bone showed distinct TIMP-1 staining (Figure 3C). Those on forming surfaces within the primary spongiosa were often positive as were osteoblasts on cortical bone surfaces. At the same sites many osteoblasts showed positive collagenase expression (Figure 3D). Some lining cells on quiescent surfaces were immunoreactive for TIMP-1 (Figure 3E). Some osteocytes in rib bone were TIMP-1 positive, whereas very few stained positively in the pathological bone samples (Table 1). TIMP-1 was detected in some mononuclear cells in the marrow spaces and primary spongiosa of rib bone. Many osteoclasts were seen in these areas, with almost half staining positively for TIMP-1 (Figure 3F), but the majority showing gelatinase-B immunoreactivity (Figure 3G). These positive cells were seen both apposed to the bone surface at sites of resorption and apparently migrating within marrow spaces (Figure 3F–H). In contrast, despite high numbers of gelatinase-B-positive osteoclasts in both heterotopic and osteophytic bone, no TIMP-1 expression was detected in these cells (Figure 3I,J) (Table 1). On serial sections these osteoclasts stained positively for TRAP.
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Discussion Our results demonstrate that MMPs are widely and differentially distributed throughout developing human bone. The staining patterns observed in both normal and pathological samples of bone were very similar. In contrast, the expression of TIMP-1 was more varied, with distinct differences between normal and pathological bone. In heterotopic bone only occasional low-level TIMP-1 expression was detected, whereas osteophytic bone showed higher levels. In both types of pathological bone no TIMP-1 could be detected in osteoclasts. The widespread distribution of TIMP-1 in normal bone is consistent with a role in the regulation of MMPs during bone development. The growth of osteophytes and heterotopic bone is characterized by endochondral and intramembranous ossification with areas of vascular invasion, cellular proliferation, differentiation, and elaboration of extracellular matrix and advancing mineralization.14,32 These changes resemble those seen in the epiphyseal growth plate of immature growing long bones, thus making these types of bone useful models for studies of bone development.1,6,7,13,26,27 However, bone formation in heterotopic and osteophytic bone is clearly a pathological event and the factors responsible for its regulation may differ from those that operate in normal bone. The pattern of MMP expression in osteophytic and heterotopic bone is consistent with our previously reported results6,7 and it was notable that MMP expression in normal neonatal rib bone was similar to that observed in pathological bone. This was also demonstrated in a previous study comparing stromelysin-1 and -2 expression in osteophytic and rib bone.5 However, in contrast to the pattern of MMP expression there were clear differences in the distribution and levels of TIMP-1 expression between the three types of bone. Several studies have indicated that an imbalance between MMPs and TIMP is associated with pathological events. Lin et al.23 suggested that widespread expression of collagenase with absence or variable levels of TIMP-1 expression could lead to radicular cyst expansion. Berend et al.3 showed that a high ratio of MMP to TIMP in human chondrosarcoma may be indicative of a more invasive and aggressive tumor. However, similar studies in human bone in vivo have not been reported. In vitro studies have demonstrated the potential for TIMP-1 to inhibit bone resorption. Shimizu et al.34 reported that addition of Table 1. Summary of the distribution of TIMP-1 TIMP-1 expression in:
Cartilage/endochondral Proliferating chondrocytes Mature chondrocytes Hypertrophic chondrocytes Mineralized bone Osteoblasts Modeling/remodeling Osteoclasts Osteoblasts Osteocytes Mononuclear cells Lining cells
Normal rib bone
Osteophytic bone
Heterotopic bone
111 111 11 11 (m) 11
11 1 1 1 (m) 1
1 2 2 1 (m) 2/1
11 111 1 11 11
2 11 2 1 2
2 1 2 2/1 2
KEY: 2, no staining or occasional staining; 1, less than 20% cells staining; 11, 20%–50% cells staining; 111, more than 50% cells staining; m, mainly matrix staining.
