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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Objective

To investigate the development of osteoarthritis (OA) after transection of the medial collateral ligament and partial medial meniscectomy in mice in which genes encoding either interleukin-1β (IL-1β), IL-1β–converting enzyme (ICE), stromelysin 1, or inducible nitric oxide synthase (iNOS) were deleted.

Methods

Sectioning of the medial collateral ligament and partial medial meniscectomy were performed on right knee joints of wild-type and knockout mice. Left joints served as unoperated controls. Serial histologic sections were obtained from throughout the whole joint of both knees 4 days or 1, 2, 3, or 4 weeks after surgery. Sections were graded for OA lesions on a scale of 0–6 and were assessed for breakdown of tibial cartilage matrix proteoglycan (aggrecan) and type II collagen by matrix metalloproteinases (MMPs) and aggrecanases with immunohistochemistry studies using anti-VDIPEN, anti-NITEGE, and Col2-3/4Cshort neoepitope antibodies. Proteoglycan depletion was assessed by Alcian blue staining and chondrocyte cell death, with the TUNEL technique.

Results

All knockout mice showed accelerated development of OA lesions in the medial tibial cartilage after surgery, compared with wild-type mice. ICE-, iNOS-, and particularly IL-1β–knockout mice developed OA lesions in the lateral cartilage of unoperated limbs. Development of focal histopathologic lesions was accompanied by increased levels of MMP-, aggrecanase-, and collagenase-generated cleavage neoepitopes in areas around lesions, while nonlesional areas showed no change in immunostaining. Extensive cell death was also detected by TUNEL staining in focal areas around lesions.

Conclusion

We postulate that deletion of each of these genes, which encode molecules capable of producing degenerative changes in cartilage, leads to changes in the homeostatic controls regulating the balance between anabolism and catabolism, favoring accelerated cartilage degeneration. These observations suggest that these genes may play important regulatory roles in maintaining normal homeostasis in articular cartilage matrix turnover.

Osteoarthritis (OA) is a degenerative condition characterized by loss of articular cartilage and joint remodeling. The degradation of cartilage is characterized by increased expression of matrix metalloproteinases (MMPs), cytokines including interleukin-1 (IL-1) and tumor necrosis factor α (TNFα) and their receptors, and nitric oxide synthase (NOS) (1). Aging mice present valuable opportunities for the study of the pathogenesis of OA. We have previously demonstrated a naturally occurring degeneration of the medial tibial cartilage in the STR/ort mouse as a model of OA (2). Accelerated development of OA in mice can be induced by partial medial meniscectomy (PMM). This results in rapidly developing tibial cartilage degeneration (3). Articular cartilage degeneration in OA is thought to result from an imbalance between anabolic and catabolic pathways involved in the turnover of its extracellular matrix (ECM) (1). Changes in the expression or activity of several cytokines and matrix-degrading enzymes, such as MMPs and aggrecanases, have been implicated in the pathogenesis (1).

Addition of IL-1 to healthy articular cartilage induces the catabolism of cartilage ECM proteoglycans (4) and type II collagen (CII) (5, 6) and suppresses the synthesis of matrix components such as CII and proteoglycan (7, 8). IL-1 is a known inducer of MMPs (9, 10), and its expression is increased at an early stage in the development of OA in the STR/ort mouse (11) and in human OA (12). IL-1β–converting enzyme (ICE) is the physiologic modulator of IL-1β generation and generates the mature 17-kd IL-1β cytokine (13). ICE is expressed in human synovial membrane and cartilage, with significantly more cells staining positive in OA tissue than in normal tissue (14). Overexpression of ICE, also known as caspase 1, can induce apoptosis in transfected cell lines (15).

The MMP stromelysin 1 (SLN-1) is thought to play a major role in cartilage metabolism due to its ability to act on a wide range of protein substrates. It has been shown to be up-regulated in OA cartilage (16). It is an activator of procollagenase (17) and progelatinases (18) and can cleave the nonhelical regions of CII (19) and the aggrecan core protein (20). It can also cleave fibronectin and the minor cartilage collagens, types IX and XI (21). Mice deficient in the gene for SLN-1 fail to exhibit cleavage of aggrecan by MMPs or collagenase cleavage of CII during antigen-induced arthritis (22); nevertheless, cartilage proteoglycans are depleted, presumably by the action of aggrecanases.

The enzyme inducible NOS (iNOS) is responsible for NO production in cartilage. Production of NO is increased in OA, as shown by elevated nitrite concentration in the synovial fluid and serum of OA patients (23). NO can inhibit the synthesis of matrix macromolecules (24–26) and enhance or suppress MMP activity (27, 28). It can also stimulate the synthesis of IL-1 receptors on chondrocytes, which, along with a decrease in IL-1 receptor antagonist production, may lead to increased matrix degradation (29). NO can also induce chondrocyte apoptosis, a recognized feature of OA (30).

Abrogation of the expression of genes encoding such factors may provide information about their role in the disease process. In this study we induced secondary OA by sectioning of the medial collateral ligament and PMM in mice in which genes encoding IL-1β, ICE, SLN-1, and iNOS had been deleted. The results of these deletions in relation to the development of OA lesions are described.

