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Abstract

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

Objective

A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage, and this process requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen-degrading matrix metalloproteinases (MMPs), membrane type 1 MMP (MT1-MMP), in synovial pannus invasiveness.

Methods

The expression and localization of MT1-MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemical analysis of RA joint specimens. The functional role of MT1-MMP was analyzed by 3-dimensional (3-D) collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, or GM6001. The effect of adenoviral expression of a dominant-negative MT1-MMP construct lacking a catalytic domain was also examined.

Results

MT1-MMP was highly expressed at the pannus–cartilage junction in RA joints. Freshly isolated rheumatoid synovial tissue and isolated RA synovial fibroblasts invaded into a 3-D collagen matrix in an MT1-MMP–dependent manner. Invasion was blocked by TIMP-2 and GM6001 but not by TIMP-1. Invasion was also inhibited by the overexpression of a dominant-negative MT1-MMP, which inhibits collagenolytic activity and proMMP-2 activation by MT1-MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1-MMP–dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix.

Conclusion

MT1-MMP serves as an essential collagen-degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1-MMP–dependent invasion may represent a novel therapeutic strategy for RA.

Rheumatoid arthritis (RA) is a systemic autoimmune disease, and one of its key pathologic features is the destruction of joint tissue, including cartilage, by inflamed synovial pannus, leading to a loss of joint function. Invading synovial pannus tissue produces proteinases that degrade components of the joint extracellular matrix (ECM) (1). Cartilage is a unique tissue comprised of a small number of resident chondrocytes and their abundant surrounding ECM, and its function is heavily dependent on the nature of the ECM (2, 3). Cartilage ECM is predominantly composed of type II collagen and aggrecan, with other minor components including small leucine-rich proteoglycans and type IX and type XI collagens (2, 3). Aggrecan is considered to be lost first in the early stages of RA, followed by collagen degradation. Aggrecan loss is primarily attributable to 2 types of metalloproteinases: aggrecanases that belong to the ADAMTS family and matrix metalloproteinases (MMPs) (1, 4). Because aggrecan maintains hydration in cartilage, it is an important ECM component endowing cartilage with compressive resistance properties. Loss of aggrecan thus abrogates cartilage function.

The proteinases responsible for collagen degradation are members of the MMP family, including collagenases (MMP-1, MMP-8, MMP-13), gelatinase A (MMP-2), and membrane type 1 MMP (MT1-MMP/MMP-14) (5). Type II collagen is the major collagen in cartilage, accounting for 90% of total collagen (2). Monomeric type II collagen is composed of 3 α1(II) chains in a triple helical structure, which form a fibrillar meshwork by cross-linking with each other and with other minor collagens. The collagen meshwork provides the tissue with structure and tensile strength. Thus, degradation of collagen results in irreversible structural and functional damage to cartilage.

Fibrillar collagens, including type II collagen, are resistant to most proteinases, due to their triple helical structure; however, 5 MMPs (MMP-1, MMP-2, MMP-8, MMP-13, and MT1-MMP) can degrade type II collagen (5). Among those MMPs, MT1-MMP has been shown to be an essential collagenolytic enzyme during postnatal development (6, 7). MT1-MMP–null mice show skeletal defects and general fibrosis, which are thought to be caused by a lack of cellular collagenolytic activity (6). Among the above-mentioned collagenases, MT1-MMP is the only one that can promote cellular invasion into a collagen matrix (8). MT1-MMP, as a cellular collagenase, has also been shown to play an essential role in angiogenesis (7, 9), tumor invasion (10–12) and growth (13), and smooth muscle cell migration in the arterial wall (14). Besides degrading type II collagen, MT1-MMP degrades type I collagen, type III collagen, fibronectin, laminin 1 and laminin 5, fibrin, and aggrecan core protein (15–17). MT1-MMP also activates proMMP-2 (11) and proMMP-13 (18), and it processes ECM receptors including CD44 (19) and syndecan 1 (20), promoting cell migration. MT1-MMP was reported to be expressed by the lining cells of human RA synovium (21, 22) and cells at the cartilage–pannus junction (22), and the potential involvement of MT1-MMP in cartilage invasion by RA synovial cells, by activation of proMMP-2 or by itself, has been reported (21–23). However, the exact role of MT1-MMP in the pathogenesis of RA and the mechanism of synovial pannus invasion are not clear at present.

