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. 2004 Jun;63(6):656–664. doi: 10.1136/ard.2003.009720

Adenovirus mediated intra-articular expression of collagenase-3 (MMP-13) induces inflammatory arthritis in mice

K Joronen 1, R Ala-aho 1, M Majuri 1, H Alenius 1, V Kahari 1, E Vuorio 1
PMCID: PMC1755025  PMID: 15140772

Abstract

Objectives: To better understand the role of collagenase-3 (MMP-13) in joint inflammation by investigating the consequences of transient overexpression of human collagenase-3 (matrix metalloproteinase-13 (MMP-13)), introduced by adenoviral gene delivery, in the mouse knee joint.

Methods: A single dose (5x107 pfu) of recombinant adenovirus coding either for ß-galactosidase (RAdLacZ) or human MMP-13 (RAdMMP-13) was injected intra-articularly into the knee joint of adult mice. The joints were analysed at frequent intervals up to 4 weeks by histology, immunohistochemistry, and RNA analysis.

Results: When RAdLacZ reporter virus was used, adenoviruses efficiently infected synovial cells, chondrocytes of articular cartilage, and hypertrophic chondrocytes of the growth plate. The infection was transient as no reporter gene activity was detected 3 weeks after the injection. After RAdMMP-13 injection into the knee joints, expression of human MMP-13 in joint tissues resulted in an arthritis characterised by recruitment of inflammatory cells and increased production of cytokines and chemokines, synovial hyperplasia, and pannus formation. After the loss of MMP-13 transgene expression at 3 weeks, these inflammatory changes began to diminish.

Conclusions: MMP-13 has a role in the onset of inflammatory reaction in synovium. However, damage to articular cartilage was only rarely detected after the short term overexpression of MMP-13.

Full Text

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Figure 1 .

Figure 1

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The in vivo transduction efficiency of RAdLacZ. Lateral view of mouse knee joints (A) 1 and (B) 2 weeks after intra-articular injection of 5x107 pfu of RAdLacZ. The joint was dissected as a block, fixed, and stained with X-Gal. The contralateral, uninjected knee exhibited no ß-galactosidase activity as shown 1 week after the injection (C). P, patella; T, tibial muscles; F, femoral muscles.

Figure 2 .

Figure 2

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Recombinant adenoviruses infect different tissues and cells in the knee joint. Immunohistochemical localisation of ß-galactosidase in mouse knee joints using a monoclonal antibody was performed 1 week after injection with either RAdLacZ (A, B, and C) or a control virus RAd66 (D, E, and F). Representative sections of synovium (A and D), articular cartilage (B and E), and growth plate (C and F) are shown. These immunohistochemical analyses were also used to measure the transduction efficiency of RAdLacZ to various cells and tissues of the knee joint. Scoring shown to the left is based on the percentage of the cells positive for ß-galactosidase: –, no positive cells; +/–, 1–24%; +, 25–49%; ++, 50–74%; +++, 75–100%.

Figure 3 .

Figure 3

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Overexpression of MMP-13 results in inflammatory synovitis. Histopathology of RAdMMP-13 treated mouse knee joints 1 week after injection. (A) Photomicrograph of a sagittal section of the inflamed knee joint; (B) qualitative analysis of synovitis based on synovial hyperplasia, fibrosis, inflammation, and necrosis in the RAdMMP-13, RAdLacZ, and RAd66 injected knees (n = 5–6 in each group). Values are the mean (SD); (C) synovial hyperplasia; (D) inflammatory reaction centre consisting of mononuclear cells; (E) invasive pannus formation; (F) superficial erosion of articular cartilage and meniscus were found in the RAdMMP-13 injected joints. The sections were counterstained with haematoxylin and the scale bar represents 800 µm (panel A), 200 µm (panels C and E), and 100 µm (for panels D and F). ac, articular cartilage; b, bone; m, meniscus.

Figure 4 .

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RAdMMP-13 injection increased MMP-13 mRNA production in the knee joint. (A) RT-PCR analysis of human MMP-13 mRNAs in RAdMMP-13 injected knee joints. RNA extracted from a squamous cellular carcinoma sample was used as a positive control. Aliquots of RNA extracted 3, 7, and 21 days after the injection were used for cDNA synthesis by reverse transcriptase reaction and subsequent amplification by PCR. The reaction products were electrophoresed on a 1% agarose gel with molecular weight markers. The expected human MMP-13 derived band of 300 bp is seen migrating close to the 300 bp fragment of the standard. (B) Compiled data from northern analysis of total RNAs extracted from intact, RAdLacZ, or RAdMMP-13 injected knee joints at 1 and 3 weeks after injection (n = 6 at each time) using a probe for murine MMP-13 and 28S ribosomal RNA. The hybridisation intensity was analysed with a phosphoimager and normalised to a constant amount of 28S RNA. Values are mean (SD). Northern analysis: *p<0.05.

Figure 5 .

Figure 5

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Immunohistochemical localisation of MMP-13 production in the joint. Immunohistochemical localisation of human MMP-13 production in the synovium (A), articular cartilage (D), and growth plate (G) 1 week after injection of RAdMMP-13 using a monoclonal antibody recognising only human MMP-13. Binding of the antibody was detected as a reddish brown precipitate using the avidin-biotin complex method. As a control (K) the monoclonal antibody against human MMP-13 was also applied on a section of uninjected control knee. Localisation of both human and mouse MMP-13 by immunohistochemistry in joint tissues 1 week after injection of RAdMMP-13 (B, E, and H) or in uninjected knee joints (C, F, and J) using polyclonal antibody (B and C synovium, E and F articular cartilage, H and J growth plate). Binding of antibodies was detected using biotin labelled secondary antibody and alkaline phosphatase conjugated streptavidin. The sections were counterstained with haematoxylin. The scale bar in panel A represents 100 µm for panel K and 50 µm for the other panels.

Figure 6 .

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Expression of chemokines and cytokines is up regulated after RAdMMP-13 injection Production of mRNAs for (A) MCP-1, (B) MCP-2, (C) MIP-1α, (D) RANTES, (E) TNFα, and (F) IL1ß mRNA production was measured in intact, RAdMMP-13, and RAdLacZ injected mouse knee joints (n = 5–6 per group) using a quantitative Taqman RT-PCR method. The results were normalised for a constant amount of 18S ribosomal RNA. For statistical analyses a Wilcoxson matched pair test was used: *p<0.05, **p<0.005 significance of mRNA levels in RAdMMP-13 treated knee joints over those injected with RAdLacZ.

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