To assess dependency (or lack thereof) of cartilage matrix breakdown on these factors, we graded histologically the severity of tibial plateau cartilage lesions in wild-type (WT) and knockout mice at different times postoperatively. Proteolytic cleavage of CII by collagenase was localized using the Col2-3/4Cshort antibody that specifically recognizes a carboxy-terminal neoepitope on the three-quarter fragment resulting from cleavage of collagen by a collagenase (31). There is evidence of enhanced collagen cleavage and of both aggrecanase- and MMP-mediated turnover of aggrecan in human OA cartilage (31, 32) and in naturally occurring murine OA (34–36), and we investigated cleavage of the matrix proteoglycan aggrecan by aggrecanase 1 or aggrecanase 2, or by MMPs, using the anti–carboxy-terminal NITEGE (32) and VDIPEN (33) neoepitope antibodies, respectively. The occurrence of tibial chondrocyte apoptosis was also assessed, using a DNA fragmentation (TUNEL) assay.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Animals and generation of gene deletions.

Adult mice were used in these studies. Mice were bred at Merck Research Laboratories. All aspects of the study were approved by the Merck Animal Care and Use Committee and were performed in accordance with institutional policy and National Institutes of Health guidelines governing the humane treatment of vertebrate animals. The mice were maintained in a barrier facility, handled in a laminar flow hood, housed 3–10 per sterile micro-isolator cage (6 × 9.25 × 18 inches), and fed a sterilized standard rodent laboratory diet and acidified (pH 2.5–3.0) autoclaved water ad libitum. Sentinel mice were evaluated quarterly as part of a health surveillance program and were determined to be specific pathogen free by Charles River Laboratory Assessment Plus profile (Wilmington, MA), endo- and ectoparasite examinations, and gross necropsy.

Gene deletions in mice were generated as described previously (37–40). The following strains were used: SLN-1–knockout F2 N2 B10.RIII (75% B10.RIII, 12.5% C57/BL6, and 12.5% 129), ICE and IL-1β–knockout F2 N3 B10.RIII (87.5% B10.RIII, 6.25% C57/BL6, and 6.25% 129), iNOS-knockout C57/BL6/129 (50% C57/BL6 and 50% 129). Full-cousin WT mice were used as controls. The mean ± SD age (weeks) of each group of mice at the time of surgery was as follows: WT 22.0 ± 3.3 (n = 16), SLN-1−/− 28.1 ± 1.5 (n = 17), ICE−/− 25.2 ± 1.7 (n = 18), iNOS−/− 27.6 ± 3.0 (n = 16), IL-1β−/− 21.9 ± 4.5 (n = 22). Only male mice were used. They were anesthetized with ketamine (2 mg/kg) and xylazine (0.12 mg/kg) intramuscularly, and the right stifle (knee) joint was destabilized by transection of the medial collateral ligament and removal of the cranial half of the medial meniscus (3). Left stifle joints served as unoperated controls. The mice were killed at 4 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after surgery, by carbon dioxide inhalation. Both stifle joints were removed, placed in 5% polyvinyl alcohol, then moved to n-hexane at −80°C, after which they were stored at −80°C until used.

Histologic analysis and grading of cartilage lesions.

Serial sagittal sections (10 μm thick) were cut across the whole joint, using a Bright bone cutting cryostat (Bright Scientific Instruments, Huntingdon, UK). Sections were mounted onto Superfrost Plus microscope slides (BDH Laboratories, Poole, UK) and stored at −70°C until use. Each unstained section was graded for histologic changes of OA on the tibial plateau, using a previously described grading system (11) in which 0 = normal articular cartilage, 1 = roughened articular surface and small fibrillations, 2 = fibrillations down to zone 2 chondrocytes just below the superficial flattened cell layer (SFCL) and some loss of SFCL, 3 = loss of SFCL and fibrillations extending to the calcified cartilage, 4 = major fibrillations and cartilage erosion to the subchondral bone, 5 = major fibrillations and erosion of up to 80% of cartilage, and 6 = >80% loss of cartilage. The overall grade of OA in the joint was defined as the most advanced grade observed in the tibial plateau in all sections from that joint.

For statistical analysis, mice assessed 4 days–2 weeks after surgery and those assessed 3–4 weeks after surgery were grouped together (separate groups for WT and each knockout). A mean OA grade was calculated for the medial and lateral plateaus in each group. OA scores for each plateau for each group were then compared for operated versus unoperated joints, by paired t-test. Mean scores in groups of knockout mice were also compared with those in control WT mice, by unpaired 2-tailed t-test. P values less than or equal to 0.05 were considered significant.

Detection of CII and aggrecan degradation.