We have previously shown that homodimerization of MT1-MMP is essential for cell surface collagen degradation (24) and proMMP-2 activation (25), and that disruption of homodimerization by overexpression of a catalytic domain deletion mutant of MT1-MMP (MT1F-ΔCat) effectively inhibits both collagenolytic activity and proMMP-2 activation (24, 25). Thus, MT1-ΔCat is a useful tool for examining the biologic significance of MT1-MMP. In this study, we observed that MT1-MMP is highly expressed in synovial cells obtained from patients with RA, particularly in the cells actively invading into cartilage. Using an adenoviral MT1-ΔCat overexpression system, we demonstrated that MT1-MMP is a key enzyme involved in synovial invasion in RA.

MATERIALS AND METHODS

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

Human RA joint specimens.

RA synovial tissue specimens were obtained, with consent, from patients undergoing joint replacement surgery at Charing Cross Hospital, London and Bath United Hospitals. Ethics approval was granted by the Riverside Research Ethics Committee. All patients met the American College of Rheumatology (formerly, the American Rheumatism Association) 1987 revised criteria for the classification of RA (26). RA joint synovia were identified by their anatomically distinct areas and were harvested directly into RPMI containing 5% fetal bovine serum (FBS)/1% penicillin/streptomycin.

Synovial cell isolation and culture.

Fresh RA synovial tissue specimens were subjected to collagenase digestion. Briefly, the diced synovium was incubated in 1% bacterial collagenase in RPMI for 90 minutes, with gentle shaking. The isolated cells were then washed, counted, and frozen in Dulbecco's modified Eagle's medium (DMEM) containing 10% DMSO and 50% FBS in liquid nitrogen until required for experiments. For Western blot analysis of MT1-MMP, cells were directly seeded onto culture plates without further passage. Synovial cells were cultured in 10% FBS/1% penicillin/streptomycin in DMEM at 37°C in a humidified chamber. For invasion assays, synovial cells passaged between 3–4 times were used. After the third passage, the majority of the cell population was fibroblast-like.

Antibodies.

Mouse monoclonal anti–MT1-MMP antibody, 222-1D8, was generated by injecting recombinant MT1-MMP expressed in Escherichia coli as described previously (25). Goat polyclonal antiactin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-FLAG M2 antibody was obtained from Sigma-Aldrich (Dorset, UK). Mouse monoclonal anti-human CD68 antibody was obtained from Dako (Cambridge, UK).

Histologic analysis of RA specimens.

Fresh RA metacarpal joint specimens obtained from patients with RA were fixed in 4% formaldehyde in phosphate buffered saline (PBS) followed by decalcification in 10% EDTA in PBS. Decalcification was monitored by radiography and was completed after ∼4 weeks. Decalcified tissue specimens were then embedded in paraffin wax for sectioning at 5 μm, using a microtome. Sections were stained for MT1-MMP using 222-1D8 antibody (0.2 μg/ml) with hematoxylin counterstaining, and sections were stained for collagen and aggrecan using fast green FCF and Safranin O, respectively. Some specimens were also subjected to staining with anti-CD68 antibody (1:250 dilution) and Masson's trichrome. Samples obtained for the cartilage invasion assay (see below) were fixed in 4% formaldehyde in PBS and then embedded in paraffin wax for sectioning at 5 μm, using a microtome. Sections were stained for MT1-MMP using 222-1D8 antibody (0.2 μg/ml) with hematoxylin counterstaining and were stained for aggrecan using Safranin O. Images were obtained using a CCD camera–equipped microscope with a 10× objective lens (Leica, Milton Keynes, UK).

Adenoviral vector.

FLAG-tagged MT1-MMP (MT1F) (the FLAG tag of DYKDDDDK was inserted immediately downstream of RRKR111) and its catalytic domain deletion mutant (MT1F-ΔCat) lacking Tyr112 to Pro312 were constructed using the polymerase chain reaction extension method, and the sequence was confirmed by DNA sequencing, as described previously (24, 25). For expression in human synovial cells, adenoviral vectors were constructed using the AdEasy system (Qbiogene, Costa Mesa, CA) according to the manufacturer's instructions. The transgene is expressed under the cytomegalovirus promoter. In addition to the recombinant adenoviruses expressing MT1F (Ad-MT1F) and MT1F-ΔCat (Ad-MT1F-ΔCat), mock virus (Ad-mock) was also constructed. High-titer virus stocks were prepared by discontinuous cesium chloride gradient ultracentrifugation, and their titer was measured using the Adeno-X titer kit (BD Biosciences, Oxford, UK).

