UV-induced reactive oxygen species production and altered cellular response to the Ro60 autoantigen

The primary aim of this project was to investigate if T cells isolated from systemic lupus erythematosus subjects were able to respond to purified human recombinant Ro60 after it has been modified by reactive oxygen species. Initial work focused on purification of soluble recombinant Ro60 after overexpression in E. coli. Ro60 was then exposed to reactive oxygen species via ultraviolet B irradiation and metal catalyzed oxidation and immunochemical techniques were applied to detect aggregation, fragmentation and carbonyl formation as markers of protein oxidation. None of these markers were induced by ultraviolet B irradiation but metal catalyzed oxidation induced carbonyl formation on a high molecular weight aggregate of Ro60. Free radical scavengers and metal ion chelators suggested the mechanism of oxidation to be a 'caged reaction'.;A method was established to measure T cell activation analysing viable interferon gamma secreting and interleukin-4 secreting leukocytes by flow cytometry using a non-specific T cell activator as a positive control. This was used to investigate T cell activation of peripheral blood mononuclear cells obtained from systemic lupus erythematosus subjects after stimulation in vitro with Ro60 that had been exposed to reactive oxygen species. In addition cells undergoing apoptotic and necrotic cell death were also detected by flow cytometry after staining with annexin V and propidium iodide. The data were compared to that from age- and sex-matched healthy controls. There was no significant difference increase or decrease in T cell activation or the level of cell death between the two groups after stimulation with reactive oxygen species modified Ro60. The data represent the highly individual response of subject's immune cells to antigen stimulation. The reactive oxygen species modified Ro60 used in these investigations represented only a model system to test the hypothesis of reactive oxygen species modification to autoantigen as an initiating factor in autoimmunity.;The methodology established may be applied to other autoantigens in systemic lupus erythematosus or other autoimmune conditions to test a whole spectrum of reactive oxygen species modifications to proteins in addition to other posttranslational modifications which may be involved in the initiation of autoantigen as non 'self'.