Expression of osteopontin at sites of bone erosion in a murine experimental arthritis model of collagen-induced arthritis: Possible involvement of osteopontin in bone destruction in arthritis

Authors


Abstract

Objective

To investigate the involvement of osteopontin (OPN) in bone destruction in a murine experimental arthritis model of collagen-induced arthritis (CIA).

Methods

The expression of OPN was examined at both the messenger RNA (mRNA) and protein levels in various arthritic lesions in mice with CIA by in situ hybridization and immunohistochemistry, respectively. In addition, the expression of αvβ3 integrin, a receptor for OPN, the ligation of which is thought to be essential for bone resorption by osteoclasts, was examined by immunohistochemistry. Plasma concentrations of OPN were measured at different time points in the course of CIA by enzyme-linked immunosorbent assay.

Results

OPN mRNA was detected mainly at sites of bone erosion in arthritic lesions, where activated osteoclasts were present; OPN protein was also detected at sites of bone erosion. In the arthritic synovium, OPN was predominantly expressed in the synovial lining layer, but not in lymphoid aggregates. In addition, αvβ3 integrin was detected coincident with OPN at sites of bone erosion (bone–pannus junction). Plasma OPN levels were markedly elevated at the time points that corresponded to arthritis flares, and higher levels were maintained during the progression of arthritis.

Conclusion

OPN may mediate bone resorption by osteoclasts in arthritis through ligation with its receptor, αvβ3 integrin. OPN may be a useful therapeutic target molecule in the prevention of bone destruction in arthritis.

Osteopontin (OPN), a secreted phosphoglycoprotein, was originally isolated from bone extracellular matrix (1, 2). OPN is expressed by various cell types, including osteoclasts, macrophages, activated T cells, smooth muscle cells, and epithelial cells, and is present in several tissues, including bone, kidney, placenta, smooth muscle, and secretory epithelia (3). OPN contains an Arg-Gly-Asp (RGD) sequence that interacts with αvβ1, αvβ3, and αvβ5 integrins and is capable of promoting cell attachment, chemotaxis, and signal transduction in several different cell types (4–6). It is now thought that OPN is involved in normal tissue remodeling processes, such as bone resorption, angiogenesis, wound healing, and tissue injury, as well as in certain diseases, such as restenosis, atherosclerosis, renal diseases, and tumorigenesis (7–10).

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by destructive polyarthritis (11). Although destruction of bone and cartilage is a hallmark of RA and one of the most critical problems clinically, the precise mechanism of this destruction remains unknown. Cumulative evidence suggests that bones are remodeled through cycling of bone resorption and new bone formation. The balance depends on the activity of 2 major cell types: osteoclasts for bone resorption and osteoblasts for new bone formation (12). An imbalance between these processes results in various pathogenic states (13). At sites of bone erosion in RA, proliferating synovium, or so-called pannus, can be seen invading the subchondral bone, and many activated osteoclasts are present at the bone–pannus junction (14, 15). These observations support the view that bone and cartilage destruction in RA is mediated through bone resorption by osteoclasts not only from inside the bone, but also from outside the bone (from the side of the proliferating synovium).

Recently, the mechanism of bone resorption by osteoclasts was clarified at the molecular level by studies using knockout mice (12). The attachment of activated osteoclasts to the bone surface is a critical step in bone resorption by osteoclasts, and this step is mediated by the ligation of OPN with αvβ3 integrin. This suggests that OPN plays a critical role in the pathogenesis of RA, particularly in bone resorption, but there have been few studies regarding OPN in RA (16).

In the present study, we investigated the involvement of OPN in bone destruction in arthritis. We examined OPN expression in a murine model of experimental arthritis, collagen-induced arthritis (CIA) in mice.

MATERIALS AND METHODS

Mice.

Animal experiments in this study were performed in accordance with the guidelines of the Animal Experimentation Committee of Osaka University. Male DBA/1J mice were purchased from Nippon Charles River, Kanagawa, Japan; all were 10–12 weeks old at the time of first immunization. All mice were bred at the Experimental Animal Center of Osaka University Medical School and housed in filter-top cages under standard pathogen-free conditions.

