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Modelling arthritis in transgenic mice

Objective

1. to characterize, develop and optimize an existing tumour necrosis factor (TNF) transgenic mouse model of arthritis in mimicking the human disease.
2. to exploit its molecular and cellular biology in order to gain further insight into the pathogenic mechanisms leading to the development of this disease in humans.
3. to use this model in the assessment of the in vivo neutralizing activity of anti-inflammatory substances or anti-inflammatory therapeutic approaches.
4. to develop, using several different TNF transgenes, additional new animal models relevant to the understanding and protection of human health.
1. Development of arthritis is independent of the presence of mature T and B cells.
- The original human TNF transgenic mice (Tg197) which develop arthritis with a 100% phenotypic penetrance at 4-5 weeks of age were bred into the DBA/1 (susceptible to collagen-induced arthritis) and RAG1{-}/- (immunodeficient) genetic backgrounds. Tg197 x DBA/1 mice develop arthritis with clinical symptoms occuring earlier (10 days to 3 weeks) and to a greater extent e.g degree of tissue damage and number of joints affected, in comparison to the Tg197 mice. The vast majority of cells infiltrating the inflammed synovium of both strains were determined as CD45(+)neutrophils, macrophages and few lymphocytes. Production of human TNF was found restricted to the synovial tissue.
- Tg197 x RAG1{-}/- mice also developed arthritis suggesting that the mechanism of induction is B and T cell independent. However, pathology evolves with a general delay of a few weeks demonstrating that mature T and B cells contribute positively in the destructive process.
2. (a) Transmembrane TNF can accumulate on the membrane of macrophages and synoviocytes and can act as a trigger for the induction of arthritis. Mutant uncleavable forms of both the human and murine 26 kD membrane-associated TNF have been engineered by the deletion of the triplets coding for the first to the twelfth aminoacid of the mature 17kappaD TNF form. Transgenic mouse lines has been obtained which express high levels of mutant Delta1-12 TNF in their joints. Typical chronic inflammatory polyarthritis similar to the arthritis triggered by wild type TNF transgenes was induced. In the murine transmembrane TNF induced arthritis high levels of transmembrane TNF were found to accumulate on macrophages and synoviocytes and arthritis could also be triggered in the absence of endogenous TNF expression as assessed in backcrosses to TNFá knockout mice. These results have important implications for a specific involvement of membrane associated TNF in the development of arthritis and question the validity of therapeutic approaches aiming at the inhibition of TNF cleavage in arthritis as for example by the use of metalloproteinase inhibitors.
(b) TNF exerts pro-inflammatory but also immunoregulatory activities. TNF knockout mice have been established by the targeted disruption of their endogenous TNFalpha gene using homologous recombination in embryonic stem cells. In the absence of TNFalpha, mice are rendered resistant to LPS-mediated toxicity but readily succumb to L. monocytogenes infection and show impaired hypersensitivity responses to contact allergens. Interestingly, TNFalpha knockout mice are found defective in the formation of primary lymphoid follicles and germinal centres (GC) upon immunisation with T cell dependent antigens. In addition, a complete absence of Follicular Dendritic Cell (FDC) networks was observed in these mice. These results establish a critical requirement for TNF in the formation of functional lymphoid follicles and in the generation of the humoural immune response. These mice will serve also as invaluable tools in the understanding of the role of TNF in many important disease processes including the pathological complications of infectious, chronic inflammatory and autoimmune diseases. To this end we are currently evaluating their susceptibility to various forms of experimentally induced arthritis and neuroinflammatory diseases.
3. Studies on the in vivo modulation of TNF expression by anti-TNF reagents and by TNF specific ribozymes. (a) The neutralizing capacity of several antibodies against TNF was assessed in both the wild type and transmembrane TNF transgenic arthritis models. In most of the cases, the results were successful establishing the in vivo ability of these reagents to neutralise TNF mediated arthritis. (b) To assess whether ribozymes can be active, in trans, as transgenes, mice expressing a TNF-specific ribozyme gene were established and backcrossed to the Tg197 arthritic mice. Unfortunately, development of arthritis could not be prevented. To optimize ribozyme structure we are currently constructing new ribozyme genes that could generate, in vivo, small ribozyme molecules with more stable hairpins at their 3 and 5 ends. New transgenic mouse lines will be generated and backcrossed to the arthritic TNF transgenic mice to assess the in vivo validity of this therapeutic approach.
4. New animal models relevant to the understanding of human health. A murine TNF transgene which was expressed ectopically, in the CNS of mice induced CNS inflammation, demyelination and degeneration, a pathology very similar to Multiple Sclerosis and other neuro-inflammatory diseases in humans. These mice which represent today important animal models of human neuroinflammatory disease should be useful in the further analysis and understanding of the pathogenesis and treatment of these disorders.
In general, the transgenic systems which were developed, optimized and characterized under this contract have already aided in the making of multiple breakthroughs in our understanding of the role played by TNF in the pathogenesis of arthritis. As a result, these systems have served and continue to serve as invaluable tools in basic research and industrial R&D on anti-TNF or other anti-inflammatory reagents.

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Coordinator

HELLENIC PASTEUR INSTITUTE
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127,Vassilissis Sofias Ave. 127
11521 ATHENS
Greece

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