Kristin A. Fenton

Lupus Nephritis : The Central Role of Nucleosomes Revealed

Systemic lupus erythematosus (SLE) is an autoimmune syndrome characterized by autoantibodies to nuclear constituents. Some of these antibodies are diagnostically important, whereas others act as disease-modifying factors. One clinically important factor is autoantibodies against dsDNA and nucleosomes, which have overlapping diagnostic and nephritogenic impact in SLE. Although a scientific focus for 5 decades, the molecular and cellular origin of these antibodies, and why they are associated with lupus nephritis, is still not fully understood. A consensus has, however, evolved that antibodies to dsDNA and nucleosomes are central pathogenic factors in the development of lupus nephritis. In contrast, no agreement has been reached as to which glomerular structures are bound by nephritogenic anti-nucleosome antibodies in vivo. Mutually contradictory paradigms and models have evolved simply because we still lack precise and conclusive data to provide definitive insight into how autoantibodies induce lupus nephritis and which specificity is critical in the nephritic process(es). In this review, data demonstrating the central role of nucleosomes in inducing and binding potentially nephritogenic antibodies to DNA and nucleosomes are presented and discussed. These autoimmune-inducing processes are discussed in the context of Matzingers danger model (Matzinger P: Friendly and dangerous signals: is the tissue in control? Nat Immunol 2007, 8:11-13; Matzinger P: The danger model: a renewed sense of self. Science 2002, 296:301-305; Matzinger P: Tolerance, danger, and the extended family. Annu Rev Immunol 1994, 12:991-1045) and Medzhitovs and Janeways (Medzhitov R, Janeway CA Jr: Decoding the patterns of self and nonself by the innate immune system. Science 2002, 296:298-300; Medzhitov R, Janeway CA Jr: How does the immune system distinguish self from nonself? Semin Immunol 2000, 12:185-188; Janeway CA Jr, Medzhitov R: Innate immune recognition. Annu Rev Immunol 2002, 20:197-216) distinction of noninfectious self (NIS) and infectious nonself (INS). The mechanisms leading to production of potentially nephritogenic anti-nucleosome antibodies and to overt lupus nephritis are interpreted in the context of these paradigms.

Glomerular Targets of Nephritogenic Autoantibodies in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibodies directed against various nuclear autoantigens, particularly against components of chromatin, such as doublestranded DNA (dsDNA), histones, nucleosomes, and ribonucleoproteins. Lupus nephritis, a severe clinical manifestation of SLE, is generally associated with high titers of antinuclear antibodies, especially antibodies against chromatin, and with immunoglobulin and complement deposits in the glomerulus. Historically, the formation of antibodies against dsDNA has been considered the serologic hallmark of SLE (1), and anti-dsDNA antibodies are found in the majority of SLE patients (2). In recent years, it has become clear that the nucleosome is the driving autoantigen in SLE (3), and some investigators claim that the presence of antinucleosome antibodies could serve as a better marker for SLE than anti-dsDNA antibodies, since the former are present in up to 90% of lupus patients (4,5). In SLE patients and in lupus-prone mice, nucleosomes can be found in the circulation; they are released from apoptotic cells and are present as a result of either disturbed apoptosis or a defective mechanism for clearing of apoptotic cells (3). Chromatin is basically defined as the entire complex of compacted DNA and associated proteins. The nucleosome is the fundamental unit of chromatin. The core nucleosome particle consists of 146 bp of dsDNA, wrapped twice around a histone octamer (2 copies of each of the core histones H2A, H2B, H3, and H4) (6). Antibodies against epitopes on the nucleosome, called nucleosome-specific antibodies, have no or very low reactivity against individual histones or naked dsDNA. The formation of nucleosome-specific antibodies precedes the appearance of anti-dsDNA and antihistone antibodies as a result of epitope spreading. Anti-dsDNA and antihistone antibodies bind to dsDNA and histones, respectively, but also to nucleosomes (7). A serious topic of debate for several decades involves the pathogenicity, and especially the nephritogenicity, of anti-dsDNA antibodies. DNA itself is not immunogenic, and immunization of mice with naked dsDNA leads to rapid removal, mainly through the liver. Nevertheless, anti-dsDNA antibody levels often show a high correlation with disease activity, especially in lupus nephritis, and elucidating their pathogenic properties is thus of great interest. Therefore, the targets of antidsDNA antibodies have been intensively studied to gain clues to mechanisms that could explain the pathogenicity of anti-dsDNA antibodies in SLE. With regard to their role in the development of lupus nephritis, many investigators postulate that antidsDNA antibodies localize in the glomerular basement membrane (GBM) due to direct cross-reactive binding to intrinsic glomerular antigens (mechanism 1), whereas, in our opinion, glomerular localization of anti-dsDNA antibodies is mediated via nucleosomes, which bind to heparan sulfate (HS) in the GBM (mechanism 2). These two hypotheses may not be mutually exclusive, and until recently, there was no unequivocal proof for either of the two proposed mechanisms. In this review, we will discuss the pros and cons of each of these mechanisms, taking into account recent pertinent observations by Supported by the Dutch Kidney Foundation (grant C05.2119), the PhD student program of the Radboud University Nijmegen Medical Centre, the Health and Rehabilitation Organization, Norway (grant 2001/2/0235), and Oslo Sanitetsforening and Skibsreder Tom Wilhelmsens Stiftelse. Casandra C. van Bavel, MSc, Johan van der Vlag, PhD, Jo H. Berden, MD, PhD: Nijmegen Centre for Molecular Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; Kristin A. Fenton, PhD: University of Tromso, Tromso, Norway; Ole P. Rekvig, MD, PhD: University of Tromso, and University Hospital of North Norway, Tromso, Norway. Address correspondence and reprint requests to Jo H. Berden, MD, PhD, Division of Nephrology (464), Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: J.Berden@nier.umcn.nl. Submitted for publication November 8, 2007; accepted in revised form April 14, 2008.