Ex vivo invasion assay.

For the 3-dimensional (3-D) ex vivo invasion assay, we used acid-soluble type I collagen without pepsin treatment (3 mg/ml Cellmatrix Type 1-A; Nitta Gelatin, Osaka, Japan). Nine parts collagen gel were mixed with 1 part 10× RPMI (Sigma-Aldrich), and the pH was adjusted to 8.0 by the addition of 1M NaOH on ice. The collagen was further diluted using DMEM, adjusting its concentration to 2 mg/ml. Synovial tissue specimens were diced into small pieces ∼2 mm3 in size and embedded within the collagen gel. Tissue specimens in the collagen gel were cultured in 10% FBS/DMEM for 5 days, and images were obtained with a CCD camera–equipped microscope (Nikon TE2000; Nikon, Surrey, UK).

In situ collagen degradation assay.

An in situ collagen degradation assay was carried out as described previously (24). Six-well culture plates were coated with a thin layer of chilled neutralized PureCol collagen (Nutacon, Leimuiden, The Netherlands) at 2.7 mg/ml in 1× RPMI medium (typically 100 μl/well) and incubated for 60 minutes at 37°C to enable fiber formation. Isolated RA synovial fibroblasts were seeded on the collagen film (1 × 105/well) and cultured for 4 days in the absence of serum at 37°C. At the end of the culture period, the remaining collagen film was exposed by removing cells, using repeated treatment with PBS containing 0.5 mg/ml trypsin and 1 mM EDTA. The collagen film was then fixed with 3% paraformaldehyde for 20 minutes at room temperature. Collagen was visualized by staining with Coomassie brilliant blue R250, and the images were captured using a 20× objective lens and a CCD camera–equipped microscope (Nikon TE2000). The degraded area was visualized as a clear unstained zone.

Microcarrier bead invasion assay.

Isolated RA synovial fibroblasts were attached to Cytodex 3 microcarrier beads (Sigma-Aldrich) by incubating trypsinized cells with sterile beads at 37°C overnight, with gentle agitation. The beads and attached cells were suspended in 2 mg/ml Cellmatrix Type 1-A collagen gel (Nitta Gelatin) and cultured for up to 72 hours. At the end of the culture period, images were obtained using the 10× objective lens on a CCD camera–equipped microscope (Nikon TE2000). The distance that cells had migrated from the bead surface was measured using Openlab software (Improvision, Coventry, UK). Median values were calculated, and statistical comparisons between the groups were performed using the Mann-Whitney U test (GraphPad Prism; GraphPad Software, San Diego, CA).

Cartilage invasion assay.

Fresh bovine nasal septum cartilage was diced to a size of ∼5 × 5 × 3 mm and treated with or without retinoic acid for 7 days in serum-free DMEM. Pieces were then frozen and thawed 3 times to kill chondrocytes. Retinoic acid–treated cartilage was further incubated in an excess volume of serum-free DMEM to remove any retinoic acid remaining in the tissue. In some experiments, fresh cartilage was immediately frozen. Isolated RA synovial cells were cultured on top of the cartilage in the presence or absence of inhibitors for 2 weeks or 4 weeks, in DMEM supplemented with 2% FBS and antibiotics.

RESULTS

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

Cartilage invasion by pannus requires the degradation of type II collagen (1). Because one of the membrane-bound MMPs, MT1-MMP, has been shown to promote cellular invasion into collagen matrix (8–10, 27), we performed Western blot analysis to examine whether synovial cells from patients with RA express MT1-MMP, using a specific anti–MT1-MMP antibody. As shown in Figure 1A, all of the tested primary synovial cells (n = 6 patients) expressed high levels of MT1-MMP. HT1080 human fibrosarcoma cells were used as a positive control, because they are well known to express MT1-MMP (11, 28). The band patterns seen in synovial cells were the same as those observed in HT1080 cells, with the active form of MT1-MMP (50 kd), a catalytic domain–processed form of ∼40 kd, and further-degraded forms of lower molecular weights being visible. It is noteworthy that the level of MT1-MMP expressed by the synovial cells is equivalent to or higher than that expressed by HT1080 cells, which are an invasive cancer cell line.

