Antibodies from systemic lupus erythematosus (SLE) sera define differential release of autoantigens from cell lines undergoing apoptosis

Abstract

SLE is an autoimmune disease characterized by a wide range of anti-cellular and anti-nuclear autoantibodies. Many of these antigens are exposed or altered during apoptosis when the nucleus is dismantled in a controlled manner by caspases. We used Western blotting techniques to demonstrate that autoantibodies in SLE sera recognize antigens released during apoptosis. Reproducible bands, not seen in the untreated cells, with the characteristics of histones were seen when staining apoptotic cell lysates with SLE sera. Normal sera recognized some of these bands but much less strongly. Different triggers of apoptosis did not produce marked differences in the antigens recognized. We also compared different cell lines (Jurkat and U937) and found that the staining differed for one autoantigen in particular. The differential release of autoantigens by apoptotic cells may have relevance to the variety of autoantibodies seen in SLE.

Fig. 1

Morphological changes that occur during the induction of apoptosis in Jurkat cells after treatment with cyclohexamide. Left panel, untreated cells, right panel apoptotic cells. Arrows indicate blebs, and nuclear condensation and fragmentation. Cytospins were stained with haematoxylin and eosin.

Fig. 2

Western blot of apoptotic (A) and untreated (U) Jurkat cell lysates stained with SLE sera and normal human serum (NHS). Apoptosis was induced by (a) UV irradiation, (b) cyclohexamide and (c) anti-Fas antibody. Arrows indicate prominent bands in apoptotic cell preparations. *Bands present in both apoptotic and untreated preparations.

Fig. 3

Western blot of UV-irradiated Jurkat cell lysates stained with normal sera (or SLE serum 21). Arrows indicate prominent bands in the apoptotic cell lysates (A) not seen in the untreated cell (U) lysates.

Fig. 4

Western blot of cyclohexamide-treated (A) and untreated (U) Jurkat cell lysates, stained with normal human serum (NHS), SLE patient's serum and commercial human serum known to contain antibodies to U1-RNP. Arrows indicate U1-RNP-specific bands.

Fig. 5

Diagrammatic representation of histone subunits as would be seen on an SDS–PAGE gel.

Fig. 6

Western blots that compare staining of apoptotic Jurkat cell lysate with histone. (a) Staining of purified histone (H), UV-irradiated Jurkat cells lysate (A) and untreated Jurkat cell lysate (U) with anti-histone antibody and SLE serum. (b) Staining of histone and apoptotic Jurkat (AJ) preparations with anti-histone antibody (−) is reduced by preincubation of antibody with purified histones (+).

Fig. 7

Western blots showing a difference in staining of apoptotic Jurkat cell lysates and U937 cell lysates with SLE sera. The soluble antigens from supernatant of Triton X-100-lysed cells (a) showed the 33-kD band in apoptotic Jurkat preparations, but this band was very weak or absent from the apoptotic U937 cell preparations. The total cell contents were lysed with reducing sample buffer (b), resulting in the previously absent band being weakly detected in the U937 cells. Prominent bands are indicated by arrows.