Anti-dsDNA antibodies promote initiation, and acquired loss of renal Dnase1 promotes progression of lupus nephritis in autoimmune (NZBxNZW)F1 mice.

Background Lupus nephritis is characterized by deposition of chromatin fragment-IgG complexes in the mesangial matrix and glomerular basement membranes (GBM). The latter defines end-stage disease. Methodology/Principals In the present study we determined the impact of antibodies to dsDNA, renal Dnase1 and matrix metalloprotease (MMP) mRNA levels and enzyme activities on early and late events in murine lupus nephritis. The major focus was to analyse if these factors were interrelated, and if changes in their expression explain basic processes accounting for lupus nephritis. Findings Early phases of nephritis were associated with chromatin-IgG complex deposition in the mesangial matrix. A striking observation was that this event correlated with appearance of anti-dsDNA antibodies and mild or clinically silent nephritis. These events preceded down-regulation of renal Dnase1. Later, renal Dnase1 mRNA level and enzyme activity were reduced, while MMP2 mRNA level and enzyme activity increased. Reduced levels of renal Dnase1 were associated in time with deficient fragmentation of chromatin from dead cells. Large fragments were retained and accumulated in GBM. Also, since chromatin fragments are prone to stimulate Toll-like receptors in e.g. dendritic cells, this may in fact explain increased expression of MMPs. Significance These scenarios may explain the basis for deposition of chromatin-IgG complexes in glomeruli in early and late stages of nephritis, loss of glomerular integrity and finally renal failure.

Nephritogenic antibodies bind in glomeruli through interaction with exposed chromatin fragments and not with renal cross-reactive antigens

Cross-reactivity of anti-double stranded DNA (anti-dsDNA) antibodies with glomerular antigens has been postulated as a key factor in the development of lupus nephritis. Because no direct proof has been presented on anti-dsDNA antibodies binding in vivo to glomerular structures, we have analysed the binding of potentially nephritogenic anti-dsDNA antibodies to α-actinin and laminin. By enzyme-linked immunosorbent assay and surface plasmon resonance (SPR) analyses, we demonstrate that monoclonal antibodies (mAbs) bind both double-stranded DNA and α-actinin at high affinity. However, when added to nephritic kidney sections they did not bind to such structures, but rather to nucleosome-containing structures within the mesangial matrix or the glomerular basement membranes (GBMs). Nucleosomes, anti-nuclear antibodies and complexes of them were tested for their binding to glomerular components such as agrin, perlecan and laminin using SPR analysis. Nucleosomes bound to laminin, marginally to agrin, but not to perlecan or heparan sulphate-depleted agrin. Anti-histone H2B and anti-nucleosome antibodies in complex with nucleosomes slightly increased the binding of nucleosomes to agrin, while binding to laminin was slightly decreased compared to nucleosomes alone. In conclusion, the availability of nucleosomal antigens and the binding of these antigens to components of the mesangial matrix and GBM seem crucial for the glomerular deposition of immune complexes.