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Figure 1. Expression of membrane type 1 matrix metalloproteinase (MT1-MMP) in rheumatoid arthritis (RA) pannus. A, Primary synovial cells were isolated from the RA pannus of different patients (patients 1–6) and plated into 6-well plates and grown until they became confluent. Cell lysates were then subjected to Western blot analysis, using a mouse monoclonal anti–MT1-MMP hemopexin domain antibody and a polyclonal antiactin antibody. HT1080 cell lysate was applied as a positive control. M = marker; pro = proMT1-MMP; 40 kDa = 40-kd processed form; ∗ = further degraded form. B, RA joint sections were stained with anti–MT1-MMP antibody (panels a and d), preimmune mouse IgG (panels b and e), and fast green FCF with Safranin O (panels c and f). Three matching representative areas are shown (a–c and d–f). Arrows indicate the cartilage–pannus junction, where MT1-MMP is highly expressed. Note that Safranin O staining is almost negative throughout the specimens. C, RA joint sections were stained for MT1-MMP using CD68 macrophage marker and Masson's trichrome, and images were captured using a 40× objective lens. The bottom row shows enlarged images of the boxed areas in the top row.

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Next, we tested whether MT1-MMP expression correlates with cartilage invasion, by performing immunohistochemical analysis of RA joint specimens stained with the same anti–MT1-MMP antibody. As shown in Figure 1B, MT1-MMP was highly expressed in pannus tissue, particularly at the pannus–cartilage junction, i.e., at the invasion edge of the pannus (panels a and d [arrows]). In these specimens, cartilage completely lacked aggrecan, as indicated by the lack of Safranin O staining (Figure 1B, panels c and f). We analyzed joint specimens from 24 different patients with RA, and similar results were obtained for all specimens (data not shown). Some RA joint specimens were also stained with anti–MT1-MMP and anti-CD68 antibodies. Most cells invading into cartilage, especially those invading deeply into the tissue, were CD68-negative and spindle shaped (Figure 1C, deep invasion). However, in some regions, CD68-positive cells were present in the pits formed on the cartilage surface (Figure 1C, superficial invasion). Nevertheless, both cell types highly expressed MT1-MMP at the invasion front.

To test the functional contribution of MT1-MMP to synovial pannus invasion, we developed an ex vivo invasion model. Freshly isolated RA synovial tissue specimens were diced into small pieces (∼2 mm3), embedded in a 3-D collagen gel, and cultivated for 5 days. During the initial 24 hours, small T cell–like cells migrated into the collagen matrix but were undetectable after 48 hours. Numerous large spindle-shaped fibroblastic cells started to invade into collagen after 48–72 hours (Figure 2). Because the invasion edge of synovial tissue consisted of large fibroblastic cells (Figure 1C), the fibroblastic cell invasion into collagen gel observed in this ex vivo model is likely to represent in vivo pannus invasion.

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Figure 2. Ex vivo invasion assay of rheumatoid arthritis (RA) pannus tissue. Fresh RA pannus tissue samples were subjected to an ex vivo invasion assay, as described in Materials and Methods, and synovial pannus invasion was monitored after 5 days. The “control wide view” represents a typical pattern of invasion from a single piece of tissue, and this image was created by combining 2 fields of view that were obtained using a 10× objective lens. Tissues were cultured in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP-1) (0.5 μM), TIMP-2 (0.5 μM), or GM6001 (10 μM). These 4 images were obtained using a 20× objective lens. Bars = 100 μm.