CII cleavage by collagenase(s) was visualized with the rabbit antibody Col2-3/4Cshort (31), using a modified detection method, as described previously (35). Control sections were incubated with Col2-3/4Cshort that had been preabsorbed with 500 μg/ml of the immunizing peptide for 1 hour at 37°C. Aggrecan degradation in the G1–G2 interglobular domain was visualized using the rabbit polyclonal neoepitope antibodies anti–carboxy-terminal VDIPEN (MMP cleavage) and anti–carboxy-terminal NITEGE (aggrecanase cleavage), as described previously for murine OA (36). The antibodies were generated at Merck Laboratories; their specificity for the neoepitopes in mouse cartilage has been demonstrated by preabsorption with the immunizing peptide (36). The murine aggrecan sequence differs slightly from the human sequence such that cleavage by aggrecanases generates an NVTEGE neoepitope rather than NITEGE. However, it is unlikely that this affects the antibody affinity for the murine neoepitope (36). Control sections in the present experiments were treated with rabbit IgG to exclude the possibility of nonspecific immunostaining.

Alcian blue staining.

Alcian blue staining (41) was performed to detect any loss of proteoglycan in the cartilage of WT and knockout mice, with or without surgery. Thawed sections were fixed in freshly prepared 4% formaldehyde in phosphate buffered saline (PBS) for 5 minutes at room temperature. After washing in PBS, sections were stained with 0.05% Alcian blue solution (8GX; BDH Laboratories) containing 0.5M magnesium chloride (BDH Laboratories) prepared in 0.025M sodium acetate (pH 5.8), overnight at room temperature. Following rinsing in distilled water, sections were cleared in butan-1-ol and mounted using DPX mounting medium (BDH Laboratories).

TUNEL analysis.

Chondrocyte cell death (apoptosis) was detected in cartilage by the TUNEL assay (Apoalert DNA Fragmentation Assay; Clontech, Palo Alto, CA), which labels low molecular weight DNA fragments as well as single strand breaks. The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes the polymerization of fluorescein-conjugated nucleotides to free 3′-hydroxyl DNA ends, which are visualized by fluorescence microscopy.

The assay was performed according to the protocol recommended by the manufacturer. Briefly, thawed sections were fixed with freshly prepared 4% paraformaldehyde (BDH Laboratories) in PBS for 5 minutes at room temperature. The permeability of the ECM was increased by treatment with 0.1% (weight/volume) proteinase K for 2 minutes at room temperature before sections were fixed again, washed, and incubated with equilibrium buffer for 15 minutes at room temperature. The TdT reaction mixture (45 μl labeling solution, 5 μl TdT enzyme solution per section; Roche Diagnostics, Mannheim, Germany) was added for 90 minutes at 37°C. Sections were placed in 1× saline–sodium citrate solution before mounting with Vectashield mounting medium (Vector, Burlingame, CA) containing propidium iodide as a nuclear counterstain.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Histopathologic findings.

Unoperated joints of WT mice showed little or no histopathologic evidence of OA (Figure 1A). Two joints had very mild lesions (grades 1–2) of the lateral tibial cartilage and 1 had a grade 1 lesion in the medial cartilage. In contrast, following surgery, WT mouse joints developed mild lesions (grades 1–3), predominantly on the medial tibial plateau (Figure 1B). Approximately 50% of mice showed no evidence of lesions during the first 2 weeks after surgery, but all developed lesions by 3–4 weeks after surgery.

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Figure 1. Osteoarthritic (OA) lesions in wild-type (WT) and gene-deleted mice before and after partial medial meniscectomy (PMM). The stifle joints of WT mice (A and B) and of mice in which the genes for stromelysin 1 (SLN-1) (C and D), interleukin-1β (IL-1β)–converting enzyme (ICE) (E and F), inducible nitric oxide synthase (iNOS) (G and H), and IL-1β (I and J) had been deleted were graded for OA lesions 4 days or 1, 2, 3, or 4 weeks after transection of the medial collateral ligament and PMM (Operated). Contralateral joints were used as controls (Unoperated). Lesions in the medial tibial cartilage and in the lateral tibial cartilage were graded on a scale of 1–6, as described in Materials and Methods. Each bar represents 1 animal.

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Examination of mice that were null for 1 of the 4 genes (SLN-1, ICE, iNOS, or IL-1β) revealed the following: In unoperated limbs, SLN-1–knockout mice showed a low incidence of lesions, similar to that observed in WT mice (Figure 1C). However, ICE-, iNOS-, and IL-1β–knockout mice had an increased incidence of lesions in unoperated joints (Figures 1E, G, and I). These lesions were generally mild (grade 1) in the ICE- and iNOS-null mice and were present predominantly on the lateral tibial plateau. In contrast, in IL-1β–knockout mice, several unoperated joints had more advanced lesions (grades 2–4), mainly on the lateral tibial plateau (Figure 1I).

All groups of null mice exhibited accelerated formation of advanced lesions (up to grade 5) on the medial tibial plateau in operated limbs (Figures 1D, F, H, and J). This occurred as early as 4 days after surgery. Milder lesions (grades 1–2) were present on the lateral tibial plateau of most operated limbs in null mice, but the incidence was similar to that found in unoperated joints. Similarly, although a higher incidence of more severe lesions (grades 2–5) was seen on the lateral tibial plateau of operated joints in IL-1β–knockout mice (Figure 1J), this was also found in unoperated joints. Typical examples of lesions are shown in Figures 2–6.

