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Functional aspects of antibodies to collagen on cartilage synthesis and degradation

Drs. Marie-Paule Van Damme and Merrill Rowley

Rheumatoid arthritis (RA) is a chronic inflammatory synovitis that results in progressive destruction of cartilage and subchondral bone. Antibodies to collagen type II (the major cartilage protein) occur in ~70% of patients with early early RA, suggesting that autoimmunity to cartilage collagen may play a part in the development of this destructive arthritis. It has been shown that antibodies to collagen II are critical for the development of the disease Collagen Induced Arthritis, an animal model widely used for human arthritis which is induced by immunisation of mice with collagen II. However, not all antibodies to collagen type II are disease-associated and it appears that there are particular regions on the collagen molecule where antibody-binding causes damage.

This project is based on the hypothesis that antibodies to collagen type II, which transfer arthritis in mice, are those that react specifically with regions of the collagen fibrils that are crucial for cartilage stability and function. This hypothesis will be tested in an "in vitro" culture system of cartilage cells (chondrocytes).

The aim of the project is to test if antibodies to collagen II from mice with Collagen Induced Arthritis interfere with the normal assembly and structure of cartilage.

Methods: Chondrocytes will be grown in culture in the presence of various antibodies under defined conditions and the matrix synthesised will be analysed after radioactive labelling of the cultures with 35S- sulphate and 3H-proline (for incorporation into proteoglycans and collagen, respectively). Macromolecules will be extracted and purified using standard purification techniques. Growth morphology of the cells will be assessed using phase contrast microscopy and the structural organization of collagen fibrils will be investigated by electron microscopoy. Cell viability will be determined by confocal microscopy using various fluoroprobes. Detailed examination of the structure of the matrix synthesised in culture will be performed by immunofluorescence, using antibodies specific for type II collagen, to determine the type of collagen synthesised.

Overall goal of the study: The study will be extended to rheumatoid arthritis, with a comparison of the regions of collagen II that react with antibodies of mouse and human origin. Showing that antibodies to collagen II are directly destructive, allowing for an understanding of their site and mode of action, would greatly advance our understanding of the cause of rheumatoid arthritis and would lead to more effective forms of treatment.