[Show abstract][Hide abstract] ABSTRACT: In systemic lupus erythematosus (SLE) apoptotic chromatin is present extracellularly, which is most likely the result of disturbed apoptosis and/or insufficient removal. Released chromatin, modified during apoptosis, activates the immune system resulting in the formation of autoantibodies. A study was undertaken to identify apoptosis-induced histone modifications that play a role in SLE.
The lupus-derived monoclonal antibody BT164, recently established by selection using apoptotic nucleosomes, was analysed by ELISA, western blot analysis and immunofluorescence staining using chromatin, cells, plasma and renal sections. Random peptide phage display and peptide inhibition ELISA were used to identify precisely the epitope of BT164. The reactivity of plasma samples from lupus mice and patients with SLE with the epitope of BT164 was investigated by peptide ELISA.
The epitope of BT164 was mapped in the N-terminal tail of histone H3 (27-KSAPAT-32) and included the apoptosis-induced trimethylation of K27. siRNA-mediated silencing of histone demethylases in cultured cells resulted in hypermethylation of H3K27 and increased nuclear reactivity of BT164. This apoptosis-induced H3K27me3 is a target for autoantibodies in patients and mice with SLE and is present in plasma and in glomerular deposits.
In addition to previously identified acetylation of histone H4, H2A and H2B, this study shows that trimethylation of histone H3 on lysine 27 is induced by apoptosis and associated with autoimmunity in SLE. This finding is important for understanding the autoimmune response in SLE and for the development of translational strategies.
Full-text · Article · Jan 2011 · Annals of the rheumatic diseases
[Show abstract][Hide abstract] ABSTRACT: Nuclear components targeted by autoantibodies are a characteristic feature of the autoimmune disease systemic lupus erythematosus (SLE). The nucleosome, a major autoantigen, is released in patients with SLE as a result of a disturbed apoptosis and/or an insufficient clearance of apoptotic debris. During apoptosis the nucleosome is modified, thereby creating more immunogenic epitopes. Subsequently, epitope spreading will lead to the formation of autoantibodies against unmodified chromatin components. However, characterization of B cell epitopes specific for apoptotic chromatin modifications is hampered by the fact that the existing monoclonal antibodies (mAbs) were originally selected on non-apoptotic chromatin. Here, we describe a novel approach for generating mAbs from lupus mice that are specific for apoptosis-induced chromatin modifications.
Hybridomas were generated from pre-diseased and diseased lupus mice using standard fusion methods. Selection occurred on isolated apoptotic chromatin. Antibodies were further characterized by ELISA, western blot and immunofluorescence staining with apoptotic and non-apoptotic chromatin/cells. In addition, reactivity was determined with subnucleosomal complexes and with nucleosomes treated with trypsin or DNase I. Finally, reactivity was determined with cells treated with the histone deacetylase inhibitor TSA.
Most generated mAbs appeared to be nucleosome specific with a clear preference for apoptotic nucleosomes compared to normal nucleosomes. Although the exact elucidation of the epitopes of these mAbs specific for apoptosis-associated nucleosome modifications remains a major challenge, the epitopes contain both DNA and histones, whereby the histone tails play a role in establishing the epitopes. Most importantly, the conformational epitopes of these nucleosome-specific antibodies seem to contain acetylated residues.
Our approach, yielding a new panel of anti-apoptotic-chromatin antibodies, should facilitate the discovery of more apoptosis-induced chromatin modifications and their identification as key autoantigens in the pathogenesis of SLE.
Full-text · Article · Nov 2010 · Molecular Immunology
[Show abstract][Hide abstract] ABSTRACT: Nucleosomes have been identified as a key autoantigen in systemic lupus erythematosus (SLE). Nucleosomes are present in the circulation due to a disturbed apoptosis and/or an insufficient clearance in SLE. During apoptosis, histones can be modified, thereby making them more immunogenic. Recently, we showed the importance of apoptosis-induced acetylation of histone H4 in the pathogenesis of SLE. The lupus-derived antibody LG11-2 was previously shown to react with the N-terminal tail of histone H2B, which contains amino acid residues that can be modified including phosphorylation of serine 14, known to occur during apoptosis. Here, we evaluate whether apoptosis-induced histone modifications on H2B exist that are targeted by LG11-2 or lupus-derived plasmas.
Immunofluorescence staining and western immunoblot analysis of control, apoptotic and trichostatin A-treated cells/chromatin were performed with monoclonal antibody LG11-2. Reactivity of LG11-2 and plasmas from lupus mice and SLE patients with acetylated and/or phosphorylated H2B peptides was determined in competition ELISA.
LG11-2 showed enhanced reactivity with apoptotic and hyperacetylated H2B compared to normal H2B. This enhanced reactivity was due to the acetylation of lysine 12 in H2B. This modification was also recognized by autoantibodies from pre-diseased lupus mice, but to a lesser extent by plasmas of diseased lupus mice and lupus patients.
The apoptosis-induced acetylation on H2BK12 is a target for autoantibodies in SLE. Since the anti-H2BK12ac reactivity was mainly found in pre-diseased lupus mice, this epitope seems important in the early phase of the anti-chromatin autoimmune response with subsequent epitope spreading to unmodified H2B.
[Show abstract][Hide abstract] ABSTRACT: Systemic lupus erythematosus (SLE) is a prototype inflammatory autoimmune disease resulting from autoimmune responses against nuclear autoantigens. During apoptosis many lupus autoantigens congregate inside the cells and are susceptible to modifications. Modified nuclear constituents are considered foreign and dangerous. Therefore, apoptotic cells have to has to be efficiently removed to avoid the accumulation of apoptotic debris and the subsequently development of autoimmune responses. Hence, apoptosis and clearance of apoptotic cells/material are considered key processes in the aetiology of SLE. Clearance deficiencies may account for the development of autoimmunity by inducing a loss of tolerance in lymphoid tissues. Furthermore, phagocytosis of apoptotic cells may lead to a pro-inflammatory response in the presence of autoantibodies. This may sustain inflammatory conditions and the pathology found in overt lupus.