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Figure 2. Localization of the collagen cleavage Col2-3/4cshort neoepitope in murine OA. A and B, Some weak cell-associated immunostaining, but no matrix staining, is evident in intact medial (A) or lateral (B) tibial cartilage. C–G, OA lesions in the medial tibial cartilage of either WT or knockout mice are accompanied by Col2-3/4cshort staining at the site of lesions. C, Inducible NOS–knockout mouse 4 weeks postsurgery; grade 2 lesion. D, ICE-knockout mouse 3 weeks postsurgery; grade 3 lesion. E, SLN-1–knockout mouse 4 days postsurgery; grade 3 lesion. F, Inducible NOS–knockout mouse 3 weeks postsurgery; grade 5 lesion. G, IL-1β–knockout mouse 2 weeks postsurgery; grade 3 lesion. H, Section adjacent to that shown in G, but which was incubated with primary antibody that had been preabsorbed with its immunizing peptide. Staining was abolished by incubation (asterisk indicates an area of loss of staining). AC = articular cartilage (see Figure 1 for other definitions).

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Figure 3. Localization of the aggrecan-cleavage VDIPEN neoepitope. A, Intact medial tibial cartilage from a WT mouse, showing pericellular/territorial immunostaining predominantly in the mid and deep zones. B, Lateral tibial cartilage from a WT mouse with a grade 1 lesion, 4 days postsurgery. VDIPEN expression is present predominantly in the midzone. C–E, Following surgery, all groups of mice developed OA lesions in the medial tibial cartilage, accompanied by VDIPEN neoepitope staining at the site of the lesions. C, SLN-1–knockout mouse 3 weeks postsurgery; grade 2 lesion. D, IL-1β–knockout mouse 2 weeks postsurgery; grade 3 lesion. E, Inducible NOS–knockout mouse 2 weeks postsurgery; grade 3 lesion. F, Inducible NOS–knockout mouse 3 weeks postsurgery; grade 4 lesion. (In advanced lesions [grades 4–5] such as the one seen here, staining was lost from much of the cartilage; asterisk indicates loss of staining.) G and H, Unoperated IL-1β–knockout mouse. Histologically normal medial cartilage shows increased pericellular and matrix VDIPEN immunostaining (H) when the lateral cartilage shows evidence of lesions (G). Arrows indicate increased immunostaining. See Figure 1 for definitions.

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Figure 4. Localization of the aggrecan-cleavage NITEGE neoepitope. A, Normal medial tibial cartilage from a WT mouse. Staining is present predominantly in the superficial and upper zones of the cartilage. B–F, With the development of OA in either WT or knockout mice, staining was increased at sites of medial tibial cartilage lesions except in advanced lesions (e.g., grade 5), where it was lost. B, ICE-knockout mouse 1 week postsurgery; grade 1 lesion. C, Inducible NOS–knockout mouse 2 weeks postsurgery; grade 2 lesion. D, SLN-1–knockout mouse 3 weeks postsurgery; grade 3 lesion. E, Inducible NOS–knockout mouse 2 weeks postsurgery; grade 3 lesion. F, Inducible NOS–knockout mouse 4 weeks postsurgery; grade 5 lesion. Note the loss of staining (asterisk). G and H, Unoperated IL-1β–knockout mouse. Histologically normal medial cartilage shows increased pericellular and matrix NITEGE immunostaining (H) when the lateral cartilage shows evidence of lesions (G). Arrows indicate increased immunostaining. AC = articular cartilage (see Figure 1 for other definitions).

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Figure 5. Alcian blue staining for proteoglycan. A and B, The extracellular matrix stained strongly and evenly for proteoglycan in intact WT mouse medial (A) and lateral (B) tibial cartilage. C–E, Proteoglycan staining was lost from lesional areas, as indicated by arrows. C, SLN-1–knockout mouse 4 days postsurgery; grade 1 lesion. D, Inducible NOS–knockout mouse 3 weeks postsurgery; grade 2 lesion. E, Inducible NOS–knockout mouse 2 weeks postsurgery; grade 3 lesion. See Figure 1 for definitions.

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Figure 6. TUNEL staining to detect chondrocyte cell death. Nuclei of cells are stained red with propidium iodide. Dead cells appear yellow where red nuclei and green fluorescein isothiocyanate staining from TUNEL labeling of DNA fragments merge (arrowheads and arrows). Very few, if any, dead cells were detected in intact cartilage, either in unoperated joints or after surgery, in A and B, WT mice or C–J, knockout mice. With the development of lesions (D–I), an increase in the number of TUNEL-positive dead cells was seen. C, Unoperated left stifle, ICE-knockout mouse 4 days following surgery on right stifle; medial cartilage, no damage (grade 0). D, Unoperated left stifle, ICE-knockout mouse 4 days following surgery on right knee; lateral cartilage, grade 1 lesion. E, IL-1β–knockout mouse 2 weeks postsurgery; medial cartilage, grade 4 lesion. F, IL-1β–knockout mouse 2 weeks postsurgery; lateral cartilage, grade 1 lesion. G, Inducible NOS–knockout mouse 3 weeks postsurgery; medial cartilage, grade 3 lesion. H, Inducible NOS–knockout mouse 3 weeks postsurgery; lateral cartilage, grade 1 lesion. I, SLN-1–knockout mouse 4 days postsurgery; medial cartilage, grade 4 lesion. J, SLN-1–knockout mouse 4 days postsurgery; normal lateral cartilage (grade 0). Asterisks indicate cartilage regions with few cells. See Figure 1 for definitions.

