1 Department of Medicine B and The Center for Autoimmune Diseases, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine and 2 Incumbent of the Laura SchwartzKipp chair for Autoimmunity, Tel Aviv University, Israel.
Correspondence to: Y. Shoenfeld, Department of Medicine B and Center for Autoimmune Diseases, Sheba Medical Center, Tel Hashomer, Israel 52621. E-mail: shoenfel{at}post.tau.ac.il
The aim of this editorial is to highlight the need for further insight into the network cascade leading to diverse serological, clinical and inflammatory manifestations and multiple organ damage in lupus, associated with anti-double-stranded DNA (anti-dsDNA) antibodies. Better understanding of the lupus network may serve as a basis to develop specific targeted therapy based on lessons from experimental lupus models.
Systemic lupus erythematosus (SLE) is an autoimmune disease of multifactorial etiology and diverse mechanisms. Although the disease is T-cell-dependent and antigen-driven, more than 100 autoantibodies were detected in the sera of lupus patients with differential correlation to disease activity [1, 2].
Many of the current autoantigen targets are derived from macromolecular structures such as chromatin (nucleosomes composed of dsDNA and histones), and small nuclear ribonucleoproteins (snRNPs) [3]. Using lupus-prone mice [(NZBxNZW)F1(BWF1) or (SWRxNZB)F1(SNF1)], Mohan et al. showed that the nucleosome is the major immunogen for pathogenic autoantibody-inducing T-cells [4]. Moreover, synthetic peptide localized in the critical autoepitopes for lupus nephritis-inducing Th cells had a tolerogenic potential [5]. The self macromolecules are normally sequestered from the immune system by virtue of their intracellular location, but can become more accessible to the immune system as a result of cell necrosis or apoptosis [6, 7]. Such accessibility can result from migration to apoptotic blebs and release into the extracellular environment. Furthermore, biochemical modifications can be induced by apoptotic effector mechanisms, cell injury and post-translational modifications such as oxidation and phosphorylation. (ii) Attempts to repair such lesions, can create neo-epitopes and induce immunogenicity [8, 9]. Several lines of evidence suggest a role for apoptotic cells in the induction of autoimmunity. Injection of a large number of apoptotic cells into naive mice evoked generation of autoantibodies to nuclear antigens [10]. The defects in Fas and FasL respectively result in incomplete elimination of peripheral autoreactive cells as this elimination occurs predominantly by Fas-mediated apoptosis [11, 12]. Injection of soluble Fas into naive mice protected from the development of autoimmune features due to blocking of Fas-induced apoptosis [13]. An increased rate of apoptosis and decreased clearance of apoptotic cells have been demonstrated in SLE animal models and patients [1418]. Recently, a deficiency of CD226 and survivin (anti-apoptotic member protein) in NK T cells from active SLE was suggested to be the molecular basis of a high sensitivity of the cells to anti-CD95-induced apoptosis [19]. The immunogenic potential of nuclear antigens exposed during apoptosis, together with considerable animal data, suggests that impaired apoptotic clearance can result in systemic lupus SLE-like autoimmunity and supports the idea that self-immunization with apoptotic debris may be one of the major driving mechanisms in lupus. The multiple roles of complement receptors, diverse scavenger receptors, phosphatidylserine-specific receptor and intermediate proteins (CRP, SAP, ß-2-glycoprotein-I) that bind to and opsonize apoptotic cells indicate a complex web of interactions leading to the clearance of apoptotic debris. Disturbances in parts of this system may lead to lupus or to lupus exacerbations.
Phagocytosis and clearance of apoptotic cells can be mediated by Mer, a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family [20]. Merkd mice with a cytoplasmic truncation of Mer had macrophages deficient in the specific clearance of apoptotic thymocytes [20]. Impaired clearance of apoptotic cells was documented in c-mer-deficient mice, which develop progressive lupus-like autoimmunity, accompanied by elevated circulating antibodies to chromatin and DNA [21]. Others pointed to the consequence of altered opsonization of apoptotic cells as a contributor to reduced removal of apoptotic cells by monocytes, and developing an autoimmune state [2225]. Serum amyloid P component (SAP) knockout mice developed antinuclear antibodies [23]. Exposure of phagocytes to SAP improved the uptake of apoptotic cells by macrophages [24]. In an another report, 25% of C1q/ mice developed glomerulonephritis with immune deposits and multiple apoptotic cell bodies [25].
One of the questions raised as a result of the defect in the clearance of apoptotic cells or altered opsonization in lupus is whether DNA may be presented as an immunogen evoking an anti-dsDNA response. Immunization of SF1 mice with nucleosomes or its major synthetic epitope triggers Th1-type autoimmune T cells that drive production of the antinuclear, anti-dsDNA and antihistone autoantibodies associated with the clinical picture of lupus [35].
