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. 2002 May;61(5):391–397. doi: 10.1136/ard.61.5.391

Characterisation of the cell type-specificity of collagenase 3 mRNA expression in comparison with membrane type 1 matrix metalloproteinase and gelatinase A in the synovial membrane in rheumatoid arthritis

P Petrow 1, D Wernicke 1, W Schulze 1, K Hummel 1, R Brauer 1, J Kriegsmann 1, E Gromnica-Ihle 1, R Gay 1, S Gay 1
PMCID: PMC1754087  PMID: 11959761

Abstract

Objective: To study the pattern and cell type-specificity of collagenase 3, membrane-type 1 matrix metalloproteinase (MT1-MMP), and gelatinase A mRNA expression in the synovial membrane in rheumatoid arthritis (RA).

Methods: The mRNA expression of collagenase 3, MT1-MMP, and gelatinase A was characterised by northern blot analysis, reverse transcriptase-polymerase chain reaction, and in situ hybridisation. In situ hybridisation was performed in combination with the immunohistochemical detection of cell type-specific antigens.

Results: Synovial membrane specimens from 19 of 21 patients with RA expressing collagenase 3 mRNA were positive for MT1-MMP and gelatinase A mRNA. In control samples from patients without destructive inflammatory joint diseases collagenase 3 mRNA was not expressed and only in two of seven cases was a coexpression of MT1-MMP and gelatinase A mRNA detected. Fibroblast-like cells of the synovial membrane were found to be the predominant source of collagenase 3, MT1-MMP, and gelatinase A mRNA expression in lining and sublining layers as well as at the synovial membrane-cartilage interface. Additionally, the expression of MT1-MMP mRNA was detected in endothelial cells. Collagenase 3 mRNA expression was found in about 5% of CD68 positive macrophages.

Conclusions: Collagenase 3 mRNA is expressed simultaneously with MT1-MMP and gelatinase A mRNA in fibroblast-like cells of the synovial membrane in RA. These results suggest (a) a broad extracellular proteolytic potential of fibroblast-like cells and (b) an important role of cell surface associated procollagenase 3 activation by MT1-MMP and gelatinase A for cartilage degradation by invading fibroblast-like cells.

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Figure 1 .

Figure 1

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In situ hybridisation of collagenase 3 and interstitial collagenase mRNA in the synovial membrane from patients with RA (NBT/BCIP colour reagent). Collagenase 3 mRNA was detected in lining and sublining layers (A), whereas the mRNA expression of interstitial collagenase was largely restricted to the synovial lining (B). These data were confirmed by analysing synovial membrane samples of six patients with RA. Scale bar 100 µm in A; 130 µm in B.

Figure 2 .

Figure 2

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Cell type-specific mRNA expression of collagenase 3 in the synovial membrane in RA. Collagenase 3 mRNA was detected by in situ hybridisation (NBT/BCIP colour reagent; black) and expressed in fibroblast-like cells (arrowheads) of the synovial membrane which were immunohistochemically labelled with mAb D7-Fib (APAAP method; red) known to recognise fibroblasts (A). Lymphocytic infiltrates (L) in the synovial membrane were negative for collagenase 3 mRNA expression (B). Scale bar 80 µm in A; 130 µm in B.

Figure 3 .

Figure 3

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mRNA expression of collagenase 3, MT1-MMP, and gelatinase A in the synovial membrane in RA. Northern blot analysis was performed using total RNA from six separate synovial membrane specimens of patients with RA by successive hybridisation with radioactively labelled cDNA probes corresponding to collagenase 3, MT1-MMP, and gelatinase A, as indicated on the left. The mRNA expression levels of MT1-MMP and gelatinase A were only slightly raised in patients with RA expressing collagenase 3 mRNA in the synovial membrane (lanes 1 and 2) compared with patients without collagenase 3 mRNA expression (lane 3–6). GAPDH was used to check the loading of RNA.

Figure 4 .

Figure 4

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Localisation of collagenase 3, MT1-MMP, and gelatinase A mRNA expression at the synovial membrane-cartilage interface. Collagenase 3 (A), MT1-MMP (B), and gelatinase A mRNA (C) are colocalised in the synovial membrane adjacent to the cartilage (arrowheads), as shown for serial sections of the synovial membrane-cartilage interface of a patient with RA by in situ hybridisation (NBT/BCIP colour reagent; black). This observation was confirmed by analysing synovial membrane-cartilage samples of six patients with RA. Scale bar 100 µm.

Figure 5 .

Figure 5

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Cell type-specific mRNA expression of MT1-MMP and gelatinase A in the synovial membrane in RA. MT1-MMP and gelatinase A mRNA were detected by in situ hybridisation (NBT/BCIP colour reagent; black). Both MMPs are expressed in fibroblast-like cells (arrowheads) of the synovial membrane, which were immunohistochemically labelled with mAb D7-Fib (APAAP method; red) as shown in (A) and (B), respectively. MT1-MMP mRNA expression (black) was detected in endothelial cells (arrows) marked with an mAb against type IV collagen of the basal membrane (red, arrowheads) (C). Scale bar 80 µm in A and B; 200 µm C.

Figure 6 .

Figure 6

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mRNA expression of collagenase 3, MT1-MMP, and gelatinase A in the synovial membrane in RA. RT-PCR was performed on total RNA from primary synovial fibroblast cultures of patients with RA using specific primers for collagenase 3, MT1-MMP, gelatinase A, and GAPDH as indicated on the left. All 10 fibroblast cell cultures expressed basal levels of MT1-MMP and gelatinase A mRNA, whereas basal collagenase 3 mRNA expression was detected only in 4/10 cultures.

Selected References

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