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A mean lesion severity score was calculated for groups of mice analyzed 4 days–2 weeks (Table 1) and 3–4 weeks (Table 2) after surgery, as described in Materials and Methods. Analysis of the data by paired t-test confirmed that there were significant increases (P < 0.05) in lesion severity in the medial cartilage of operated joints compared with unoperated contralateral joints in each group of knockout mice, 4 days–2 weeks postsurgery (Table 1). No significant differences were observed in the lateral cartilage of operated and unoperated joints, nor were there any significant differences in mean lesion score in either the medial or lateral cartilage of operated versus unoperated joints in WT control mice at this stage. Comparison of the mean scores of medial cartilage lesions in operated joints of each group of knockout mice with those of WT mice (Table 1) confirmed that the former were significantly increased compared with the latter at this early postoperative stage (P < 0.05 for each knockout group, by unpaired t-test).

Table 1. Mean ± SD lesion severity scores in tibial plateau cartilage from unoperated and operated joints of wild-type and gene-deficient mouse groups, 4 days–2 weeks postsurgery
Mouse group, cartilage*UnoperatedOperated
  • *

    SLN-1 = stromelysin 1; ICE = interleukin-1β (IL-1β)–converting enzyme; NOS = nitric oxide synthase.

  • P < 0.05 versus unoperated joint, by paired t-test.

Wild-type  
 Medial0.1 ± 0.30.4 ± 0.7
 Lateral0.1 ± 0.30.2 ± 0.4
SLN-1–knockout  
 Medial0.2 ± 0.42.2 ± 1.6
 Lateral0.5 ± 1.30.4 ± 0.7
ICE-knockout  
 Medial0.2 ± 0.41.2 ± 0.9
 Lateral0.8 ± 0.61.0 ± 0.6
Inducible NOS–knockout  
 Medial0.3 ± 0.72.7 ± 1.2
 Lateral0.3 ± 0.50.6 ± 0.5
IL-1β–knockout  
 Medial0.3 ± 0.61.9 ± 1.4
 Lateral1.7 ± 1.61.6 ± 1.3
Table 2. Mean ± SD lesion severity scores in tibial plateau cartilage from unoperated and operated joints of wild-type and gene-deficient mouse groups, 3–4 weeks postsurgery
Mouse group, cartilage*UnoperatedOperated
  • *

    SLN-1 = stromelysin 1; ICE = interleukin-1β (IL-1β)–converting enzyme; NOS = nitric oxide synthase.

  • P < 0.05 versus unoperated joint, by paired t-test.

Wild-type  
 Medial0.0 ± 0.02.2 ± 0.8
 Lateral0.3 ± 0.80.3 ± 0.8
SLN-1–knockout  
 Medial0.0 ± 0.03.3 ± 1.0
 Lateral0.3 ± 0.80.7 ± 1.0
ICE-knockout  
 Medial0.4 ± 0.52.4 ± 1.4
 Lateral1.0 ± 1.01.0 ± 0.6
Inducible NOS–knockout  
 Medial0.6 ± 0.53.3 ± 1.6
 Lateral1.1 ± 0.41.0 ± 0.0
IL-1β–knockout  
 Medial0.6 ± 0.93.2 ± 1.1
 Lateral0.2 ± 0.41.0 ± 1.0

At 3–4 weeks postsurgery, comparisons between the mean severity score for medial cartilage lesions in operated and unoperated joints revealed significantly increased scores in the WT mice, as well as in each of the knockout groups (P < 0.05 for each group) (Table 2). There were no significant differences in lesion severity between the lateral cartilage of operated joints and the lateral cartilage of unoperated joints in any group of mice at this stage. However, the mean severity scores for lesions in the medial cartilage of operated joints of the SLN-1–, iNOS-, and IL-1β–knockout mice were significantly higher than they were for operated joints of WT mice (P < 0.002 for each, by unpaired t-test).

Following surgery, the lesions in joints of WT mice and SLN-1–, ICE-, and iNOS-null mice were usually focal and located toward the anterior aspect of the medial tibial plateau. The cartilage posteriorly was intact and of normal appearance. In contrast, in IL-1β–knockout mice, lesions often occurred throughout the medial tibial plateau, with few or no regions of intact cartilage. Overall, lesions showed a similar range of severity scores in all groups of knockout mice at all postoperative time points.

The frequency of histopathologic OA lesions following surgery in either WT or gene-deficient mice was much lower in the femoral condyle cartilage than in the tibial cartilage. Moreover, since the femoral cartilage is thin, it is more difficult to grade lesions at this site with the same accuracy as in the tibial cartilage. Similarly, there was very little evidence of osteophyte formation or other pathologic change on the margins of the joint. Thus, this report focuses on OA in the tibial plateau.

Cleavage of CII by collagenase: Col2-3/4Cshort neoepitope immunostaining in WT and gene-deleted mice.