Mammalian DNA was generally considered to be non-immunogenic and unable to induce a specific response under the usual immunization conditions, including complexation with a protein carrier and administration in Freund's complete adjuvant [26, 27]. Qiao et al., in the current issue of Rheumatology, show that immunization of naive mice with genomic DNA derived from concanavalin A-stimulated syngeneic splenocytes evoked anti-dsDNA antibody production and glomerulonephritis, while genomic DNA from non-activated splenocytes failed to be an immunogen. Previously, anti-DNA monoclonal antibodies were prepared using an in vitro immunization method. BALB/c mouse splenocytes were immunized with HeLa cell nuclear extract; whether this antibody had any association with lupus was not analysed [28].
DNA has unique immunological properties that may affect its immunogenicity. Thus, depending on sequence and base methylation, DNA can be stimulatory, inhibitory or neutral with respect to mitogenic activity and cytokine induction. CD4+ T-cell DNA hypomethylation may contribute to the development of drug-induced and idiopathic human lupus. Inhibiting DNA methylation in mature CD4+ T cells caused autoreactivity specific to the major histocompatibility complex in vitro. The lupus-inducing drugs hydralazine and procainamide also inhibit T-cell DNA methylation and induce autoreactivity, and T cells from patients with active lupus had hypomethylated DNA and a similarly autoreactive T-cell subset [29]. Further, the pathological significance of the autoreactivity induced by inhibiting T-cell DNA methylation had been tested by treating murine T cells in vitro with drugs that modify DNA methylation, then injecting the cells into syngeneic female mice. Mice receiving CD4+ T cells demethylated by a variety of agents, including procainamide and hydralazine, developed a lupus like disease [29].
Bacterial DNA, due to its higher frequency of unmethylated CpG sequences in a particular base context (CpG motif), and synthetic CpG oligodeoxynucleotides can directly stimulate dendritic cells, macrophages and B cells in a Toll-like-receptor-9 (TLR-9)-dependent fashion [26, 27, 3034]. In B cells, bacterial DNA and CpG oligodeoxynucleotides provide protection against spontaneous and B-cell receptor-triggered apoptosis associated with increased c-Myc, c-Jun and BclXL expression and induce polyclonal B-cell proliferation, immunoglobulin secretion and isotype switching [31, 35]. Lupus-prone Palmerston North (PN) mice showed a defective response to CpG oligodeoxynucleotides, as reflected by decreased IL-12p40 and IL-6 secretion compared with controls, and higher IL-10 secretion associated with accelerated lupus pathogenesis [36]. Recently, using lupus-prone mice models (BWF1, PN), Brummel and Lenert [37] showed that CpG oligodeoxynucleotides induced marginal-zone B-cell activation, costimulatory molecule expression and polyclonal immunoglobulin secretion. The authors suggested that, through increased IL-10 secretion, marginal-zone B cells may also modify the activity of other cell types, particularly dendritic cells and macrophages [37].
Antibodies binding dsDNA play an important role in the pathogenesis and activity of lupus. Although anti-dsDNA antibodies are present in the damaged area, such as the kidney, in lupus nephritis, it is not widely accepted that they are pathogenic. Anti-ds DNA deposition associated with kidney damage was shown in mice infused with the immunoglobulins [3844]. SCID mice implanted with human hybridoma cells secreted anti-dsDNA IgG that was deposited in the kidney, causing proteinuria [38, 39]. Some anti-dsDNA monoclonal antibodies bound to the membranous structures of the glomerulus while others penetrated into the cells and bound to nuclei in vivo [38]. Penetration of anti-dsDNA into living murine renal tubular cells in vivo indicated that cellular penetration requires the presence of DNA or the binding of antibodies to a membrane determinant precisely resembling DNA [40]. An additional example is the cross-reacting anti-dsDNA/P-ribosomal protein causing cellular damage [41]. In an additional set of experiments, when radiolabelled murine anti-DNA antibodies were injected into BALB/c mice there was renal deposition of anti-dsDNA antibodies combined with proteinuria [42]. A synthetic peptide with the consensus sequence DWEYS, identified by a peptide phage display library, protected the kidneys from renal deposition of the anti-dsDNA in both BALB/c and SCID mice injected with anti-dsDNA [42, 43]. This peptide binds the anti-dsDNA antibodies in or near the dsDNA binding site [43]. Immunization of naive mice with DWEYS DNA mimetic in a branched form induced the generation of mouse anti-dsDNA associated with renal damage [44]. It has been suggested that cross-reactivity of anti-DNA antibodies plays a central role in the development of lupus nephritis. A case of molecular mimicry between cross-reacting anti-dsDNA antibodies with the glomerular protein -actinin was identified by mass spectroscopy [45].
-Actinin is an actin-binding protein and is a major structural component of glomerular podocytes and mesangial cells, and is known to play a prominent role in several experimental glomerulonephritis models. The anti-
-actinin antibodies were colocalized with in vivo-deposited anti-DNA monoclonal antibodies in the RAG-1-deficient mouse and were deposited in the mesangium of BWF1 kidneys [45]. In the lupus-prone MRL-lpr/lpr mouse and in a non-autoimmune BALB/c mouse subjected to the pathogenic anti-DNA antibody R4A, anti-
-actinin was eluted from the affected kidneys [46].