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Next, we performed this invasion assay in the presence of tissue inhibitor of metalloproteinases 1 (TIMP-1) (0.5 μM), TIMP-2 (0.5 μM), or GM6001 (10 μM). It is known that the activity of transmembrane-type MT-MMPs, including MT1-, MT2-, MT3-, and MT5-MMP, cannot be inhibited by TIMP-1 but is inhibited by TIMP-2 and GM6001 (29, 30). As shown in Figure 2, invasion was completely inhibited by TIMP-2 and GM6001 but not by TIMP-1. We performed this invasion assay using joint synovial tissue specimens obtained from 5 different patients, and the results were identical (data not shown). Among TIMP-1–insensitive MT-MMPs, MT1-MMP and MT2-MMP have been shown to promote cell invasion into type I collagen matrix (8). However, we could not detect MT2-MMP in any of the synovial cells tested by Western blotting (data not shown), which supports the results of previous studies (21, 31) showing either no expression or inconsistent, low expression of MT2-MMP compared with MT1-MMP. Therefore, these data suggest that MT1-MMP may be a key collagenolytic enzyme promoting synovial invasion.

We recently reported that MT1-MMP requires homodimerization to degrade collagen, and inhibition of this interaction by the expression of a catalytic domain deletion mutant of MT1-MMP (MT1F-ΔCat) effectively inhibited collagenolytic activity on the cell surface (24). Therefore, we constructed an adenoviral vector for expression of MT1F-ΔCat (Ad-MT1F-ΔCat) to test the role of MT1-MMP in synovial fibroblast invasiveness (Figure 3A). We also constructed Ad-MT1F to overexpress the enzyme (Figure 3A). When isolated RA synovial fibroblasts were cultured on a collagen film, they degraded collagen, leaving unstained clear areas, and this activity was inhibited by TIMP-2 and GM6001 but not by TIMP-1 (Figure 3B). This suggests that collagen degradation by these cells was caused by MT1-MMP and not by any soluble MMPs, including MMP-1, MMP-2, MMP-8, and MMP-13.

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Figure 3. In situ collagen-degrading assay showing that membrane type 1 matrix metalloproteinase (MT1-MMP) is the cellular collagenase in rheumatoid arthritis synovial cells. Passaged synovial cells were subjected to an in situ collagen-degrading assay for 3 days, as described in Materials and Methods. A, A schematic representation of MT1-MMP constructs is shown. B, Synovial cells were subjected to an in situ collagen-degrading assay in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP-1) (0.5 μM), TIMP-2 (0.5 μM), or GM6001 (10 μM), as described in Materials and Methods. Darkly stained areas represent remaining collagen, and clear areas represent the area where collagen has been degraded. C, Synovial cells were infected with adenovirus constructs of mock virus (Ad-mock), the catalytic domain (Cat) deletion mutant of FLAG-tagged MT1-MMP (Ad-MT1F-ΔCat), or MT1F (Ad-MT1F), and cell lysates were subjected to Western blot analysis for MT1-MMP, FLAG tag, and actin. D, Synovial cells infected with the viruses described above at different multiplicities of infection (MOIs) were subjected to an in situ collagen-degrading assay. Pro = propeptide; FLAG = FLAG tag; L1 = linker 1 region; Hpx = hemopexin domain; TM = transmembrane domain; CP = cytoplasmic tail; Zn = catalytic zinc atom. Bar = 100 μm.

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We then tested the effect of adenoviral expression of MT1F and MT1F-ΔCat. As shown in Figure 3C, these exogenous genes were overexpressed in synovial fibroblasts. It was noted that most of the MT1F expressed in cells processed to a 40-kd species and lost its catalytic domain containing FLAG tag (Figure 3C). Such processing has been reported previously and suggests that the functional activity in these cells is high (30, 32, 33). The collagen-degrading activity was not affected by infection of the cells with mock viruses (Ad-mock) with different multiplicities of infection but could be inhibited in a dose-dependent manner by Ad-MT1F-ΔCat (Figure 3D). Degradation was further accelerated in a dose-dependent manner by infection with Ad-MT1F. Taken together, these data suggest that MT1-MMP is the cellular collagenase in human synovial cells.

We next examined whether MT1F-ΔCat can inhibit synovial cell invasion, using an ex vivo collagen invasion assay. To establish effective adenoviral infection of human RA synovial tissue, we carried out dose-dependent infection with a recombinant adenovirus that expresses green fluorescent protein and found that a virus titer of 108 plaque-forming units/ml was minimally toxic and maximally effective for expression of the gene, although, in this system, it is inevitable that some cells will not be infected, especially in deeper areas of the tissue. As shown in Figure 4, cells from RA synovial tissue infected with mock virus invaded into the collagen gel, and this invasion was largely suppressed by GM6001. In contrast, pannus infected with Ad-MT1F-ΔCat showed significantly reduced invasion, which was further inhibited by the addition of GM6001. Infection with Ad-MT1F slightly enhanced invasiveness in terms of invasion distance compared with Ad-mock–infected cells, and GM6001 again strongly inhibited invasion. In order to obtain a more quantitative measurement of synovial cell invasion, we carried out microcarrier bead invasion assays, using isolated synovial fibroblasts.

