Apoptosis and NET formation in the pathogenesis of SLE.

Systemic Lupus Erythematosus is an autoimmune disease characterized by the formation of anti-nuclear autoantibodies, particularly anti-chromatin. Although the aetiology of the disease has not yet been fully elucidated, several mechanisms have been proposed to be involved. Due to an aberrant apoptosis or decreased removal of apoptotic cells, apoptotic blebs containing chromatin are released. During apoptosis, chromatin is modified that increases its immunogenicity. Myeloid dendritic cells (myDC) can take up apoptotic blebs and stimulate autoreactive T helper cells, and subsequently the formation of autoantibodies by autoreactive B cells. Immune complexes formed by anti-chromatin autoantibodies and modified chromatin deposit on basal membranes, and incite a local inflammation, but can also stimulate plasmacytoid dendritic cells to produce IFN-α. In addition to apoptotic blebs, neutrophil extracellular traps released by dying neutrophils, in a process called NETosis, may serve as a source of autoantigens as well. In this review, we describe the role of both apoptosis and NETosis in the pathogenesis of SLE, and show how both processes may interact with each other.

Apoptosis-induced histone H3 methylation is targeted by autoantibodies in systemic lupus erythematosus.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Lupus-derived monoclonal autoantibodies against apoptotic chromatin recognize acetylated conformational epitopes.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Apoptosis-induced acetylation of histones is pathogenic in systemic lupus erythematosus.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Apoptosis-induced acetylation of nucleosomes may represent an important driving force in the development of lupus.

A prospective study of anti-chromatin and anti-C1q autoantibodies in patients with proliferative lupus nephritis treated with cyclophosphamide pulses or azathioprine/methylprednisolone.

OBJECTIVE: To study the prevalence and course of anti-chromatin (anti-nucleosome, anti-double-stranded (ds) DNA and anti-histone) and anti-C1q autoantibodies in patients with proliferative lupus nephritis (LN), treated in a randomised controlled trial with either cyclophosphamide or azathioprine plus methylprednisolone. METHODS: Autoantibody levels were measured and analysed in 52 patients with proliferative LN, during their first year of treatment. Levels in both treatment arms were compared and associations with clinical, serological and outcome parameters were studied. RESULTS: At study entry, prevalences for anti-nucleosome, anti-dsDNA, anti-histone and anti-C1q autoantibodies were 81%, 96%, 23% and 65%, respectively. Anti-chromatin autoantibodies correlated with each other, but not with anti-C1q levels. If patients were divided for their autoantibody titre at the start of treatment above or below the median, the only significant differences were higher SLE disease activity index with higher anti-nucleosome, and higher creatinine with higher anti-C1q autoantibodies. During the first year, a comparable rapid decline in the levels of anti-nucleosome, anti-dsDNA and anti-C1q autoantibodies was seen in both treatment arms. Anti-histone autoantibody levels were low and did not change. Renal flares were not preceded by rises in autoantibody titres. CONCLUSIONS: These results indicate that measurement of anti-chromatin and anti-C1q autoantibodies is useful for diagnosing LN, but not for monitoring disease course.

Asymmetry in histone H3 variants and lysine methylation between paternal and maternal chromatin of the early mouse zygote.

In mammalian fertilization, the paternal genome is delivered to the secondary oocyte by sperm with protamine compacted DNA, while the maternal genome is arrested in meiotic metaphase II. Thus, at the beginning of fertilization, the two gametic chromatin sets are strikingly different. We elaborate on this contrast by reporting asymmetry for histone H3 type in the pre-S-phase zygote when male chromatin is virtually devoid of histone H3.1/3.2. Localization of the histone H3.3/H4 assembly factor Hira with the paternal chromatin indicates the presence of histone H3.3. In conjunction with this, we performed a systematic immunofluorescence analysis of histone N-tail methylations at position H3K4, H3K9, H3K27 and H4K20 up to the young pronucleus stage and show that asymmetries reported earlier are systematic for virtually all di- and tri-methylations but not for mono-methylation of H3K4 and H4K20, the only marks studied present in the early male pronucleus. For H4K20 the expanding male chromatin is rapidly mono-methylated. This coincides with the formation of maternally derived nucleosomes, a process which is observed as early as sperm chromatin decondensation occurs. Absence of tri-methylated H3K9, tri-methylated H4K20 and presence of loosely anchored HP1-beta combined with the homogenous presence of mono-methylated H4K20 suggests the absence of a division of the paternal chromatin in eu- and heterochromatin. In summary the male, in contrast to female G1 chromatin, is uniform and contains predominantly histone H3.3 as histone H3 variant.