Induction of CIA

CIA was induced by established methods, as previously described (17, 18). Briefly, mice were immunized by intradermal injection at the base of the tail with 100 μg of bovine type II collagen (Cosmo Bio, Tokyo, Japan) in 0.1M acetic acid, emulsified with an equal volume of Freund's complete adjuvant (Difco, Detroit, MI). Twenty-one days later, mice received a booster injection by the same method.

Clinical assessment of arthritis

Every week after the first immunization, 10 mice were assessed for signs of arthritis by 2 different observers throughout the study period (11 weeks). The severity of arthritis was graded on a scale of 0–4, where 0 = normal, 1 = swelling and/or redness in 1 joint, 2 = swelling and/or redness in >1 joint, 3 = severe swelling and redness in the entire paw, and 4 = deformity and/or ankylosis. Each paw was graded, and the 4 scores were summed (maximum possible score 16 per mouse). The mean arthritis score was calculated by dividing the total score in all mice by the total number of mice examined.

Tissue preparation

Six weeks after the first immunization, 12 mice were anesthetized with pentobarbital and killed. The arthritis severity in the hind paws was graded and an arthritis score was calculated as described above. Paws representative of each arthritis score (2–4 per score) were removed and fixed in 4% paraformaldehyde in phosphate buffered saline for 2 hours. After decalcification for 10 days with EDTA, the paws were embedded in paraffin. All paraffin sections were used for hematoxylin and eosin staining, for tartrate-resistant acid phosphatase (TRAP) staining using a commercial acid phosphatase leukocyte kit (Sigma, St. Louis, MO), and for immunohistochemical studies.

In situ hybridization

Digoxigenin-11-UTP–labeled single-stranded RNA probes were prepared with a digoxigenin RNA labeling kit (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions. In situ hybridization was performed according to the method of Nomura et al (19).

Immunohistochemistry

Immunohistochemistry was performed on paraffin sections according to the avidin–biotin method, as previously described (8, 20). Briefly, 3-μm deparaffinized sections were pretreated with 10 mM citrate buffer, pH 6.0, in a microwave oven for 20 minutes for antigen retrieval. Rat monoclonal IgG anti-mouse OPN antibody (OPN2.2) (20), hamster monoclonal IgG anti-mouse CD51 (αv integrin) antibody (H9.2B8; PharMingen, San Diego, CA), and hamster monoclonal IgG anti-mouse CD61 (β3 integrin) monoclonal antibody (HMb3; PharMingen) were employed as primary antibodies. An equal concentration of either purified rat IgG or hamster IgG (both from PharMingen) was used as a negative control.

Paraffin sections were treated with 0.3% H2O2, then blocked with 10% rabbit serum and incubated with either the primary or control antibodies overnight at 4°C. Sections were then washed and reacted for 1 hour at room temperature with either biotin-conjugated goat anti-rat or anti-hamster IgG (both from Vector, Burlingame, CA) as a second antibody. After washing, sections were incubated with avidin–peroxidase complex for 1 hour, and the enzyme reaction was developed with diaminobenzidine tetrahydrochloride. All sections were counterstained with hematoxylin.

Measurement of plasma concentrations of OPN by enzyme-linked immunosorbent assay (ELISA)

Plasma from 10 mice with CIA was obtained before immunization and 2, 5, 8, and 11 weeks after the first immunization. Concentrations of OPN were measured using a sandwich ELISA system established by our laboratory, as previously described (21). Briefly, we immunized rabbits with either a synthetic peptide corresponding to the internal sequence of mouse OPN, L17PVKVTDSGSSEEKLY32, or K155SRSFGVSDEQYPDATDE172. Each rabbit serum was purified as OPN-1 or OPN-3. OPN-1 antibody was used as the coating antibody, and OPN-3 antibody was peroxidase-labeled and used as the detecting antibody. Standard samples were affinity-purified recombinant fusion proteins consisting of glutathione S-transferase and mouse OPN.