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Figure 4. Effect of MT1F-ΔCat expression on pannus cell invasion. Fresh synovial pannus tissue specimens were infected with adenovirus constructs for mock virus (Ad-mock), MT1F-ΔCat (Ad-MT1F-ΔCat), or MT1F (Ad-MT1F) at 108 plaque-forming units/ml and subjected to an ex vivo invasion assay for 5 days in the presence or absence of GM6001 (10 μM). See Figure 3 for other definitions. Bar = 100 μm.

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As shown in Figure 5, synovial cells attached to microcarrier beads invaded from the beads into the collagen gel very efficiently. This invasion was inhibited by TIMP-2 and GM6001 but not by TIMP-1 (Figure 5A). TIMP-1 actually enhanced invasion in this particular experiment, but the effect was observed in only 1 of 3 experiments using different synovial cells. When these cells were infected with Ad-MT1F-ΔCat prior to the assay, invasion was almost completely inhibited, whereas the expression of MT1F enhanced invasion (Figure 5B). These data suggest that MT1-MMP is indeed a crucial collagenolytic proteinase enabling these synovial cells to invade into a collagen matrix.

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Figure 5. Synovial cell invasion assay using microcarrier beads. Isolated synovial cells were subjected to microcarrier bead invasion assay, as described in Materials and Methods. A, Scatterplot representation of the migration distance from the bead surface, showing the effects of tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, and GM6001. The number of cells analyzed is shown for each treatment. The top row shows representative images of the assayed bead for each treatment. B, Scatterplot representation of the migration distance from the bead surface of cells treated or infected as indicated, showing the effect of adenoviral infection with Ad-mock, Ad-MT1F-ΔCat, or Ad-MT1F. The number of cells analyzed is shown for each treatment. The top rows show representative images of the assayed beads (left) and Western blot analysis of cell lysates from cells infected with Ad-mock, Ad-MT1F-ΔCat, or Ad-MT1F using 222-1D8 (MT1-MMP) (right). Bars in scatterplots show the median. ∗ = P < 0.0199; ∗∗∗ = P < 0.0001. See Figure 3 for other definitions.

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Next, we examined the invasion of synovial fibroblasts into the cartilage matrix. For this assay, we chose bovine nasal cartilage, because it is possible to dissect out pieces of reproducible size and quantity for different treatments. Fresh bovine nasal cartilage was frozen and thawed 3 times to kill the chondrocytes and diced into 3 × 5 × 5–mm pieces. Human synovial cells were then cultured on the cartilage surface for up to 4 weeks. As shown in Figure 6A, synovial cells invaded into the cartilage in a time-dependent manner. This invasion was not inhibited by TIMP-1 but was completely inhibited by TIMP-2 and GM6001. We attempted adenoviral infection in this experimental system but were hampered by the 4-week length of the assay, because adenoviral transgene expression lasts only up to 10 days.

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Figure 6. Invasion of synovial cells into cartilage. A, Fresh bovine nasal cartilage was frozen and thawed 3 times to kill the chondrocytes and cut into pieces of ∼5 × 5 × 3 mm. Isolated human synovial cells were then cultured on the cartilage pieces in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP-1) (0.5 μM), TIMP-2 (0.5 μM), or GM6001 (10 μM) for 2 weeks or 4 weeks. Cartilage pieces were then fixed with 4% formaldehyde in phosphate buffered saline, and paraffin sections were stained with the anti–membrane type 1 matrix metalloproteinase (anti–MT1-MMP) antibody, 222-1D8. B, Fresh bovine nasal cartilage pieces were treated with or without retinoic acid (10 μM) to remove aggrecan by inducing the expression of aggrecanases, for 7 days prior to freezing and thawing. Synovial cells were then cultured on the cartilage pieces for 4 weeks, and paraffin sections were stained with Safranin O or 222-1D8. C, The numbers of cells at various depths were counted and expressed as a histogram. Note that synovial cells invaded much deeper into retinoic acid–treated cartilage compared with untreated cartilage.

