Immunogenicity of singlet oxygen modified human DNA: implications for anti-DNA antibodies in systemic lupus erythematosus.

Reactive oxygen species (ROS)-modified DNA has been shown to be a better and more discriminating immunogen than native DNA (nDNA) for the production of anti-DNA autoantibodies in SLE (systemic lupus erythematosus). Among ROS, the role of hydroxyl radical (.OH) in the induction of damage and modification of nDNA has been extensively studied while such documentation implicating singlet oxygen ((1)O(2)) in inducing immunogenicity in nDNA leading to the production of anti-double-stranded (ds) DNA autoantibodies in SLE is not yet available. This prospective study was undertaken to evaluate the immunogenicity of healthy human dsDNA modified with (1)O(2) generated by methylene blue plus radiant light. Female rabbits were immunized with (1)O(2)-modified human dsDNA to raise anti-dsDNA antibodies. (1)O(2)-modified anti-dsDNA rabbit immune sera and the (1)O(2)-modified anti-dsDNA rabbit purified immunoglobulin G (IgG) were tested against a variety of dsDNA antigenic substrates through direct enzyme-linked immunosorbent assay (ELISA). The immunogenicity of (1)O(2)-modified human dsDNA was further evaluated by studying its immunoreactivity with SLE patients' sera and SLE patients' purified anti-dsDNA IgG. As compared to healthy human sera, (1)O(2)-modified anti-dsDNA rabbit immune sera as well as the (1)O(2)-modified anti-dsDNA rabbit purified IgG demonstrated a strong affinity towards (1)O(2)-modified human dsDNA(.)(1)O(2)-modified human dsDNA proved to be potentially more immunogenic against SLE patients' whole sera and SLE patients' purified IgG as compared to healthy human sera. Our findings suggest that (1)O(2) may also be inducing immunogenicity in native dsDNA resulting in the production of anti-dsDNA autoantibodies as seen in SLE patients.

Immunogenicity of DNA modified by singlet oxygen: implications in systemic lupus erythematosus and cancer.

Activation, proliferation and programmed cell death of immune cells are dependent on controlled production of ROS (reactive oxygen species). However, under chronic inflammatory conditions, large amounts of ROS generated are a major cause of many human degenerative diseases. ROS are known to cause DNA damage, leading to strand breaks, base damage and conformational changes. (1)O(2) (singlet oxygen), being one of the most potent ROS, is known to selectively react with the deoxyguanosine moiety in DNA. The effect of (1)O(2), generated by UV irradiation of Methylene Blue, on plasmid DNA was studied by UV spectroscopy and electrophoresis. The antibodies raised against the modified DNA in experimental animals induced a high titre. IgG was purified on a Protein A-Sepharose matrix, and oxidative lesions in DNA of SLE (systemic lupus erythematosus) and cancer patients were probed using anti-((1)O(2)-plasmid DNA) [anti-((1)O(2)-modified plasmid DNA)]. The DNA isolated from the sera of SLE and cancer patients was found to inhibit anti-(1)O(2)-plasmid DNA IgG activity, reiterating the results obtained with serum samples. These binding results indicate the presence of oxidative lesions in the SLE and cancer patients' genome caused by excessive production of ROS. The results confirm the damaging effect ROS has on DNA. The excessive production of reactive oxygen intermediates may be one of the factors responsible for the induction of autoimmune response seen in SLE and cancer.