Statistical analysis

The Mann-Whitney U test was used to assess the significance of differences in plasma OPN levels before and after immunization. Data are expressed as the mean ± SEM. P values less than 0.05 were considered significant.

RESULTS

Expression of OPN mRNA. Twelve mice with CIA were killed 6 weeks after immunization, and each hind paw was graded for arthritis on a 0–4 scale. Paws representative of each score were fixed, decalcified, and embedded in paraffin, and in situ hybridization was performed. As shown in Table 1, cells positive for OPN mRNA were detected in some samples with an arthritis score ≥3, whereas no cells positive for OPN mRNA were detected in any sample with an arthritis score <3. In addition, OPN mRNA–positive cells were detected predominantly at sites of bone erosion (bone–pannus junction) and in the synovial lining layers rather than the sublining area. No OPN mRNA–positive cells were observed in lymphoid aggregates.

Table 1. Expression of osteopontin (OPN) mRNA at different histologic sites in each hind paw of mice with collagen-induced arthritis, by arthritis score and percentage of positive cells*
Histologic site, arthritis scoreNo. of samples positive for OPN mRNA
0% positive cells≤5% positive cells5–20% positive cells>20% positive cells
  • *

    * Twelve mice with collagen-induced arthritis were killed 6 weeks after immunization, and each paw was scored (0–4 scale) for the presence of arthritis. Representative paws for each score were fixed, decalcified, and embedded in paraffin, and in situ hybridization was performed using an OPN-specific probe as described in Materials and Methods. A total of 1 × 103 cells were randomly selected from each histologic site and evaluated by microscopy to calculate the percentage of cells that were positive for OPN mRNA expression. NA = not applicable.

Synovial lining layer
 Arthritis score
  02000
  12000
  23000
  32110
  42010
Synovial sublining
 Arthritis score
  0NANANANA
  12000
  23000
  33100
  43000
Lymphoid aggregates
 Arthritis score
  0NANANANA
  12000
  23000
  34000
  43000
Bone erosions (bone–pannus junction)
 Arthritis score
  0NANANANA
  1NANANANA
  2NANANANA
  30121
  42010

Figure 1 shows a representative sample of the histologic features and in situ hybridization results in paws with an arthritis score of 3. Figures 1A and B show sections from a bone erosion site. OPN mRNA expression was detected at the bone–pannus junction, coincident with the presence of multinucleated giant cells. These cells showed positive staining for TRAP, suggesting that they were activated osteoclasts (data not shown). Figures 1C and D show proliferating synovium, where there are many osteoclast-like cells with a morphologic appearance of multinucleated giant cells. OPN mRNA was detected coincident with these cells. Thus, activated osteoclasts expressed OPN mRNA, particularly at sites of bone erosion (bone–pannus junction) and in proliferating or activated synovium.

Figure 1.

Expression of osteopontin (OPN) mRNA in arthritic paws (arthritis score 3) from mice with collagen-induced arthritis at 6 weeks after immunization. Paws were fixed, decalcified, and embedded in paraffin. Sections were stained with hematoxylin and eosin (A and C), and in situ hybridization (B and D) was performed using a primer specific for OPN mRNA (see Materials and Methods). A and B, In sections from a bone erosion site, OPN mRNA expression was detected at the bone–pannus junction, coincident with the presence of multinucleated giant cells. Those cells stained positive for tartrate-resistant acid phosphatase, suggesting that they were activated osteoclasts (data not shown). C and D, In sections from a proliferating or activated synovium (pannus), many osteoclast-like cells with a morphologic appearance of multinucleated giant cells were also observed, and OPN mRNA was detected coincident with those cells. (Original magnification × 400.)

