picogreen and Lupus-Erythematosus--Systemic

Nucleosomes occur in the blood of patients with systemic lupus erythematosus and are thought to result from in vivo cell death. To determine the conditions for the release of nucleosomes into the blood, normal mice were treated with four agents that have the potential to induce apoptosis or immune cell activation in vivo: LPS, CpG DNA, anti-Fas antibody, and dexamethasone. Blood nucleosomes were measured by a capture ELISA immunoassay, with the DNA component assessed by fluorimetry with the dye PicoGreen. Following treatment with LPS and a monoclonal anti-Fas antibody, nucleosomes and DNA appeared in the plasma in a dose-dependent fashion. In contrast, dexamethasone treatment, despite causing significant thymocyte loss, did not elicit plasma nucleosomes. Similarly, CpG DNA, while inducing an IL-12 response comparable to that of LPS, also did not elicit plasma nucleosomes. These results suggest that plasma nucleosome levels reflect specific patterns of cell death and are not an invariable consequence of in vivo apoptosis or immune cell activation.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; Apoptosis; CpG Islands; Dexamethasone; Enzyme-Linked Immunosorbent Assay; fas Receptor; Female; Fluorescent Dyes; Immunosuppressive Agents; Lipopolysaccharides; Lupus Erythematosus, Systemic; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred MRL lpr; Nucleosomes; Organic Chemicals; Spleen; Thymus Gland

Antibodies to DNA (anti-DNA) play an important role in the pathogenesis of systemic lupus erythematosus (SLE). In blood, these antibodies may exist in a free, unbound state or as part of complexes with DNA. Furthermore, circulating DNA may be either complexed or free. Because of the central role of these immunoreactants (anti-DNA and DNA) in the disease, monitoring of their levels could provide valuable information for both clinical and investigative purposes. In these studies, we have explored the use of a DNA-binding dye, PicoGreen, for the detection of circulating DNA, either total or immune complex bound. In addition, we have used this dye for Farr-type antibody assays. Using autoimmune MRL/lpr mice as a model, we have shown that, while the levels of free DNA in the plasma of these mice were comparable with those of normal BALB/c mice, the amounts in complexes precipitable by ammonium sulfate were significantly greater. Furthermore, we showed that Farr assays using PicoGreen reliably detect levels of free anti-DNA, with values correlated with anti-DNA levels by an enzyme-linked immunosorbent assay. Together, our results suggest that a fluorometric dye can accurately monitor DNA and anti-DNA antibody levels in SLE and may provide important information on immunopathogenesis.

Topics: Ammonium Sulfate; Animals; Antibodies, Antinuclear; Blood Chemical Analysis; Chemical Precipitation; DNA; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique; Fluorescent Dyes; Lupus Erythematosus, Systemic; Mice; Mice, Inbred BALB C; Mice, Inbred MRL lpr; Organic Chemicals