Pure anti-dsDNA mAbs need chromatin structures to promote glomerular mesangial deposits in BALB/c mice

The glomerular targets for nephritogenic antibodies have been identified as membrane-associated chromatin fragments. The processes responsible for their deposition are poorly understood. To determine early events in antibody-mediated nephritis, we injected highly pure anti-dsDNA mAbs into BALB/c mice. Mice receiving one dose of anti-dsDNA mAbs were sacrificed 6 or 24 h later. No direct binding of mAbs to glomerular membranes or to the mesangial matrix was observed by immune electron microscopy. In contrast, repeated injections of the same antibodies over 4 weeks resulted in deposition of electron dense structures predominantly in the mesangial matrix. These structures contained mAbs and chromatin fragments as determined by co-localization immune electron microscopy. Biotinylated anti-dsDNA mAbs, injected into nephritic (NZB × NZW)F1 or MRLlpr/lpr mice were detected in newly formed electron dense structures within glomerular capillary membranes. There were no correlation between mAb affinity for DNA, as determined by surface plasmon resonance analyses, and ability to bind chromatin fragments in vivo. No direct binding of mAbs to inherent membrane antigens was observed. Quantification of DNA in sera before and after one single injection of antibodies revealed increased DNA levels at 6 h after injection of anti-dsDNA mAb, and lower levels after 24 h. Repeated injections of anti-dsDNA caused an increase in circulating DNA. These results indicate that availability of chromatin fragments, presumable in circulation, is important for glomerular mesangial matrix deposition of anti-dsDNA antibody-containing immune complexes in context of lupus nephritis.

Heparin exerts a dual effect on murine lupus nephritis by enhancing enzymatic chromatin degradation and preventing chromatin binding in glomerular membranes.

OBJECTIVE Association of nucleosome-IgG immune complexes with glomerular basement membranes (GBMs) is an important event in the development of lupus nephritis. Preventing this binding and/or increasing nuclease sensitivity of nucleosomes may be viable strategies for the prevention of the disease. Theoretically, heparin may alter nucleosomal structure and increase sensitivity to proteinases and nucleases, and may also inhibit binding of nucleosomes and nucleosome-IgG complexes to basement membrane structures. The aim of this study was to investigate whether and eventually how heparin prevents murine lupus nephritis. METHODS Surface plasmon resonance was used to analyze if heparin inhibits binding of nucleosomes to laminin and collagen. The effect of heparin on nuclease- and proteinase-mediated degradation of nucleosomes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel electrophoresis. In vitro results were compared with analyses in vivo in heparin-treated (NZB × NZW)F(1) mice. Anti-double-stranded DNA antibody production, deposition of nucleosome-IgG complexes in GBMs, and development of proteinuria were monitored, and circulating chromatin fragments were quantified using quantitative polymerase chain reaction. RESULTS In vitro studies demonstrated that heparin increased enzymatic degradation of nucleosomes and almost completely inhibited binding of nucleosomes to laminin and collagen. (NZB × NZW)F(1) mice treated with heparin demonstrated delayed or no antibody production and higher variation of circulating chromatin levels compared with untreated control mice. This effect was accompanied by highly reduced nucleosome-IgG complexes in GBMs and delayed development of nephritis. CONCLUSION Increasing the degradation of nucleosomes, reducing their immunogenicity, and preventing binding of nucleosome-IgG complexes in glomeruli together provide an alternative basis for the treatment of lupus nephritis.

