Search

Summary This thesis can be roughly divided into two parts. In the first three chapters, we discussed and studied the role of Hsp70 in retinal pigment epithelial (RPE) cells, and the innate and adaptive immune response. We reviewed the relationship of T cell-mediated diseases and HSPs, and pointed out that the induction of Hsp70 may contribute to therapeutic tolerance (chapter 2). Then, we explored a new Hsp70 co-inducer, leucinostatin, and its role in canine RPE cells (chapter 3). Further, we studied the anti-inflammatory effects of Hsp70 in canine macrophages (chapter 4). In the last two chapters, we focus on various differently activated canine macrophage subsets originating from both primary monocytes and a monocyte-like cell line (030D cell) (chapter 5 and 6). We successfully polarized canine monocyte-derived macrophages (MDMs) into M1 and M2 cells and thoroughly characterized their features (chapter 5). Meanwhile, we demonstrated that 030D cells can be differentiated into M1 and M2 macrophages and that each subset shares the characteristics of the corresponding canine monocyte-derived macrophage subset.

Chronic autoimmune diseases are driven by cells that respond to tissue components of the body. Inflammation in diseases like rheumatoid arthritis, diabetes or multiple sclerosis, can be suppressed by drug therapy. However, the broad range of immunosuppressive action of these drugs often does not restrict to the autoimmune response, but increases the risk of serious infection. Therefore, therapies that restrict to suppression of only the auto-immune response need to be developed. CD4+ T cells that recognize cartilage are responsible for induction of arthritis because they direct their actions to cartilage in the joints. Such T cells are present in the joints of arthritis patients. Therefore, we hypothesized that T cells specific for the cartilage-derived antigen proteoglycan can be used to target arthritis with suppressive agents after introduction of genes that express suppressive agents in these T cells. For our studies we used cartilage proteoglycan-induced arthritis, which is a chronic arthritis in mice. This arthritis represents many features of rheumatoid arthritis, and is mediated by Th1 cells, B cells and antibodies. To enable studies on cartilage-specific T cells, we generated a transgenic mouse expressing a proteoglycan-specific T cell receptor on its T cells. Due to the high number of cartilage-specific T cells, transgenic mice were highly susceptible for arthritis. Moreover, the T cell response of transgenic mice with arthritis shifted to an excessive Th1-phenotype. Therefore, these transgenic mice were useful donors for arthritogenic CD4+ T cells. To explore gene therapy of inflammation with T cells, we provided proteoglycan-specific T cells with different genes encoding immunosuppressive proteins. The genes were isolated from mouse cells and inserted into T cells by retroviruses. Retroviral transduction of T cells resulted in up to 80% of cells expressing the transgenes (IL-4, IL-10, TNF-alpha-Receptor-Ig, IL-1 receptor antagonist). T cells expressing the inserted genes were sorted by flow cytometry and transferred to mice with arthritis. Especially T cells that were manipulated to express IL-10 were able to induce significant suppression of joint inflammation. Moreover, only the IL-10-producing T cells that expressed the cartilage-specific T cell receptor suppressed arthritis, indicating specificity of therapy. In addition, manipulated T cells suppressed production of pro-inflammatory proteins like TNF-alpha, IL-17, IL-2 and PG-specific IgG2a antibodies. Strikingly, we discovered that propagation of IL-10 production in cells of the treated recipient was the mechanism that was used by the transferred IL-10-producing T cells to suppress arthritis. Among the recipient’s cells, T cells and B cells were found to express increased levels of IL-10. Because interleukin-10 is an immunomodulatory cytokine expressed by cells in order to naturally prevent exacerbation of inflammation in healthy individuals, these results indicate that T cells producing IL-10 restore natural immunosuppressive immune responses in arthritis. In conclusion, manipulation of pro-inflammatory T cells that recognize tissue components is a powerful approach to specifically target inflammation in chronic autoimmune diseases. In addition, moving focus from suppression of pro-inflammatory mediators to propagation of immunosuppressive functions of cells may provide a more comprehensive insight in mechanisms that support specific regulatory capacities of the immune system.