Mimotopes for lupus-derived anti-DNA and nucleosome-specific autoantibodies selected from random peptide phage display libraries: facts and follies.

Autoantibodies against chromatin are the most characteristic serological feature in SLE patients. Anti-dsDNA and nucleosome-specific antibodies are associated with glomerulonephritis, the most serious manifestation of SLE. Identification of peptides mimicking conformational epitopes (so-called mimotopes) on the nucleosome recognized by these antibodies is of considerable interest. Using an approach similar to that used previously to characterize mimotopes for anti-DNA autoantibodies, we have selected and identified a mimotope for a nucleosome-specific autoantibody (#32) by screening a random peptide phage display library. However, the reactivity of monoclonal antibody (mAb) #32 with the selected mimotope (MIMO#0) in ELISA was dependent on the blocking reagents used. Using nonfat dry milk (5%), mAb #32 clearly bound to MIMO#0, but using fetal bovine calf serum (FCS) (5%), there was no binding. Furthermore, again dependent on the blocking reagent used in ELISA, the selected mimotope MIMO#0 was not only recognized by the selecting antibody mAb #32, but also by a large number of other monoclonal anti-DNA, anti-histone and nucleosome-specific autoantibodies (NSA). We could demonstrate that the selected mimotope was able to bind directly to nucleosomal material (DNA/histone complexes) and labeled DNA. This finding was extended to other previously identified mimotopes for anti-DNA autoantibodies. We conclude that nucleosomal material (DNA/histone complexes), derived from reagents used during the mimotope selection procedure, resulted in the selection of DNA-binding peptides from the phage display library, rather than mimotopes. In addition, we could demonstrate that blocking reagents greatly influence the reactivity of anti-DNA, anti-histone and nucleosome-specific autoantibodies in ELISA. Development of blocking reagents devoid of nucleosomal material (DNA/histone complexes) is urgently needed for assay systems in which anti-nuclear autoantibodies are tested.

Albuminuria in mice after injection of antibodies against aminopeptidase A: role of angiotensin II.

It has been shown that injection of combinations of anti-aminopeptidase A (APA) monoclonal antibodies (mAb) that inhibit the enzyme activity induces an acute albuminuria in mice. This albuminuria is not dependent on inflammatory cells, complement, or the coagulation system. APA is an important regulator of the renin-angiotensin system because it is involved in the degradation of angiotensin II (Ang II). This study examined the potential role of glomerular Ang II in the induction of albuminuria. The relation among renal Ang II, glomerular APAX enzyme activity, and albuminuria was examined first. Injection of the nephritogenic combinations ASD-3/37 and ASD-37/41 in BALB/c mice induced albuminuria, whereas the non-nephritogenic combination ASD-3/41 had no effect. There was no clear relation between the inhibition of glomerular APA activity and albuminuria, yet it was evident that intrarenal Ang II levels were significantly increased in albuminuric mice and not in nonalbuminuric mice. As a next step, anti-APA mAb were administered to angiotensinogen-deficient mice that do not produce Ang II, and kidney morphology and albuminuria were determined. Angiotensinogen-deficient mice also developed albuminuria upon ASD-37/41 administration. Altogether, these findings clearly demonstrate that Ang II is not required for the induction of albuminuria upon injection of enzyme-inhibiting anti-APA mAb.