Circulating chromatin–anti-chromatin antibody complexes bind with high affinity to dermo-epidermal structures in murine and human lupus nephritis

Murine and human lupus nephritis are characterized by glomerular deposits of electron-dense structures (EDS). Dominant components of EDS are chromatin fragments and IgG antibodies. Whether glomerular EDS predispose for similar deposits in skin is unknown. We analysed (i) whether dermo-epidermal immune complex deposits have similar molecular composition as glomerular deposits, (ii) whether chromatin fragments bind dermo-epidermal structures, and (iii) whether deposits in nephritic glomeruli predispose for accumulation of similar deposits in skin. Paired skin and kidney biopsies from nephritic (NZBxNZW)F1 and MRL-lpr/lpr mice and from five patients with lupus nephritis were analysed by immunofluorescence, immune electron microscopy (IEM) and co-localization TUNEL IEM. Affinity of chromatin fragments for membrane structures was determined by surface plasmon resonance. Results demonstrated (i) presence of EDS containing chromatin fragments and IgG in both organs in nephritic patients, (ii) chromatin fragments possessed high affinity for dermo-epidermal laminins and collagens, (iii) glomerular immune complex deposits did not predict similar interstitial deposits in skin, although such complexes were present in capillary lumina in glomeruli and skin of all nephritic individuals. Thus, chromatin-IgG complexes accounting for lupus nephritis seem to reach skin through circulation, but other undetermined factors are required for these complexes to deposit within skin membranes.

The effect of cell death in the initiation of lupus nephritis

Cell death and the release of chromatin have been demonstrated to activate the immune system producing autoantibodies against nuclear antigens in patients with systemic lupus erythematosus (SLE). Apoptosis, necrosis, necroptosis, secondary necrosis, autophagy and the clearance of dying cells by phagocytosis are processes believed to have a role in tolerance avoidance, activation of autoimmune lymphocytes and tissue damage by effector cells. The released chromatin not only activates the immune system; it also acts as antigen for the autoantibodies produced, including anti‐dsDNA antibodies. The subsequent immune complex formed is deposited within the basement membranes and the mesangial matrix of glomeruli. This may be considered as an initiating event in lupus nephritis. The origin of the released chromatin is still debated, and the possible mechanisms and cell sources are discussed in this study.

Tertiary lymphoid structures are associated with higher tumor grade in primary operable breast cancer patients

BackgroundTertiary lymphoid structures (TLS) are highly organized immune cell aggregates that develop at sites of inflammation or infection in non-lymphoid organs. Despite the described role of inflammation in tumor progression, it is still unclear whether the process of lymphoid neogenesis and biological function of ectopic lymphoid tissue in tumors are beneficial or detrimental to tumor growth. In this study we analysed if TLS are found in human breast carcinomas and its association with clinicopathological parameters.MethodsIn a patient group (n = 290) who underwent primary surgery between 2011 and 2012 we assessed the interrelationship between the presence of TLS in breast tumors and clinicopathological factors. Prognostic factors were entered into a binary logistic regression model for identifying independent predictors for intratumoral TLS formation.ResultsThere was a positive association between the grade of immune cell infiltration within the tumor and important prognostic parameters such as hormone receptor status, tumor grade and lymph node involvement. The majority of patients with high grade infiltration of immune cells had TLS positive tumors. In addition to the degree of immune cell infiltration, the presence of TLS was associated with organized immune cell aggregates, hormone receptor status and tumor grade. Tumors with histological grade 3 were the strongest predictor for the presence of TLS in a multivariate regression model. The model also predicted that the odds for having intratumoral TLS formation were ten times higher for patients with high grade of inflammation than low grade.ConclusionsHuman breast carcinomas frequently contain TLS and the presence of these structures is associated with aggressive forms of tumors. Locally generated immune response with potentially antitumor immunity may control tumorigenesis and metastasis. Thus, defining the role of TLS formation in breast carcinomas may lead to alternative therapeutic approaches targeting the immune system.

A Central Role of Nucleosomes in Lupus Nephritis

Abstract:  Lupus nephritis is characterized by the presence of subendothelial and subepithelial immune complexes and thickening of the glomerular basement membranes (GBM). Electron‐dense structures (EDS) in mesangium and GBM have been demonstrated to constitute target structures for nephritogenic autoantibodies in vivo. Whether these antibodies bind nucleosomal antigens within the EDS or cross‐react with components of the GBM has not been resolved. Data recently published point at intra‐GBM‐associated nucleosomes as target for the nephritogenic autoantibodies. Colocalization IEM has demonstrated that autoantibodies and experimental antibodies against DNA, histones, or transcription factors like TATA box‐binding protein colocalize in the EDS. By using terminal transferase in situ nick‐end labeling in combination with immune electron microscopy to detect DNA specifically in human and murine SLE kidneys, we were able to detect DNA within the EDS of nephritic glomeruli that corresponded with the detected autoantibodies.