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OP0073 Histone release into the cytoplasm of human pbmcs and preapoptotic lymphoblasts
  1. C Gabler1,
  2. T Hieronymus1,
  3. N Blank1,
  4. M Schiller1,
  5. JH Berden2,
  6. S Winkler1,
  7. JR Kalden1,
  8. HM Lorenz1
  1. 1Department of Medicine III, Institute for Clinical Immunology, Erlangen, Germany
  2. 2University Hospital St. Radboud, Division of Nephrology, Nijmegen, The Netherlands

Abstract

Background Systemic lupus erythematosus (SLE) is an autoimmune disease characterised by a wide range of antibodies reacting with nuclear antigens. Abnormalities in the apoptotic cell death process and apoptotic cell clearance may play an important role in generating these antibodies. Especially antibodies to histones in serum are frequently associated with SLE.

Objectives The aims of this study were to evaluate the presence of histones in the cytoplasm of fresh peripheral blood mononuclear cells (PBMCs) and in activated lymphoblasts. The activated lymphoblasts cultured without IL-2 served as a model for apoptosis induction.

Methods Human PBMCs were isolated from heparinized peripheral blood of normal donors (n = 6) and activated with 1 μg/ml phytohemagglutinin (PHA) and 10 U/ml IL-2 for the first 5 days. The following two days, the cells were expanded in IL-2 containing medium (0 h). Lymphoblasts were then incubated in IL-2 free medium for 8 or 24 h as well as for 24 h with IL-2 (10 U/ml). Furthermore, lymphoblasts were treated with IL-2 after 8 or 24 h IL-2 deprivation. PBMCs and lymphoblasts were lysed in a modified RIPA-buffer, 20 μg of protein were subjected to denaturing SDS-PAGE. Western blot detection was performed with antibodies against all 5 histones H1, H2A, H2B, H3 and H4 as well as against phosporylated H3 and acetylated H4.

Results Only very weak signals of the histones H2A, H2B, H3 and H4 were detected in the cytoplasm of freshly isolated PBMCs, but H1 was undetectable. The activated lymphoblasts showed elevated amounts of all five histones compared to PBMCs. Furthermore, we observed an increased amount of the histones H2A, H2B, H3 and H4 in the cytoplasm in cell culture after another 8 or 24 h without IL-2. Interestingly, the signal intensity of these histones in the cytoplasm decreased significantly after addition of IL-2 to the lymphoblasts after 8 or 24 h IL-2 deprivation.

However, no difference of the detectable amount of H1 was noted in activated lymphoblasts compared to preapoptotic lymphoblasts incubated for another 8 or 24 h without IL-2. In addition, for H1 no effect of addition of IL-2 was observed.

Lymphoblasts activated with IL-2 for 24 h showed the same signal intensities for the histones H2A, H2B, H3 and H4 as observed for the cells at 0 h.

No bands for acetylated H4 or phosporylated H3 were detected in cytoplasm samples in contrast to strong signals in nuclear preparations.

Conclusion These results suggest that histones were released from the nucleus into the cytoplasm of preapoptotic human lymphoblasts. The mechanism of the transport of the histones into the cytoplasm is until now unknown. Acetylated H4 and phosporylated H3 were only detected in nuclear extracts which excludes nuclear membrane fragility and nuclear contamination as cause for our findings. In addition, our data indicate that IL-2 can reduce the release of histones from the nucleus into the cytoplasm.

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