The ECM of undamaged, intact cartilage showed no immunostaining for the CII cleavage neoepitope in any of the mice studied, but weak, cell-associated staining was sometimes present (Figures 2A and B). In contrast, in all groups, much stronger Col2-3/4Cshort staining was detected in cartilage matrix wherever lesions were present (Figures 2C–F). In the case of grade 1 lesions, staining was restricted to the region of roughened cartilage surface. With higher-grade lesions, the Col2-3/4Cshort neoepitope was present on both the surface and in the ECM of the cartilage adjacent to the lesions. This staining occurred throughout the adjacent matrix down to the lowest level of the lesion, but no staining was observed in the calcified zone, even where lesions were full depth. Weak cell-associated staining was also evident for some chondrocytes, as in normal cartilage. However, histologically normal cartilage more distant from the lesion showed no matrix immunostaining with the antibody, indicating the focal nature of collagenase activity in the lesional areas. The same pattern of Col2-3/4Cshort staining occurred whenever lesions were present in either WT or gene-deficient mice, in both medial and lateral cartilage, and was independent of surgery or the gene deleted.

In all cases, both cell-associated and matrix staining were abolished by incubating with Col2-3/4Cshort antibody that had been preabsorbed with the immunizing peptide (Figures 2G and H). Thus, staining was not due to nonspecific binding of the antibody.

Cleavage of aggrecan core protein by MMPs: VDIPEN neoepitope immunostaining in WT and gene-deleted mice.

Unoperated WT mice exhibited pericellular and territorial VDIPEN staining throughout the hyaline cartilage in both the medial and lateral tibial plateaus, with the mid and deep zones of the cartilage staining most strongly (Figure 3A). Little or no specific staining was evident in the calcified cartilage. VDIPEN staining in undamaged cartilage of SLN-1–, ICE-, and iNOS-null mice was similar in pattern and intensity to that in WT mice (results not shown). Following surgery, the VDIPEN staining pattern was unchanged in histologically normal WT cartilage, but around lesions, staining followed a pattern similar to that in null mice with lesions, as described below.

When surface roughening was present (grade 1), as occurred frequently in the lateral cartilage of the unoperated ICE- and iNOS-knockout mice and occasionally in WT mice (Figure 1), more prominent pericellular VDIPEN staining was often seen in the midzone, with less marked staining in the deep zone (Figure 3B). With more advanced lesions (grades 2–3), which occurred generally in the medial cartilage of operated animals, VDIPEN staining was variable. In some cases the surface and midzone pericellular areas were positive (Figure 3C), while in others there was more prominent pericellular and ECM staining throughout the full depth of the hyaline cartilage (Figures 3D and E), but without specific staining in the calcified zone. However, the increased staining was restricted to the cartilage adjacent to the lesion, with histologically normal areas having a pattern indistinguishable from that in unoperated WT mice (Figure 3A). With progression to advanced lesions (grades 4–5), VDIPEN neoepitopes were often much decreased in both pericellular and ECM locations (Figure 3F).

A number of unoperated IL-1β–knockout mice had well-advanced lesions in the lateral tibial cartilage (Figures 1I and 3G). Interestingly, undamaged medial cartilage in this group showed a pattern of VDIPEN neoepitope staining different from that in the WT joint, with more intense immunostaining in both the pericellular and ECM throughout the medial cartilage (Figure 3H). The pattern of VDIPEN immunostaining in the medial cartilage of operated IL-1β–knockout mice was the same as that of other groups in which lesions developed.

Cleavage of aggrecan core protein by aggrecanases: NITEGE neoepitope immunostaining in WT and gene-deleted mice.

In unoperated WT mice, NITEGE staining was present predominantly in the upper zones of the cartilage in pericellular sites and at the articular surface (Figure 4A). In lateral tibial cartilage, immunostaining was even more restricted to the upper zones (results not shown). Following surgery, the pattern of NITEGE staining in histologically normal cartilage of WT mice was similar to that in unoperated joints. In regions of focal OA lesions, the intensity of NITEGE immunostaining was increased in the adjacent area (results not shown).

In knockout mice, histologically normal cartilage exhibited an NITEGE staining pattern similar to that in WT mice (results not shown), while immunostaining in damaged cartilage was markedly increased in the area of the lesion and toward the cartilage surface (Figures 4B–E). There was no specific staining in the calcified cartilage. In cases of severe damage, NITEGE staining in the hyaline cartilage was frequently lost (Figure 4F), as with VDIPEN staining. The distribution of the NITEGE neoepitope in damaged cartilage was similar in unoperated IL-1β–knockout mice (Figure 4G) to that in cartilage with lesions in operated mice. Moreover, as with the VDIPEN staining, where the lateral cartilage had lesions, undamaged medial cartilage in unoperated IL-1β–null mice showed increased NITEGE, with staining throughout the cartilage depth (Figure 4H). Staining was still strongest in the upper zones. This increased level of NITEGE was also seen in undamaged contralateral medial cartilage of some ICE-knockout mice (results not shown).

The pattern and intensity of VDIPEN and NITEGE staining in all groups of mice correlated with the grade of OA lesions rather than with the length of time since surgery. In all cases, only very faint background staining occurred when tissue sections were incubated with rabbit IgG (results not shown).

