OP0075 Chromatin clearance from necrotic cells by a concerted action of dnase 1 and serine proteases resembles apoptotic dna-degradation

Background Systemic lupus erythematosus (SLE) is an autoimmune disease characterised by the presence of antinuclear antibodies (ANA) which tend to accumulate as immunodeposits in different tissues and cause inflammations. One main feature of SLE-pathogenesis seems to be a reduced elimination of cellular debris after cell death.1,2 The recently generated mice deficient for the endonuclease Dnase 1 has underscored the important influence of defects of chromatin clearence for the onset of SLE.3 Objectives Methods Cell nuclei of MCF-7 cells were isolated and either incubated with serum from Dnase 1 wildtype and Dnase 1 deficient mice or with recombinant human DNASE 1 alone and in combination with different serine proteases. Subsequently the DNA was isolated and analysed by agarose gel-electrophoresis. DNA-degradation of MCF-7 cells incubated with different amounts of native or heat inactivated bovine serum in DMEM was analysed under apoptotic (2 μM staurosporin) and necrotic conditions (1 mM H2O2) or after complement lysis due to the application of 6.7% (v/v) rabbit complement. Subsequently the DNA was isolated and analysed. Results Recombinant human Dnase 1 could induce an apoptotic-like DNA-fragmentation pattern in isolated MCF-7 cell nuclei by a concerted action with different serine proteases like plasmin, trypsin, a-chymotrypsin, thrombin and kallikrein. Dnase 1 alone only induces an unspecific, time-retarded DNA-degradation. Serum from Dnase 1 wildtype mice also induced DNA-laddering in cell nuclei whereas serum from knockout mice did not due to the lack of Dnase 1. The laddering capacity of wildtype serum could be inhibited by serine protease inhibitors. In contrast to apoptotic cells native bovine serum and not heat-inactivated serum induces internucleosomal DNA-laddering in MCF-7 cells lysed by rabbit complement or by the cytotoxic action of H2O2. Conclusion This study gives insight into a new function of Dnase 1 which could be linked with its protective role towards the induction of an anti-DNA immunoreaction and subsequently towards SLE-pathogenesis.2,3 Dnase 1 in connexion with serine proteases is able to effectively degrade the genomic DNA of nuclei and of cells lysed by complement or the cytotoxic action of H2O2. In contrast apoptotic cells were not influenced by serum Dnase 1 and serine proteases. Since apoptotic cells underly a cell-autonomous intracellular DNA-degradation process one can conclude that Dnase 1 in connexion with serine proteases fullfills this process for necrotic cells. References Vanholder R, De Keyser F, Kips J, Praet M, Naeyaert JM. The pathophysiology of lupus erythematosus. Eur J Dermatol. 1998;1:4–7 Walport MJ. Lupus, DNase and defective disposal of cellular debris. Nat Genet. 2000;25:135–6 Napirei M, Karsunky H, Zevnik B, Stephan H, Mannherz HG, Moroy T. Features of systemic lupus erythematosus in Dnase 1-deficient mice. Nat Genet. 2000;25:177–81