Mikko Liljeström

OBJECTIVE: To measure cartilage pH in patients with osteoarthritis (OA) and to analyze the presence of cathepsin K, the recently discovered acidic endoproteinase, in phenotypically altered chondrocytes. METHODS: Intraoperative measurements of the pH of clinically normal, fibrillated, superficially fissured, and deeply fissured cartilage surfaces (grades 0-3, respectively) in OA patients undergoing primary hip replacement surgery were performed with the use of a sting electrode sterilized with microbicidic plasma. Fluorescent pH probes were used for in situ assessment of cartilage matrix pH. Cathepsin K was assessed using quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry methods. RESULTS: The pH of grade 0 cartilage surfaces was 7.1 +/- 0.4 (mean +/- SD), compared with 6.2 +/- 0.9 (P < 0.05), 5.7 +/- 1.0 (P < 0.001), and 5.5 +/- 1.0 (P < 0.001) for grades 1-3 cartilage surfaces, respectively. Fluorescent pH probes and acid-dependent autocatalytic conversion of cathepsin K into its active, low molecular weight form in cartilage confirmed these findings. Cathepsin K messenger RNA levels increased in relation to the severity of OA, and the number of cathepsin K-containing chondrocytes increased from a mean +/- SD of 12 +/- 3 in grade 0 cartilage surfaces to 47 +/- 7, 50 +/- 6, and 100 +/- 12 in grades 1-3 cartilage surfaces, respectively (P < 0.001 for all comparisons). CONCLUSION: Acid-activated, but pharmacologically inhibitable, cathepsin K is induced in phenotypically altered chondrocytes in OA. The findings suggest that cathepsin K, rather than neutral matrix metalloproteinases, degrades the superficial gliding surfaces of the articular hyaline cartilage in OA.

Normal bone remodeling and pathological bone destruction have been considered to be osteoclast-driven. Osteoclasts are able to attach to bare bone surface and produce an acidic subcellular space. This leads to acid dissolution of hydroxyapatite, allowing cathepsin K to degrade the organic type I collagen-rich osteoid matrix under the acidic condition prevailing in Howship lacunae. Using a sting pH electrode, the interface membrane around a loosened total hip replacement prosthesis was found to be acidic. Confocal laser scanning disclosed irregular demineralization of the bone surface in contact with the acidic interface. Cathepsin K, an acidic collagenolytic enzyme, was found in interface tissue macrophages/giant cells and pseudosynovial fluid. Tissue extracts contained high levels of cathepsin K messenger RNA (mRNA) and protein. These observations suggest the presence of an acid- and cathepsin K-driven pathological mechanism of bone resorption, mediated not by osteoclasts in subosteoclastic space, but rather by the uncontrolled activity of macrophages in extracellular space.

OBJECTIVE: To investigate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) in the synovial membrane of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Mouse monoclonal antibody against human EMMPRIN was applied according to an avidin-biotin-peroxidase complex method to reveal EMMPRIN expression. Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed to check for the presence of EMMPRIN protein and messenger RNA (mRNA). RESULTS: EMMPRIN immunoreactivity was more intense in RA than in OA synovial membrane (P < 0.01). EMMPRIN staining was more widespread in RA than in OA, especially in association with macrophage infiltrates. RT-PCR of synovial membrane samples disclosed the presence of EMMPRIN mRNA. Nucleotide sequencing of the PCR amplification products confirmed the identity of the amplified bands. Immunoblot analysis revealed 55-kd glycosylated EMMPRIN bands, which were particularly prominent in RA samples. CONCLUSION: The expression of EMMPRIN is upregulated in the rheumatoid synovial membrane. EMMPRIN can induce local production of at least MMPs 1, 2, and 3, and can thereby play a role in joint destruction in RA.

OBJECTIVE: Synovial inflammation in rheumatoid arthritis (RA) leads to pannus tissue invasion and destruction of cartilage/bone matrix by proteinases. Our intention was to analyze some of the key matrix metalloproteinases (MMPs) in pannus tissue overlying evolving cartilage erosions in RA. METHODS: Frozen tissue samples of pannus and synovium from advanced RA and synovium from osteoarthritic patients were used for immunohistochemical, western blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of MMP-1, -3, -13 and -14. Synovial fibroblast cultures, stimulated with tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta), were analyzed with enzyme-linked immunosorbent assays (ELISA) and quantitative RT-PCR. RESULTS: MMP-3 was highly expressed in pannus tissue compared with significantly lower expression levels of MMP-1, -13 and -14. In fibroblast cultures IL-1beta was a potent stimulus for MMP-3, whereas TNF-alpha was more potent for MMP-1. CONCLUSION: This is the first study to demonstrate quantitatively in real time that MMP-3 mRNA expression is clearly higher in advanced RA pannus tissue compared to parallel RA or osteoarthritic synovium. MMP-3 mRNA levels were also clearly overexpressed in RA pannus compared to MMP-1, -13 and -14. Advanced RA has previously been found to overexpress IL-1beta. The high expression of MMP-3 in pannus and IL-1beta, mediated stimulation of MMP-3 suggest that MMP-3 plays a significant role in the progression of erosions through the proteoglycan-rich cartilage matrix.