Alcian blue staining.

Alcian blue staining was evenly distributed throughout the cartilage depth in WT mice (Figures 5A and B) and in histologically normal cartilage in all groups of knockout mice (results not shown). Loss of Alcian blue staining was often detected as soon as roughening of the cartilage was seen in either WT or null mice (Figure 5C), indicating that significant proteoglycan depletion occurred at an early stage in the development of OA. Marked loss of staining occurred in areas where more advanced OA lesions were present in the cartilage, indicating massive depletion of matrix proteoglycan at such sites (Figures 5D and E).

TUNEL assay for cell death.

Very few, if any, TUNEL-positive chondrocytes were detected in the intact cartilage of WT mice, either with or without surgery (Figures 6A and B). Intact cartilage in all knockout groups also showed few, if any, TUNEL-positive cells (Figures 6C and J). However, positive cells were clearly present when there was roughening of the cartilage surface (grade 1; Figures 6D, F, and H). Moreover, following surgery, an increase in the number of TUNEL-positive cells was observed when more advanced lesions developed, in all knockout mouse groups (Figures 6E, G, and I). In cases of severe cartilage loss, areas of acellular cartilage were observed (Figure 6E, G, and I).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Unexpectedly, ICE-, iNOS-, and IL-1β–null mice that underwent surgery in one limb developed OA lesions in the lateral tibial plateau of the contralateral unoperated limb as well as in the operated limb. Since such lesions occurred only rarely in unoperated limbs of WT and SLN-1–null mice, it is unlikely that they are due solely to mechanical changes following contralateral surgery. Moreover, whereas both WT and all gene-deficient mouse groups developed OA lesions in the medial tibial plateau of meniscectomized joints, these appeared more rapidly and were of greater overall severity in the knockout mice. These two observations suggest that each of the deleted genes, though encoding factors favoring catabolism, is important physiologically in maintaining the balance between anabolism and catabolism of cartilage matrix. If one of these genes is knocked out, other less well-regulated catabolic factors/pathways may take over (see below), leading to changes in cartilage metabolism and an abnormal matrix being laid down.

Changes in immunostaining for the VDIPEN, NITEGE, and Col2-3/4short neoepitopes provided direct evidence of enhanced CII and aggrecan catabolism in lesional areas. Although VDIPEN and NITEGE epitopes are present in normal murine cartilage (36), they, together with the Col2-3/4Cshort neoepitope, which is not seen in normal cartilage (35), are evident in increased amounts in focal areas around and within even the earliest histopathologic OA lesions. There is also associated loss of Alcian blue staining, denoting a depletion of proteoglycan which is mainly aggrecan.

Recently it was reported that AOAMTS-4 (aggrecanase 1), while cleaving aggrecan core protein primarily at the Glu373/Ala374 site, is also capable of cleaving it slowly and secondarily at the Asn341/Phe342 site, potentially creating VDIPEN neoepitopes, which were thought previously to be produced exclusively by MMPs (42). However, it is noteworthy that strong immunostaining for the aggrecanase neoepitope NITEGE was observed at the surface of normal tibial cartilage in this study (Figure 4A) and a previous investigation (36), in contrast with the absence of staining for VDIPEN neoepitope at this site (Figure 3A). This suggests that such secondary cleavages of aggrecan by aggrecanase do not necessarily occur in vivo, or if they do, only very low levels of VDIPEN neoepitope are produced.

The distribution patterns of aggrecan and collagen neoepitopes associated with osteoarthritic lesions were the same for all mice, irrespective of whether the limbs were unoperated (e.g., IL-1β–knockout mice) or operated, and independent of the gene deletion or WT mouse group investigated. These results are consistent with previous findings in the STR/ort mouse (35, 36) and in C57BL/6 and BALB/c mice (34) and indicate the focal nature of the biochemical as well as the histopathologic changes in murine OA, especially in the early stages of the disease. These focal changes are very different from the widespread changes in neoepitope levels seen in inflammatory arthritides (34, 35). OA, irrespective of its initiating cause, may be a disorder, at least initially, of localized groups of chondrocytes, whereas inflammatory arthritis involves widespread chondrocyte responses to signals originating outside the cartilage. Similar neoepitope expression is seen in human OA (31, 32).

Another emerging feature of OA is its association with chondrocyte death, irrespective of the initiating cause. This occurs in human OA (30, 43, 44), in idiopathic OA in the STR/ort mouse (2), and, in the present study, in the tibial cartilage of operated limbs of WT and knockout mice and in contralateral unoperated limbs of iNOS-, ICE-, and IL-1β–knockout animals. One consequence must be that no new cartilage matrix would be synthesized in the absence of living chondrocytes in areas already subjected to enhanced degradation, leading to increased compromise of the mechanical function of cartilage and the accelerated development of lesions.

Why should deletion of genes for catabolic factors cause increased susceptibility to lesion formation? IL-1β–deficient mice undergo normal growth (45), implying normal cartilage matrix synthesis in the absence of IL-1β signaling. However, compensatory changes in the expression of other genes involved in cartilage catabolism, such as TNFα or IL-1α, may occur. Also, since binding of IL-1β to its receptor activates several kinases in the cell (46), total deficiency of the cytokine may change the overall kinase activity profile in chondrocytes, with downstream effects on the cartilage matrix.