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In RA cartilage, aggrecan is thought to be lost in the early stages of the disease, and indeed, not much aggrecan remains at the late stage of the disease, as shown in Figure 1B. Pratta et al (34) reported that aggrecan protects collagen against degradation by MMP-1 in vitro. Therefore, we next examined whether removal of aggrecan from the cartilage would affect synovial cell invasion. To remove aggrecan, live cartilage was treated with 10 μM retinoic acid for 7 days to stimulate aggrecanase production and aggrecan removal. The cartilage was then subjected to freezing and thawing 3 times to kill the chondrocytes. As shown in Figure 6B, this treatment depleted aggrecan from the cartilage, as indicated by the lack of Safranin O staining. Untreated cartilage maintained staining, although some loss of staining was noted at the edge of the tissue specimen. With both treatments, synovial cells clearly invaded into the cartilage within 4 weeks. However, synovial fibroblasts invaded much deeper into retinoic acid–treated cartilage. To analyze this finding quantitatively, the cells at different depths from the surface of the cartilage were counted. As shown in Figure 6C, in untreated cartilage, most synovial cells invaded up to 90 μm from the cartilage surface, whereas in retinoic acid–treated cartilage more cells were present in deeper areas. The proportion of cells that had invaded deeper than 90 μm was 17% for nontreated cartilage and 39% for retinoic acid–treated cartilage. These data suggest that aggrecan indeed protects collagen in cartilage, and aggrecan loss may be an important initial step in promoting synovial invasion into cartilage in RA.

DISCUSSION

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

Synovial pannus is formed as a result of inflammation, which causes proliferation of synovial lining cells and infiltration of monocytes and lymphocytes that produce high levels of inflammation mediators such as tumor necrosis factor α (TNFα) and interleukin-1 (IL-1) (35). It is thought that the major cause of sustained inflammation is overproduction of cytokines, and indeed anti-TNFα therapy has been proven to be very effective in RA (36). However, the expression of MT1-MMP does not correlate with the area where inflammatory cells reside but rather with the invasion edge, suggesting that inflammatory cytokines may not stimulate expression of MT1-MMP. In fact, IL-1 and TNFα treatment did not modify MT1-MMP expression in isolated synovial fibroblasts (Miller MC, Itoh Y: unpublished observations). This is not surprising, because the promoter region of MT1-MMP lacks activator protein 1 and NF-κB responsible elements and a TATA box, which are present in many inflammatory cytokine–inducible genes (37, 38). It has been shown that treatment of fibroblasts with concanavalin A (39), cytochalasin D (40), and collagen (41), and transformation of epithelial cells with v-src (42) induce MT1-MMP expression. However, it is not known what regulates MT1-MMP gene expression in pathologic conditions such as RA. Nevertheless, there appeared to be a strong correlation between high levels of MT1-MMP expression and synovial invasion in RA.

To investigate whether there is a functional link between MT1-MMP expression and pannus invasion, we examined the invasion of freshly isolated synovial tissue specimens or isolated synovial fibroblasts into collagen gels or cartilage. In every assay, our results indicated that synovial cells use MT1-MMP as a collagenolytic enzyme to invade into the matrix. Invasion was almost completely inhibited by TIMP-2 and GM6001 but not by TIMP-1. The concentrations of these inhibitors used in the experiments were sufficient to achieve almost complete inhibition of the enzymes (0.5 μM for TIMPs and 10 μM for GM6001). Thus, soluble collagenases, including MMP-1, MMP-2, MMP-8, and MMP-13, are unlikely to be involved. These results are consistent with the previous report by Hotary et al (8) that overexpression of these soluble collagenases does not enhance cell invasion. Furthermore, collagen invasion was effectively inhibited by adenoviral expression of MT1F-ΔCat, suggesting that MT1-MMP is the responsible enzyme. However, several reports in the past have shown that with cytokine treatment, MMP-1 and MMP-13 are highly up-regulated in RA synovial fibroblasts and in RA joint tissues (43–50). Therefore, what is the potential role of these soluble MMPs? These soluble collagenases are likely to degrade collagens and other matrix components in cartilage, such as aggrecan (51), in a wider area, which may in turn compromise the integrity of the tissue and promote pannus invasion indirectly. Nonetheless, further work is required to understand the exact roles of these soluble collagenases in the pathogenesis of RA.