The consensus sequence Asp/Glu-Trp-As/Glu-Tyr-Ser/Gly (a molecular mimic of dsDNA), was observed also in subunits of the glutamate receptors NR2a and NR2b [47]. Anti-dsDNA antibodies binding the peptide, injected intrathecally into naive mice, could mediate excitotoxic neuronal death [47].
Anti-DNA activity can be evoked indirectly by idiotypic manipulation using anti-DNA antibodies [4858] or by lymphocyte transfer [5961]. Based on Jerne idiotype network theory, immunization with a human anti-dsDNA antibody (Ab1) will evoke anti-idiotypic antibody (Ab2) and later anti-anti-dsDNA antibody (Ab3), which resembles the immunizing Ab1, namely mouse anti-dsDNA antibodies [4850]. Immunization of naive mice with anti-DNA carrying the 16/6 idiotype induced experimental lupus manifesting as leucopenia, increased sedimentation rate, glomerulonephritis and proteinuria [51]. A peptide based on the complementarity-determining region (CDR)1 of a monoclonal murine anti-DNA antibody that bears the common idiotype 16/6Id was synthesized and used to treat 16/6-induced lupus mice [52]. Amelioration of the clinical manifestations of an established experimental lupus correlated with a decrease in TNF- secretion, elevated levels of TGF-ß, and immunomodulation of the Th1- and Th2-type cytokines to levels close to those observed in healthy mice [52]. This peptide is currently at the stage of clinical trials. Other groups were able to repeat this successfully using polyclonal and monoclonal anti-DNA antibodies in mice and rabbits [5356]. Others failed and explained the differences as a dependency on environmental factors [57].
Splenocytes from BWF1 mice, passively transferred to SCID, mice evoked anti-dsDNA generation combined with glomerulonephritis [59]. SCID mice were engrafted either with peripheral blood lymphocytes (PBL) of patients with SLE, or lethally irradiated BALB/c mice radioprotected with bone marrow of SCID mice, to which human PBL were transferred (human/mouse chimera) [60]. The engrafted mice developed anti-dsDNA, proteinuria, human IgG complex deposits as well as deposits of murine complement C3. Treatment with a synthetic peptide derived from the anti-DNA 16/6 CDR1 resulted in significant amelioration of the clinical features of SLE [61].
Data from experimental models point to the possibility of diverse origins for anti-dsDNA antibodies. In addition to an increased number of circulating apoptotic cells and impaired clearance, the possible infectious origin was documented [62, 63]. Cross-reactive antibodies to dsDNA and phosphatidylcholine were raised in mice upon lethal pneumococcal infection and were found to be protective in naive mice on the one hand and to display pathogenic autoreactivity on the other hand [62]. Genetic susceptibility to the effects of B-cell infection with EpsteinBarr virus leads to an increased number of latently infected autoreactive memory B cells, which lodge in organs where their target antigen is expressed, and act there as antigen-presenting cells [63, 64]. When CD4+ T cells that recognize antigens within the target organ are activated in lymphoid organs by cross-reactivity with infectious agents, they migrate to the target organ but fail to undergo activation-induced apoptosis because they receive a costimulatory survival signal from the infected B cells. The autoreactive T cells proliferate and produce cytokines, which recruit other inflammatory cells, with resultant target organ damage and chronic autoimmune disease [63, 64]. Thus, these infected B cells might be a source of long-lived B cells in lupus. Double-transgenic BALB/c mice expressing both the R4A-2b heavy chain and the anti-apoptotic bcl-2 gene in the B-cell compartment were used to study whether bcl-2 overexpression differentially affects the anergic and deleted B cells. The double-transgenic mice (R4A/bcl-2) expressed elevated serum titres of both high- and low-affinity anti-dsDNA antibodies and displayed rescue of autoreactive B cells, which are normally either deleted or anergized. Despite the presence of anti-dsDNA antibodies in their serum, R4A/bcl-2-transgenic mice did not develop nephritis, demonstrating that overexpression of bcl-2 is not by itself sufficient to allow disease progression [65]. Analysis of the lifespan of anti-dsDNA-secreting cells in the spleens of BWF1 mice showed that fewer than 60% of the splenic autoantibody-secreting cells were short-lived plasmablasts, whereas 40% were non-dividing, long-lived plasma cells with a half-life of >6 months. In NZB/W mice and D42 Ig heavy chain knock-in mice, a fraction of DNA-specific plasma cells were also long-lived. Although antiproliferative immunosuppressive therapy depleted short-lived plasmablasts, long-lived plasma cells survived and continued to produce autoantibodies. Thus, long-lived, autoreactive plasma cells are a relevant target for researchers aiming to develop curative therapies for autoimmune diseases [66].
Lupus experimental models, such as the one presented in this issue by Qiao et al., contribute to our understanding of the mechanisms of pathogenicity in this disease. This knowledge is essential for developing new therapeutic modalities for treating lupus patients, a disease with no breakthrough treatment until now.
References
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