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Figure 3. (A, B) Serial cryosections of osteophytic bone showing osteoblasts at a forming surface of bone (b). (A) A section immunolocalized with anti-TIMP-1 antibody shows no positive staining in osteoblasts (arrow). (B) Distinct stromelysin expression is detected in osteoblasts (arrow) close to the bone surface. (C, D) Serial sections of neonatal rib showing osteoblasts on a forming surface of bone (b). (C) TIMP-1 immunolocalized in osteoblasts (arrow). (D) Adjacent section stained for collagenase shows positive expression in some osteoblasts (arrow) but absence in others (open arrow). (E) TIMP-1 expression detected in bone (b) lining cells (arrows) on a quiescent surface of neonatal rib. (F, G) Serial cryosections of neonatal rib show osteoclasts in the primary spongiosa. (F) TIMP-1 staining in one osteoclast (arrow) but not in the second osteoclast (open arrowhead) nor in mononuclear cells (arrowhead). (G) Positive gelatinase-B expression in two osteoclasts (arrow and open arrowhead) and in some mononuclear cells (arrowhead). (H) An area of the primary spongiosa in neonatal rib shows positive TIMP-1 staining immunolocalized in a multinucleate osteoclast (arrow) and in some adjacent mononuclear cells (arrowhead). (I, J) Non-counterstained serial cryosections of heterotopic bone (b). (I) Section immunolocalized for TIMP-1 shows no staining in an osteoclast on the bone surface (arrow) or in the marrow space (open arrow). (J) Adjacent section shows gelatinase-B expression immunolocalized in both osteoclasts (arrow and open arrow). Bars 5 20 mm.
exogenous TIMP to bone cultures inhibited bone resorption. Everts et al.,16 using a selective inhibitor of MMPs (CI-I), showed an increase in the amount of nondigested bone matrix in the resorption area facing the ruffled border of osteoclasts, further indicating a role for TIMP as a regulator of bone resorption. Similar experiments by Ellis et al.15 demonstrated that exogenous TIMP and synthetic MMP inhibitors prevented cytokine-induced matrix degradation by MMPs in bovine nasal
cartilage. Our studies demonstrating the presence of TIMP-1 in osteoclasts in normal bone are in agreement with these in vitro experiments, providing in vivo support for a role for TIMP in regulating bone modeling and remodeling. Our results also suggest that TIMP may play a dual role in controlling bone turnover. Immunoreactive gelatinase-B, stromelysin-2, and occasional collagenase were detected in osteoclasts in all types of bone, both on and adjacent to bone surfaces, suggesting that MMPs may
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facilitate the migration of osteoclasts to sites of bone resorption. The presence of TIMP-1 in osteoclasts in normal bone would be consistent with regulation of this migration prior to resorption. In support of this theory, Sato et al.33 demonstrated that isolated osteoclasts were able to migrate through collagen-coated invasion chambers—this process being prevented by the addition of MMP inhibitors. They found no evidence of any involvement of serine or cysteine proteinases, suggesting a unique role for MMPs in this process. In conclusion, this study demonstrates distinct patterns of localization of TIMP-1 and MMPs in different types of human bone in vivo. The presence of MMPs with only low levels of TIMP-1 in pathological bone could result in excessive degradation of the extracellular matrix, and therefore contribute to the poorly organized bone formation seen in heterotopic and osteophytic bone in several ways: first, low levels of TIMP-1 expression in chondrocytes would allow uncontrolled MMP degradation of proteoglycans and induce premature calcification; second, collagenase present in lining cells may play a role in activation of bone remodeling,7 and the absence of TIMP-1 in cells could contribute to increased turnover; and third, the absence of TIMP-1 in osteoclasts would facilitate increased MMP-assisted migration to sites of resorption. In contrast, the widespread expression of TIMP-1 in normal developing bone suggests that it may play a role in controlling MMP activity and thus contribute to the regulation of bone modeling and remodeling in developing human bone. Acknowledgments: J.E.C. is supported by the Wellcome Trust. R.M.H. is funded by the Medical Research Council. The authors are indebted to Chris Constant, Dr. Nick Coleman, and Dr. Wilf Kelsall for their invaluable supply of human bone.
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Date Received: May 12, 1998 Date Revised: October 27, 1998 Date Accepted: October 27, 1998