MT1-MMP plays an important role in postnatal development, as evidenced by the phenotype of MT1-MMP–null mice (6, 7). A lack of the MT1-MMP gene causes skeletal dysplasia accompanied by severe runting, osteopenia, and fibrosis of soft tissue, and such mice die ∼7–12 weeks after birth (6, 7). An interesting additional phenotype is the development of severe generalized arthritis accompanied by the presence of hypercellular and vascularized synovial tissues, degrading articular cartilage in all joints (6, 7). This apparently contrasts with our finding that MT1-MMP promotes synovial invasion into cartilage in RA. However, the cell types involved in cartilage degradation appear to be different in each case. In MT1-MMP–null mice, cells degrading cartilage are tartrate-resistant acid phosphatase (TRAP)–positive osteoclast-like giant cells, which dominate the inflamed synovium (6, 7). We observed no apparent TRAP-positive osteoclast-like cells at the cartilage–pannus junction in the joint specimens from patients with RA examined. We did occasionally find CD68-positive macrophages in pits on the cartilage surface, but cells invading deeply into the cartilage were negative for factor VIII (endothelial marker), CD3 (T cell marker), and CD68 (Figure 1C, and Miller MC, Itoh Y: unpublished observations). Thus, it is likely that fibroblast-like cells are the major cartilage-invading cell type in human RA (52). Therefore, the arthritic phenotype of MT1-MMP–null mice is likely to be caused by a different mechanism.

Besides type II collagen, aggrecan is the other major component of cartilage, endowing cartilage with compressive resistance properties by retaining water molecules in the tissue (3). In RA and osteoarthritis (OA), aggrecan is depleted from cartilage by the action of aggrecanases belonging to the ADAMTS family (1, 4). It has been reported that deletion of the ADAMTS-5 gene in mice protects against aggrecan depletion and cartilage degradation in experimental OA and inflammatory arthritis models (53, 54). Pratta et al (34) have reported that removing aggrecan makes cartilage more susceptible to collagen degradation by MMP-1 in vitro, and Little et al (55) have recently shown that mutation of the aggrecanase cleavage sequence in the interglobular domain of aggrecan by gene knock-in prevents aggrecan loss and cartilage erosion. These reports suggest that aggrecan may protect against collagen degradation. In the current study, we confirmed this protective effect of aggrecan. Removing aggrecan by pretreating cartilage with retinoic acid made cartilage more susceptible to synovial cell invasion. Because aggrecan is completely lost in advanced RA, this may be one of the mechanisms promoting synovial pannus invasion.

The mechanism by which aggrecan protects against collagenolytic action remains unclear. It is possible that the negatively charged sulfated polysaccharide chains of aggrecan that fill the collagen fibril gaps may prevent enzyme–collagen interactions. Without retinoic acid treatment, synovial cells were still able to invade into cartilage, but they appeared to be unable to migrate further than 60 μm from the cartilage surface. During the course of the experiments, we observed that Safranin O staining near the cartilage surface became weaker during an incubation period of 2–4 weeks, even in the absence of viable cells (data not shown). This is presumably attributable to diffusion of aggrecan into culture media. It is thus possible that the loss of aggrecan up to 60 μm might have been sufficient to allow the invasion of synovial cells.

In summary, we have provided evidence supporting the idea that MT1-MMP is a key proteinase that promotes pannus invasion into cartilage. MT1-MMP also plays an important role in monocyte transmigration through the endothelium (56) and in angiogenesis (7, 9, 57), both of which contribute to RA progression. We thus believe that MT1-MMP can be a target molecule for potential therapeutic intervention in RA.

AUTHOR CONTRIBUTIONS

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

Dr. Itoh had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Itoh.

Acquisition of data. Miller, Manning, Jain, Troeberg, Dudhia, Essex.

Analysis and interpretation of data. Miller, Sandison, Seiki, Nanchahal, Nagase, Itoh.

Manuscript preparation. Miller, Manning, Troeberg, Dudhia, Seiki, Nanchahal, Nagase, Itoh.

Statistical analysis. Miller.

REFERENCES

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