It is noteworthy that IL-1β plays a crucial role in the propagation of joint inflammation (47) and IL-1β–knockout mice are resistant to collagen-induced arthritis (48) and to zymosan-induced arthritis (49). However, this may not be important in OA, in which, at least in the early stages, inflammation is absent. It is interesting that overall, more severe OA lesions occurred in the lateral tibial plateau of unoperated limbs of IL-1β–null mice than in other knockout groups. Mechanical load on the lateral cartilage is likely to differ from that on the medial side, but the intracellular signaling pathways in chondrocytes which respond to load are not understood. It is possible that there is cross-talk between them and other pathways, including that triggered by IL-1β binding to its receptor. Absence of IL-1β would change the pattern of cross-talk, leading to downstream effects. This concept may also help to explain why development of OA in the medial cartilage after surgery is accelerated in IL-1β–null mice compared with WT mice, since the surgery must induce marked changes in loading on that cartilage, in addition to the existing cytokine deficiency.

Although IL-1β is a known inducer of MMPs (7, 10) and of aggrecanase activity (50), the absence of IL-1β did not affect the pattern or intensity of VDIPEN and NITEGE staining in either unoperated or operated knockout mice. Collagenase cleavage of CII was also evident in IL-1β–null mice. Thus, factors other than IL-1β must be involved in stimulating the activity of all of these enzymes in murine cartilage.

SLN-1–knockout mice have a phenotype that is apparently normal (38). SLN-1 cleaves aggrecan to produce the VDIPEN epitope (33), but since the pattern and intensity of VDIPEN immunostaining were the same in SLN-1–null mice as in WT mice, other enzymes must also do this. VDIPEN staining was unchanged in SLN-1–knockout mice with collagen-induced arthritis (38), but was eliminated in the antigen-induced arthritis model, where it was absent even in control mice of another strain (22). Col2-3/4Cshort staining was unaffected in our knockout mice despite the fact that SLN-1 can activate procollagenase (17). It seems likely that SLN-1 may be replaced by SLN-2 (MMP-10) or another MMP. SLN-2 has proteoglycanase activities that are indistinguishable from those of SLN-1 and can activate zymogen forms of MMP-1 and MMP-8 (51). Little is known about SLN-2 in articular cartilage, but expression in human neonatal rib chondrocytes has been reported (52).

ICE-knockout mice also have an overtly normal phenotype (39). Several other proteases can convert the IL-1β precursor to its active form and appear to do so in synovial fluid of patients with inflammatory arthritis (53). Fas ligand also induces IL-1β processing independently of ICE (54). Presumably, the reason advanced lesions did not form in the unoperated limbs of ICE-knockout mice as frequently as in IL-1β–knockout mice is related to IL-1β activation by other proteases. ICE is clearly not essential for stimulating MMP and aggrecanase activity in the surgical model as indicated by VDIPEN, NITEGE, and Col2-3/4Cshort immunostaining, but, as discussed above, IL-1β is also not obligatory for these activities. Neither does ICE play an essential role in chondrocyte apoptosis in advanced OA, since TUNEL-positive cells are seen in the damaged cartilage of ICE-null mice. Accelerated OA in these animals probably occurs for the same reasons as in IL-1β–null mice.

The bones and joints of iNOS-knockout mice have been reported to be normal (40). However, iNOS is up-regulated in chondrocytes after sectioning of the canine anterior cruciate ligament (55). Nitric oxide is produced by chondrocytes stimulated with IL-1β, possibly mediating the suppressive effect of the cytokine on matrix synthesis (24–26, 56). Thus, we anticipated that deletion of iNOS may retard development of OA in the PMM model. However, development of OA was accelerated. One speculation is that the other two isoforms of NOS, neuronal NOS and endothelial cell NOS, contribute to cartilage degradation. Cohen et al reported that reduced NO accumulation, and subsequent matrix preservation, in arthritic human cartilage explants following treatment with methylene blue, an NOS inhibitor, is due to the down-regulation of all 3 isoforms (57). Thus, iNOS deficiency in chondrocytes of gene-deleted mice may be compensated for by increased NO synthesis by the other 2 NOS isoforms. However, it has also been shown that NOS inhibitors can enhance proteoglycan catabolism occurring in response to IL-1 (25, 28). Thus NO may also normally regulate MMP expression.

In conclusion, each deleted gene in this study encodes a factor which, in theory, has an important role in the development of OA. However, our results show accelerated development of OA in the medial tibial plateau cartilage after surgery in all knockout groups and, in the iNOS, ICE, and IL-1β knockouts, a higher level of OA lesions in the lateral cartilage of unoperated mice than is found in WT mice. One possible explanation for this is that these genes encode molecules that are essential for homeostasis in healthy cartilage, balancing anabolism and catabolism of the cartilage matrix. Another is that overcompensation of like genes may have taken place during embryonic development, resulting in the knockouts becoming more susceptible to cartilage degradation.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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