[Show abstract][Hide abstract] ABSTRACT: In systemic lupus erythematosus (SLE), inadequate removal of apoptotic cells may lead to challenge of the immune system with immunogenic self antigens that have been modified during apoptosis. We undertook this study to evaluate whether apoptosis-induced histone modifications are targets for the immune system in SLE.
The epitope of KM-2, a monoclonal antihistone autoantibody derived from a lupus mouse, was mapped by random peptide phage display. The reactivity of KM-2 and plasma with (acetylated) histone H4 (H4) peptides and with nonapoptotic, apoptotic, and hyperacetylated histones was determined by immunofluorescence staining, enzyme-linked immunosorbent assay, and Western blotting.
KM-2 recognized apoptosis-induced acetylation of H4 at lysines 8, 12, and 16. The majority of plasma samples from SLE patients and lupus mice showed higher reactivity with triacetylated H4 peptide (residues 1-22) and with hyperacetylated and apoptotic histones than with nonacetylated H4 peptide and normal histones. Importantly, administration of triacetylated H4 peptide to lupus-prone mice before disease onset accelerated the disease by enhancing mortality and aggravating proteinuria, skin lesions, and glomerular IgG deposition, while the nonacetylated H4 peptide had no pathogenic effect. The delayed-type hypersensitivity response in lupus mice against the triacetylated H4 peptide was higher than that against the nonacetylated H4 peptide. Bone marrow-derived dendritic cells (DCs) cultured in the presence of hyperacetylated nucleosomes showed increased expression/production of CD40, CD86, interleukin-6 (IL-6), and tumor necrosis factor alpha compared with DCs cultured in the presence of normal nucleosomes. Finally, DCs cultured in the presence of hyperacetylated nucleosomes were able to activate syngeneic T cells, because IL-2 production increased.
Apoptosis-induced acetylation of nucleosomes may represent an important driving force in the development of lupus.
Full-text · Article · Jun 2007 · Arthritis & Rheumatology
[Show abstract][Hide abstract] ABSTRACT: The binding of anti-double-stranded DNA (anti-dsDNA) autoantibodies to the glomerular basement membrane (GBM) in lupus nephritis can be explained by two mechanisms: (1) direct crossreactive binding to intrinsic glomerular antigens; (2) nucleosome-mediated binding to heparan sulfate in the GBM. Kalaaji et al. demonstrated using novel techniques that glomerular in vivo-bound antoantibodies bind to nucleosomes/dsDNA derived from apoptotic cells and not to intrinsic glomerular structures.
Full-text · Article · May 2007 · Kidney International
[Show abstract][Hide abstract] ABSTRACT: In recent years the analysis of anti-nucleosome antibodies has been added to the diagnostic armamentarium for systemic lupus erythematosus (SLE). This was because of the fact that it became increasingly clear that the nucleosome is the driving autoantigen in SLE. An insufficient removal of apoptotic cells leads to the release of auto-antigens including nucleosomes. During apoptosis these autoantigens can be modified, rendering them more immunogenic. Immunogenic presentation by dendritic cells then induces a T-cell response. These nucleosome-specific T-cells induce not only anti-nucleosome antibodies but also antidsDNA and anti-histone antibodies. Complexes of nucleosomes and anti-nuclear antibodies are nephritogenic because these complexes can bind via their positively charged histone tails to the negatively charged heparan sulfate in the glomerular basement membrane. Anti-nucleosome antibodies comprise autoantibodies that can bind to all accessible components of the nucleosome, that is dsDNA, histones and conformational epitopes formed between DNA and histones. These latter are called nucleosome-specific antibodies. Anti-nucleosome autoantibodies are more prevalent in SLE (55-70%) than antidsDNA antibodies. They have a better correlation with SLE disease activity and lupus nephritis. They can be detected in 40% of the SLE patients who are anti-dsDNA negative. Their sensitivity for SLE is 63%. Their specificity is 85% compared with non-lupus disease controls and 100% compared with healthy controls. The usefulness of anti-nucleosome antibodies for disease monitoring and prediction of flares is not clear yet. It has been proposed that a positive anti-nucleosome reactivity should replace a positive anti-dsDNA reactivity in the American College of Rheumatology (ACR) classification criteria for SLE.
[Show abstract][Hide abstract] ABSTRACT: To evaluate the binding of lupus-derived autoantibodies, double-stranded DNA and nucleosomes to the positively charged C-terminal SmD1(residues 83-119) peptide and the full-length SmD protein.
The binding of lupus-derived monoclonal antibodies, sera from patients with systemic lupus erythematosus, rheumatoid arthritis and systemic sclerosis, dsDNA and nucleosomes to the SmD1(83-119) peptide or the full-length SmD protein was determined using different ELISA methods.
Monoclonal anti-dsDNA antibodies and the serum of patients with systemic lupus erythematosus that are positive for anti-dsDNA antibodies react with the SmD1(83-119) peptide in ELISA. However, DNaseI treatment of the blocking reagents leads to a decreased reactivity. Purified dsDNA and nucleosomes bind to the SmD1 peptide but not to the full-length SmD protein.
The SmD1(83-119) peptide is able to bind dsDNA and nucleosomes, and dsDNA or nucleosomes in applied reagents lead to an apparent reactivity of anti-dsDNA, anti-histone or nucleosome-specific antibodies with the SmD1(83-119) peptide in ELISA.
Full-text · Article · Dec 2006 · Annals of the Rheumatic Diseases