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. 2006 Jun;168(6):1779-92.
doi: 10.2353/ajpath.2006.051329.

Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells

Manar Kalaaji  1 , Elin Mortensen, Leif Jørgensen, Randi Olsen, Ole Petter Rekvig
Affiliations

Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells

Manar Kalaaji et al. Am J Pathol. 2006 Jun.

Abstract

Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus.

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Figures

Figure 1
Figure 1
Autoantibody profiles determined over 33 weeks in 14 (NZBxNZW)F1 mice. Antibodies were determined at regular intervals against dsDNA (A), nucleosomes (B), and α-actinin (C). Four antibody patterns were detected before or at the time proteinuria was developing, decreasing (D), stable (E), or increasing (F) antibody levels. In one mouse, the antibodies increased, still without proteinuria (B/W15; G). The symbols given for individual mice in A are representative for the symbols in B and C, whereas the symbols in D are representative for those in E–G.
Figure 2
Figure 2
Morphological and apoptosis studies of murine glomeruli. Glomeruli of BALB/c (A, F, and K), non-nephritic young B/W52 (B, G, and L), anti-dsDNA-positive, nonproteinuric B/W15 (C, H, and M), nephritic B/W2 (D, I, and N), or the nephritic B/W16 (E, J, and O) were analyzed for morphological changes by light microscopy (A–E). Intraglomerular apoptosis was analyzed by TUNEL assay (F–J) or by caspase-3 assay (K–O). K–O: Caspase-3-positive cells are brown; caspase-3-negative ones are blue. Circles in D and E indicate areas with wire-loop lesions and extracellular chromatin fragments.
Figure 3
Figure 3
Morphological analyses of nephritic glomeruli of B/W16 by TEM and IEM and by confocal microscopy and colocalization IEM. Morphology is analyzed by TEM (A) and localization of autoantibody deposits by IEM (B). EDDs in A are indicated by arrows. B: Autoantibody deposits (shown as 5-nm gold) are confined to these structures. Confocal microscopy (C–H) demonstrates that autoantibody deposits (displayed in green; C) and bound anti-collagen IV antibodies (red; D) colocalize topographically and are in certain areas in confocus (yellow; E). F–H: Autoantibody deposits (green; F) and bound anti-laminin-β2 antibodies (red; G) are partly in confocus (yellow; H). By IEM analyses (I and J), autoantibodies are displayed by 5-nm gold particles (I) and anti-laminin antibodies by 10-nm gold particles (J). Colocalization IEM analyses (K) demonstrate autoantibodies (5-nm gold) and anti-laminin-β2 antibodies (10-nm gold) in EDDs and GBMs, respectively.
Figure 4
Figure 4
TEM, IEM, and two-step colocalization IEM of BALB/c glomeruli. A: TEM analysis was performed to assess glomerular morphology. B: Sections are incubated with RaM IgG/PAG-5 nm to search for in vivo-bound autoantibodies (step 1). In step 2, colocalization IEM (B) was performed with an irrelevant control antibody (anti-T-antigen mAb Ab-2). Absence of 10-nm gold demonstrates that IgG or PAG conjugates do not bind nonspecifically (B). Next, step 2 was performed with anti-dsDNA mAb 163p77 instead of Ab-2. GBM is not stained by 5- or 10-nm gold particles (C, enlarged in D), whereas the nuclei are stained by anti-dsDNA mAb, reflected by 10-nm gold particles (C, enlarged in E). For experimental details on colocaliztion IEM used in Figures 4 to 7, see Materials and Methods.
Figure 5
Figure 5
TEM, IEM, and two-step colocalization IEM of nephritic B/W 2 glomeruli. A: TEM analyses revealed confluent podocytes and electron deposits in GBMs (arrows). By standard IEM (step 1), autoantibody deposits are traced by 5-nm gold (B, enlarged in C). Colocalization IEM (step 2) was performed using the negative control anti-T-ag antibody Ab-2 (D, enlarged in E) or anti-histone H1 (F and G), anti-TBP (H and I), or the anti-dsDNA 163p77 (J–M) monoclonal antibodies. Autoantibodies and the three chromatin-specific experimental antibodies (the three latter antibodies traced by 10-nm gold) are colocalized in EDDs (enlarged in G, I, and M, for anti-H3, anti-TBP, and anti-dsDNA antibodies, respectively). Anti-T antigen did not bind (absence of 10-nm gold in D and E).
Figure 6
Figure 6
TEM, IEM, and two-step colocalization IEM of nonproteinuric B/W15 glomeruli. A: TEM analysis demonstrates partially confluent podocytes, and GBM-associated EDDs (arrows). By IEM (step 1), autoantibody deposits are traced by 5-nm gold (B, enlarged in C). Colocalization IEM (step 2) was performed using the anti-dsDNA mAb 163p77, and binding was traced by 10-nm gold (D). Autoantibodies and the anti-dsDNA mAb colocalize in the GBM-associated EDDs (5- and 10-nm gold in D, enlarged in E).
Figure 7
Figure 7
TEM, IEM, and two-step colocalization IEM of nephritic B/W16 glomeruli. A: TEM analysis demonstrates confluent podocytes and GBM-associated EDDs (arrows). B: IEM analysis (step 1) demonstrates autoantibodies in EDDs (5-nm gold). Colocalization IEM (step 2) was performed using the anti-dsDNA mAb 163p77. The anti-dsDNA mAb (10-nm gold) colocalizes with autoantibodies (C, enlarged in D). In the nucleus, the anti-dsDNA mAb is detected (C, enlarged in E).
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