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. 2009 Mar;60(3):686-97.
doi: 10.1002/art.24331.

Membrane type 1 matrix metalloproteinase is a crucial promoter of synovial invasion in human rheumatoid arthritis

Mary-Clare Miller  1 , Hugh B Manning, Abhilash Jain, Linda Troeberg, Jayesh Dudhia, David Essex, Ann Sandison, Motoharu Seiki, Jagdeep Nanchahal, Hideaki Nagase, Yoshifumi Itoh
Affiliations

Membrane type 1 matrix metalloproteinase is a crucial promoter of synovial invasion in human rheumatoid arthritis

Mary-Clare Miller et al. Arthritis Rheum. 2009 Mar.

Abstract

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.

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Figures

Figure 1
Figure 1. Expression of MT1-MMP in RA pannus
A. Primary synovial cells were isolated from the RA pannus of different patients 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 anti-actin antibody. #1 to #6 indicate samples from different patients. HT1080 cell lysate was applied as a positive control. Annotations are: Pro, proMT1-MMP; Active, active form; 40 kDa, 40 kDa processed form; *, further degraded form; actin, actin. B. RA joint sections were stained with anti-MT1-MMP antibody (a, d), pre-immune mouse IgG (b, e), and Fast Green FCS with Safranin O (c, 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, CD68 macrophage marker and Masson’s Trichrome, and images captured using 40× objective lens. Regions marked with a square box are enlarged and shown in the bottom of each image. A representative deep invasion area (left) and superficial invasion area (right) are shown.
Figure 2
Figure 2. Ex vivo invasion assay of RA pannus tissue
Fresh RA pannus tissues were subjected to an ex vivo invasion assay as described in the Methods section, and invasion was monitored after 5 days. “Control wide view” represents a typical pattern of invasion from a single piece of tissue, and this image was created by combining two fields of view that were obtained using 10× objective lens. Tissues were cultured in the presence or absence of TIMP-1 (0.5 µM), TIMP-2 (0.5 µM) or GM6001 (10 µM). These images were taken using a 20 × objective lens.
Figure 3
Figure 3. MT1-MMP is the cellular collagenase in RA synovial cells
Passaged synovial cells were subjected to an in situ collagen degrading assay for 3 days as described in the Methods section. A. Schematic representation of MT1-MMP constructs. Pro, propeptide; FLAG, FLAG-tag; Cat, catalytic domain; L1, linker-1 region; Hpx, hemopexin domain; L2, linker-2 region; TM, transmembrane domain; CP, cytoplasmic tail; and Zn, catalytic zinc atom. B. Synovial cells were subjected to an in situ collagen degradation assay in the presence or absence of TIMP-1 (0.5 µM), TIMP-2 (0.5 µM) or GM6001 (10 µM) as described in the Methods section. Dark 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 (Ad-Mock), MT1F- ΔCat (Ad- MT1F- ΔCat) or MT1-F (Ad-MT1F), and cell lysates were subjected to Western blot analysis for MT1-MMP, FLAG tag and actin. D. Synovial cells infected with the above viruses at different multiplicity of infection (MOI) were subjected to the in situ collagen degradation assay.
Figure 4
Figure 4. Effect of MT1F-ΔCat expression on pannus cell invasion
Fresh synovial pannus tissues were infected with adenovirus constructs for Mock (Ad-Mock), MT1F- ΔCat (Ad- MT1F- ΔCat) or MT1-F (Ad-MT1F) at 108 pfu/ml, and subjected to the ex vivo invasion assay for five days in the presence or absence of GM6001 (10 µM).
Figure 5
Figure 5. Synovial cell invasion assay using microcarrier beads
Isolated synovial cells were subjected to a microcarrier bead invasion assay as described in the Methods. The effects of TIMP-1, TIMP-2 and GM6001 are shown in A, and the effect of adenoviral infection with Ad-Mock, Ad- MT1F-ΔCat or Ad-MT1F are shown in B. A. Scatter plot representation of the migration distance from the bead surface. The number of cells analyzed is shown for each treatment. ***p<0.0001. Representative pictures of the assayed bead in each treatment are shown on the top. Control represents cells without any inhibitors. B. Scatter plot representation of the migration distance from the bead surface of cells treated or infected as indicated. Number of the cells analyzed is shown for each treatment. Control represents cells without any inhibitors or adenoviral infection. *p<0.0199; and ***p<0.0001. Representative pictures of the assayed beads and Western blot analysis of cell lysates from infected cells with Ad-Mock, Ad- MT1F- ΔCat or Ad-MT1F using 222-1D8 (MT1-MMP) are shown on the top. Control represents cells without any inhibitors or virus infection.
Figure 6
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 pices of about 5 × 5 × 3 mm. Isolated human synovial cells were then cultured on the cartilage pieces in the presence or absence of 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 PBS, and paraffin sections were stained with 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 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 for Safranin O or with the 222-1D8 antibody (MT1-MMP). C. Number 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 to non-treated cartilage.
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