Expression of OPN protein. Expression of OPN protein was examined by immunohistochemistry of paraffin sections of paws from mice with CIA. The localization of OPN protein was similar to that of OPN mRNA. At sites of bone erosion, some activated osteoclasts with a morphologic appearance of multinucleated giant cells were observed (Figure 2A). These cells showed positive staining for TRAP (Figure 2B). OPN was detected coincident with these cells (Figure 2D). No significant staining with a control antibody was obtained in a section of the same lesion (Figure 2C). The expression of OPN protein was also observed predominantly in synovial lining cells (Figure 3). No significant OPN expression was detected in lymphoid aggregates (data not shown).

Figure 2.

Expression of osteopontin (OPN) in activated (tartrate-resistant acid phosphatase [TRAP]–positive) osteoclasts at sites of bone erosion. Immunohistochemistry was performed as described in Materials and Methods using a monoclonal antibody specific for OPN (OPN2.2). Shown are serial sections stained with A, hematoxylin and eosin, B, TRAP, C, a control antibody, and D, OPN2.2. Bone became detached from the sections in C and D during the immunohistochemistry procedure. At sites of bone erosion, some activated osteoclasts were observed. These had A, a morphologic appearance of multinucleated giant cells and were B, TRAP positive, and D, OPN was detected coincident with those cells. No significant staining with a control antibody was observed in the section of the same lesion shown in C. OC = osteoclast. (Magnification × 400.)

Figure 3.

Expression of osteopontin (OPN) protein in proliferating or activated synovium from a representative arthritic paw (arthritis score 3) at 6 weeks after immunization. Immunohistochemistry was performed as described in Materials and Methods using a monoclonal antibody specific for OPN (OPN2.2). Sections were stained with A, OPN2.2 and B, a control antibody. OPN protein was expressed predominantly in the synovial lining cells rather than the sublining cells. (Original magnification × 200.)

Expression of OPN and αvβ3 integrin at sites of bone erosion (bone—pannus junction). Immunohistochemistry was performed on paraffin sections from sites of bone erosion in mice with CIA to examine the expression of both OPN and αvβ3 integrin, an OPN receptor that is thought to mediate the attachment of osteoclasts to bone through ligation with OPN. We used both anti–αv integrin (CD51) and anti–β3 integrin (CD61) monoclonal antibodies to detect αvβ3. A representative staining pattern is shown in Figure 4.

Figure 4.

Expression of osteopontin (OPN) and αvβ3 integrin at sites of bone erosion (bone–pannus junction) from a representative arthritic paw. Immunohistochemistry of paraffin sections was performed as described in Materials and Methods using monoclonal antibodies specific for B, OPN (OPN2.2), C, anti–αv integrin (CD51), and D, anti–β3 integrin (CD61), as well as A, a control antibody. B, Many OPN-positive (brown-stained) cells, were detected in both the pannus and bone. C, Anti–αv integrin–positive (brown-stained) cells were also observed at the bone–pannus junction. D, A similar staining pattern was observed with anti–β3 integrin antibody on an adjacent serial section. (Original magnification × 200.)

Many OPN positive (brown-stained) cells were detected at both the pannus and bone areas (Figure 4B). In addition, αv integrin–positive (brown-stained) cells were observed at the bone–pannus junction (Figure 4C). A similar staining pattern was seen with anti-β3 antibody treatment of adjacent serial sections (Figure 4D). These results demonstrate the coexistence of αv and β3, indicating that αvβ3 integrin was expressed at sites of bone erosion (bone–pannus junction). No significant staining for CD51 or CD61 was detected at any other site in the synovium (data not shown).

Changes in plasma concentrations of OPN. Plasma concentrations of OPN were measured throughout the course of CIA by an ELISA system. As shown in Figure 5, plasma OPN levels were markedly elevated, particularly at the onset of arthritis, and high levels were maintained during the progression of arthritis. Beginning at 5 weeks after immunization, plasma OPN levels were significantly elevated compared with the levels before immunization.

Figure 5.

