Date of Award

5-2008

Level of Access Assigned by Author

Open-Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

Stephen C Pelsue

Second Committee Member

Kenneth Ault

Third Committee Member

Keith Hutchison

Abstract

Ttc7fsn/fsn mice exhibit systemic autoimmunity characterized by hyperactivated B cells, increased interleukin-4, autoantibodies, kidney disease and reduced lifespan. Because the pathology is similar to systemic lupus erythematosus, Ttc7fsn/fsn mice are a useful model with which to study early events that lead to autoimmune disease. Although the Ttc7fsn mutation has been identified the gene function is unknown. The phenotype of Ttc7fsn/fsn mice mimics the Th2 autoimmunity of the IL-4 transgenic (Erb et al 1997). It was previously unknown whether the over-production of IL-4 was an intrinsic defect of Ttc7fsn/fsn lymphocytes that led to autoimmunity, or, whether excess IL- 4 was produced as a result of hyper-activated lymphocytes, which then drove pathology. Because the transcription factor, signal transducer and activator of transcription (Stat)-6 is unique to the IL-4 pathway and influences both IL-4 production and lymphocyte activation, we evaluated the regulation of Stat6 in B cells as an indication of IL-4 signaling. We examined the activation status and IL-4 production in 4-and 10-week T cells to determine if activation preceded the IL-4-driven pathology. Immunoprecipitation and Western blot revealed that Stat6 appeared constitutively activated in Ttc7fsn/fsn B cells, however, ex vivo functional analysis of Stat6 with confocal microscopy determined that when the IL-4 signal was removed, Stat6 became deactivated. The use of actinomycin D in mRNA expression studies revealed that IL-4 mRNA stability was increased in Ttc7fsn/fsn T cells which resulted in IL-4 production in unstimulated cells and extended IL-4 production after transcription was blocked as determined by ELISA. Using cyclosporine A in the same assays suggested that IL-4 production was dependent on TCR stimulation. Flow cytometry established that Ttc7fsn/fsn mice have more activated and memory T cells at 4-weeks, suggesting that activation occurs before the over-production of IL-4. Genetically deleting Stat6 from Ttc7fsn/fsn mice unexpectedly did not improve the disease phenotype but increased the percentage of immature B and activated T cells. The neutralization of IL-4 improved renal pathology but did not alter the status of Ttc7fsn/fsn lymphocytes. Therefore, we suggest that the Ttc7fsn mutation results in autoreactive lymphocytes that are induced to produce IL-4, leading to systemic autoimmunity.

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