Cartilage invasion by pannus requires type II collagen degradation (1
). Since one of the membrane-bound MMPs, MT1-MMP has been shown to promote cellular invasion into a collagen matrix (8
), we examined whether human rheumatoid synovial cells from patients with RA express MT1-MMP by Western blot analysis using a specific anti-MT1-MMP antibody. As shown in , all of the tested primary synovial cells expressed high levels of MT1-MMP (n=6). HT1080, human fibrosarcoma cells, were taken as a positive control as they are well known to express MT1-MMP (11
). Band patterns seen in synovial cells are the same as in HT1080, with the active form of MT1-MMP (50 kDa), a catalytic domain-processed form of around 40 kDa, 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 in HT1080, an invasive cancer cell line. Next, we tested if MT1-MMP expression correlates with cartilage invasion by immunohistochemical analysis of rheumatoid joint specimens stained with the same anti-MT1-MMP antibody. As shown in , MT1-MMP was highly expressed in pannus tissue, particularly at the pannus-cartilage junction, i.e. at the invasion edge of the pannus (see a, d arrows). In these specimens, cartilage completely lacks aggrecan as indicated by the lack of Safranin O staining (). We have analysed joint specimens from 24 different RA patients and similar results were obtained with 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 (, deep invasion). However, in some regions, CD68 positive cells were present in the pits formed on the cartilage surface (, superficial invasion). Nevertheless, both cell types highly express MT1-MMP at the invasion front.
Expression of MT1-MMP in RA pannus
To test the functional contribution of MT1-MMP to synovial pannus invasion, we developed an ex vivo
invasion model. Freshly isolated RA synovial tissues were diced into small pieces about 2 mm3
, embedded into a 3-D collagen gel and cultivated for 5 days. In the initial 24 h, small T-cell-like cells migrated into the collagen matrix, but they were undetectable after 48 h. Numerous large spindle-shaped fibroblastic cells started to invade into collagen after 48–72 h (). Because the invasion edge of synovial tissue consists of large fibroblastic cells (), the fibroblastic cell invasion into collagen gel observed in this ex vivo
model is likely to represent in vivo
pannus invasion. Next, we performed this invasion assay in the presence of tissue inhibitor of metalloproteinases (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
). As shown in , invasion was completely inhibited by TIMP-2 and GM6001, but not by TIMP-1. We have performed this invasion assay using joint synovial tissues 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 a type I collagen matrix (8
). However, we could not detect MT2-MMP in any of the synovial cells we tested by Western Blotting (data not shown) which supports previous reports (21
) showing either no expression or inconsistent low expression of MT2-MMP compared to MT1-MMP. Therefore, these data suggest that MT1-MMP may be a key collagenolytic enzyme promoting synovial invasion.
Ex vivo invasion assay of RA pannus tissue
We have recently reported that MT1-MMP requires homo-dimerization to degrade collagen, and inhibition of this interaction by 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 (). We also made Ad-MT1F to over-express the enzyme as well (). 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 (). 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. We then tested the effect of adenoviral expression of MT1F and MT1F-ΔCat. As shown in , these exogenous genes were overexpressed in synovial fibroblasts. It was noted that most of MT1F expressed in the cells processed to a 40 kDa species and lost its catalytic domain containing FLAG tag (). Such processing has been reported previously and suggests that functional activity is high in these cells (30
). The collagen-degrading activity was not affected by infection of the cells with different MOI of mock viruses (Ad-Mock), but could be inhibited in a dose-dependent manner by Ad-MT1F-ΔCat (). Degradation was further accelerated in a dose-dependent manner by infection with Ad-MT1F. Taken together, the data suggest that MT1-MMP is the cellular collagenase in human synovial cells.
MT1-MMP is the cellular collagenase in RA synovial cells
We next examined whether MT1F-ΔCat can inhibit synovial cell invasion using an ex vivo collagen invasion assay. To establish an 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 titre of 108 pfu/ml was minimally toxic and maximally effective for expression of the gene, although, in this system, it is inevitable that there will be some non-infected cells especially in deeper areas of the tissue. As shown in , cells from RA synovial tissue infected with Mock viruses 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, and addition of GM6001 further inhibited invasion. Infection with Ad-MT1-F slightly enhanced invasiveness in terms of invasion distance compared to Ad-Mock infected cells, and GM6001 again strongly inhibited invasion. In order to obtain more quantitative measurement of synovial cell invasion, we carried out microcarrier bead invasion assays using isolated synovial fibroblasts. As shown in , 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 (). TIMP-1 rather enhanced invasion in this particular experiment, but this effect was observed in one out of three 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 MT1-F enhanced the invasion (). These data suggest that MT1-MMP is indeed a crucial collagenolytic proteinase enabling these synovial cells to invade into a collagen matrix.
Effect of MT1F-ΔCat expression on pannus cell invasion
Synovial cell invasion assay using microcarrier beads
We next examined the invasion of synovial fibroblasts into the cartilage matrix. For this assay, we chose bovine nasal cartilage as it is possible to dissect out pieces of reproducible size and quantity for different treatments. Fresh bovine nasal cartilage was frozen and thawed three 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 , 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 have attempted adenoviral infection in this experimental system, but were hampered by the 4-week length of the assay since adenoviral transgene expression lasts only up to 10 days.
Invasion of synovial cells into cartilage
In RA cartilage, aggrecan is thought to be lost in the early stages of the disease, and indeed there is not much aggrecan left at the late stage of the disease, as shown in . Pratta et al
) 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 three times to kill the chondrocytes. As shown in , 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. In both treatments, synovial cells clearly invaded into the cartilage in 4 weeks. However, synovial fibroblasts invaded much deeper into retinoic acid-treated cartilage. To analyze this quantitatively, we counted a number of the cells at different depths from the surface of the cartilage. As shown in , 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 non-treated 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.