Changes in plasma levels of osteopontin (OPN) in mice with collagen-induced arthritis (CIA). Plasma concentrations of OPN were measured throughout the course of CIA using an enzyme-linked immunosorbent assay system as described in Materials and Methods. Plasma OPN levels were significantly elevated, particularly at the onset of arthritis, and high levels were maintained during the progression of the arthritis. = P < 0.05 versus time 0.

DISCUSSION

In the present study, we used in situ hybridization and immunohistochemistry methods to demonstrate the expression of OPN at both the mRNA and protein levels in activated osteoclasts at sites of bone erosion in a murine model of CIA. OPN expression was also observed predominantly in the lining layers of activated synovium, but not in lymphoid aggregates. In addition, immunohistochemistry revealed that αvβ3 integrin, an OPN receptor, was expressed predominantly at sites of bone erosion (bone–pannus junction) and coincident with the expression of OPN. Taken together with the fact that the ligation of OPN with αvβ3 integrin is essential for bone resorption by osteoclasts, these results suggest that the bone resorption of arthritis may also be caused by activated osteoclasts from the proliferating synovium (or pannus) and that the attachment of osteoclasts to the bone is mediated through the ligation of OPN with αvβ3 integrin.

The expression of OPN correlated with the severity of arthritis, and no significant OPN expression was detected in nonarthritic joints (arthritis score 0). Moreover, plasma OPN levels were markedly elevated during the progression of arthritis. These results suggest that OPN expression is inducible and that OPN might be involved in the progression of arthritis. Matsumoto et al (22) demonstrated the induced expression of the nuclear factor for the interleukin-6 gene (NF-IL6), a member of the CCAAT/enhancer binding protein family of transcription factors, increased OPN gene expression by the subtraction cloning method (22). We have also demonstrated by immunohistochemistry and electrophoretic mobility shift assay that NF-IL6 is expressed and activated in the rheumatoid synovium (23). Interestingly, the distribution of NF-IL6 is similar to that of OPN in the rheumatoid synovium.

Taken together, these results suggest that the production of OPN may be regulated in part by NF-IL6 and its upstream molecules, especially proinflammatory cytokines such as IL-1 and IL-6. In fact, we also found that synovial OPN levels in RA patients were elevated and correlated with serum C-reactive protein levels (Ohshima S, et al: unpublished observations). These observations suggest that OPN may be induced by proinflammatory cytokines, such as IL-6 and IL-1β.

In addition to its function as an extracellular matrix protein, OPN is also known to act as a cytokine (Eta-1), to be involved in the polyclonal activation of B cells, and to induce the production of immunoglobulins (24). OPN also induces both T cell and macrophage chemotaxis at sites of inflammation (25, 26). Giachelli et al (26) reported that subcutaneous injection of OPN induced a focal accumulation of macrophages, and anti-OPN treatment inhibited the macrophage recruitment induced by FMLP (26). Moreover, OPN plays an important role in regulating the cellular immune response as an early component of type 1 immunity (24). Ashkar et al (24) also recently demonstrated that OPN directly induced IL-12 production and inhibited IL-10 production by lipopolysaccharide in murine macrophages.

Considering that arthritis is a typical Th1 disease (27), OPN may also contribute to the initiation/onset of arthritis by polarizing Th1 cytokine responses in addition to bone resorption by osteoclasts. Indeed, plasma OPN elevations preceded arthritis, indicating that OPN may participate in the initiation/onset of arthritis. Other investigators (16) have recently demonstrated similar OPN expression in the rheumatoid synovium. Therefore, OPN might be involved in multiple aspects of the pathogenesis of RA.

In conclusion, the present study provides new evidence for the involvement of OPN in the pathogenesis of arthritis, especially in bone destruction by osteoclasts. Moreover, OPN might be a useful predictor of arthritis progression, especially bone destruction, as well as a possible therapeutic target in the prevention of bone destruction in arthritis, although further investigations are necessary.

Acknowledgements

We would like to thank Prof. Steffen Gay for useful comments and suggestions, Mr. A. Fukuyama and Mr. K. Morihana for excellent technical assistance, and Ms R. Ishida for secretarial assistance.

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