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

Reactive oxygen species have been implicated in the induction of autoimmunity via the initiation of protein oxidation reactions leading to the recognition of antigen as non ‘self. In systemic lupus erythematosus, the source of reactive oxygen species capable of inducing damage to antigens may be cellular but could also involve those generated by ultraviolet in sunlight. A key feature of systemic lupus erythematosus is the development of photosensitive skin lesions and these have been positively associated with the presence of R0 6 O autoantibodies. The autoantibody response to R0 6 O in systemic lupus erythematosus sera has been characterised and is a high affinity antibody of predominantly IgGl subclass. This implicates a T cell dependent process involving antigen presentation to T helper cells via the MHC class II antigen-presenting pathway. To date, there has been no work reported on T cell responses to R0 6 O or systemic lupus erythematosus autoantigens modified by reactive oxygen species in systemic lupus erythematosus subjects. 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 R0 6 O after it has been modified by reactive oxygen species. In addition to cell activation, cell death also plays an important role in antigen activated immune responses and dysfunctions in cell death regulation have been implicated in systemic lupus erythematosus. Therefore in addition to antigen induced cell activation, cell death was also investigated. Initial work focused on purification of soluble recombinant R0 6 O after overexpression in E coli. R0 6 O 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 R0 6 O. Free radical scavengers and metal ion chelators suggested the mechanism of oxidation to be a ‘caged reaction’ as opposed to a reaction involving free hydroxyl radical formation. 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 R0 6 O 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 ageand 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 R0 6 O. The data represent the highly individual response of subject’s immune cells to antigen stimulation. The reactive oxygen species modified R0 6 O 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.