Cytokines are important mediators of immune functions in humans and animals, interleukin (IL)-15 is a proinflammatory cytokine, which is mainly produced by monocytes. It shares many of its functions with IL-2, which is partly due to the shared use of receptor subunits on target cells, and serves as a growth and survival factor for T lymphocytes. The type IIL-15 receptor is composed of the IL-2R β and γ subunits, which form a trimeric complex with the high affinity IL-15Rα chain. The expression of IL-15 is tightly regulated both at the transcriptional and translational level. The production of IL-15 is associated with immune responses against bacterial and parasitic pathogens but has also been associated with the pathology of human autoimmune diseases, in particular Rheumatoid Arthritis (RA). RA is characterized by chronic inflammation within the synovial membrane accompanied by infiltration of lymphocytes leading to progressive, erosive destruction of cartilage and underlying bone. The severity of RA is associated with the overexpression of proinflammatory cytokines within the synovial tissue, in particular tumor necrosis factor alpha (TNF α) which is thought to play a central role in maintaining the inflammatory processes within the arthritic joint. So far, little is known about the processes that initiate and perpetuate RA. IL-15 was found in the synovial tissue of RA patients where it stimulated the production of TNFα, placing IL-15 in a central position orchestrating the cytokine cascade that causes inflammation and pathology in RA. Antagonists to IL-15 may therefore have an important therapeutic potential for the treatment of RA in humans. A major aim of this project has been to clone and express a recombinant IL-15 antagonist to use as a therapeutic agent in a murine model of RA closely related to the human disease, collagen-induced arthritis (CIA). A soluble IL-15Rα was cloned from a murine macrophage cell line and expressed in a bacterial expression system. The resulting protein has a molecular weight of 26kD and bound to IL-15 specifically. It also had a neutralizing effect on IL-15-induced proliferation of T cell lines. Administration of soluble IL-15Rα to mice prevented the onset of CIA and had a suppressive effect on disease severity and incidence. Mice treated with the recombinant IL-15Rα also showed reduced serum cytokine production and altered humoral responses against collagen. These results consolidate the therapeutic potential of IL-15 antagonists for the treatment of inflammatory diseases. To further enhance the therapeutic properties of recombinant IL-15Rα, a second expression construct has been cloned fusing the extracellular region of native IL-15Rα to the constant region of the murine immunoglobulin heavy chain. This construct was expressed in a mammalian expression system and results in a product of 66kD, which also bound to IL-15. The generation of knockout mice by gene targeting is a powerful tool to study the function of gene products in vivo. The Cre/lox system provides a novel strategy to generate inducible and tissue specific genomic alterations that allow the detailed analysis of gene function. The second part of this project was concerned with the generation of a mouse model lacking IL-15Rα in a tissue specific way by conditional mutagenesis in embryonic stem (ES) cells. Using cDNA encoding the extracellular domain of IL-15Rα as a radiolabeled probe, a murine genomic library was screened. Two clones containing part of the gene encoding IL-15Rα were characterized. A DNA construct was cloned to target the IL-15Rα gene in murine ES cells. Homologous recombination of the construct with the target locus resulted in the flanking of the critical regions of the IL-15Rα-gene by loxP sites. Cre-mediated recombination in vitro caused the deletion of loxP site flanked sequences within the genome of the targeted clone. Using this technique, two ES cell clones have been generated that allow the generation of mice that either lack IL-15Rα in all tissues or are suitable for conditional mutagenesis mediated by Cre recombinase. The resulting model may provide a useful tool to study the effects of IL-15 in inflammatory processes in vivo.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:312128 |
| Date | January 2000 |
| Creators | Ruchatz, Holger |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/30895/ |
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