Time between onset of apoptosis and release of nucleosomes from apoptotic cells: putative implications for systemic lupus erythematosus

Abstract

Objective: To investigate the kinetics of nucleosome leakage from apoptotic cells in an in vitro system and extrapolate the results to autoimmune disease, in particular systemic lupus erythematosus.

Methods: A sensitive nucleosome enzyme linked immunosorbent assay (ELISA) was developed, using a monoclonal antibody (mAb) against histone 3 and an mAb against nucleosomes. Nucleosome release during apoptotic cell death was studied in Jurkat cells. AnnexinV binding (early apoptosis) and propidium iodide positivity (late apoptosis) of the cells were compared with nucleosome release at different times after apoptosis induction.

Results: Nucleosomes appeared in culture supernatant of Jurkat cells 24 to 48 hours after apoptosis induction, when the cells had been late apoptotic for more than 12 hours.

Conclusion: Nucleosomes are released from late apoptotic Jurkat cells, with a 12 hour delay from the appearance of AnnexinV binding cells. This result suggests that in vivo scavenger mechanisms have 12 hours to remove apoptotic material from the circulation.

Figure 1

Antihistone activity of mAbs CLB-ANA/58 and CLB-ANA/60. The mAbs were tested by ELISA against a mixture of histones H1, H2A, H2B, H3, and H4. Units were calculated by comparison with a standard antihistone mAb, which was defined as 1000 antihistone U/ml.

Figure 2

Epitope determination of mAb CLB-ANA/60 on histone immunoblot. (A) 20% SDS-PAA gel with histones, silver staining. (B) Histone immunoblot incubated with 2.5 µg/ml mAb CLB-ANA/60. Lane M, 100 kDa protein marker; lane 1, histone 1; lane 2, histone 2A; lane 3, histone 2B; lane 4, histone 3; lane 5, histone 4.

Figure 3

Epitope determination of mAb CLB-ANA/58. Test of mAb on several combinations of histones or DNA, or both.

Figure 4

Validation of the nucleosome ELISA using Jurkat cell supernatant incubated 24 hours with 200 µM etoposide. 100% of the cells were late apoptotic.

Figure 5

Determination of the time between apoptosis induction and nucleosome release. Apoptosis was induced by addition of 5 µg/ml anti-Fas. Apoptosis was measured by AnnexinV (early apoptosis) and PI (late apoptosis) FACS staining, nucleosomes were measured by ELISA.