Complement and systemic lupus erythematosus

Complement is implicated in the pathogenesis of systemic lupus erythematosus (SLE) in several ways and may act as both friend and foe. Homozygous deficiency of any of the proteins of the classical pathway is causally associated with susceptibility to the development of SLE, especially deficiency of the earliest proteins of the activation pathway. However, complement is also implicated in the effector inflammatory phase of the autoimmune response that characterizes the disease. Complement proteins are deposited in inflamed tissues and, in experimental models, inhibition of C5 ameliorates disease in a murine model. As a further twist to the associations between the complement system and SLE, autoantibodies to some complement proteins, especially to C1q, develop as part of the autoantibody response. The presence of anti-C1q autoantibodies is associated with severe illness, including glomerulonephritis. In this chapter the role of the complement system in SLE is reviewed and hypotheses are advanced to explain the complex relationships between complement and lupus.     

Anti-C1q Antibodies as a Follow-Up Marker in SLE Patients

In cross-sectional studies autoantibodies against complement C1q (anti-C1q) were found to be highly associated with active lupus nephritis. The aim of this retrospective study was to determine the value of anti-C1q as follow-up marker of disease activity and renal involvement in patients with systemic lupus erythematosus (SLE). Fifty-two patients with SLE and a minimum of three anti-C1q measurements during follow-up were analyzed. Anti-C1q levels correlated with global disease activity scores. In subgroup analyses, patients without renal involvement did not show a significant correlation between anti-C1q levels and disease activity. In contrast, in patients with renal involvement, anti-C1q levels correlated well with global disease activity. In addition, a positive correlation with the urine protein-to-creatinine ratio and anti-dsDNA antibody levels as well as a negative correlation with complement levels was observed. Anti-C1q antibodies were found to strongly correlate with parameters of SLE disease activity during follow-up, in particular with regard to renal involvement.     

T cell activation by terminal complex of complement and immune complexes

T cell hyperactivation and complement consumption are prominent features of the immunopathology of systemic lupus erythematosus. Although complement activation is secondary to autoantibodies that form immune complexes (ICs), the trigger for alterations in human peripheral blood T cells is poorly understood. To study the impact (on T cells) of several types of preformed ICs and terminal complement complex, also referred to as C5b-9, we incubated these immune reactants with peripheral blood naive CD4(+) T cells as well as Jurkat cells and analyzed their effects on cellular behavior. We first assembled the C5b-9 in situ on the membrane and observed its assembly primarily on a single site where it promoted aggregation of membrane rafts and recruitment of the CD3 signaling complex. However, C5b-9 alone did not initiate proliferation or commencement of downstream signaling events associated with T cell activation. When T cells were treated with ICs together with nonlytic C5b-9, changes associated with T cell activation by possible antigen engagement then occurred. T cell antigen receptor signaling proteins, including ζ-chain, ZAP-70, Syk, Src, and Lck, were phosphorylated and organized in a synapse-like structure. The cytoskeleton formed F-actin spindles and a distal pole complex, resulting in a bipolar distribution of phosphorylated ezrin-radixin-moesin and F-actin. Furthermore, ICs and nonlytic C5b-9 induced T cell proliferation and IFN-γ production. These results raise the possibility that ICs and the nonlytic C5b-9 modulate T cell-mediated responses in systemic lupus erythematosus and other related chronic inflammatory disorders.     

Fcgamma receptors in the initiation and progression of systemic lupus erythematosus

Systemic lupus erythematosus, a systemic autoimmune disorder, is characterized by the production of autoantibodies to nuclear constituents and inflammatory lesions in multiple organ systems. Although the pathogenesis of the disease is largely unknown, recent studies have suggested that disturbances in apoptosis and/or clearance of apoptotic cells may play an important role in the induction and perpetuation of autoantibody production. When autoantibodies subsequently complex to autoantigens present on apoptotic cells, ligation of Fcgamma receptor will result in inflammation and disease development. Indeed, mice deficient in activating Fcgamma receptors were protected against inflammation in models of immune complex-mediated autoimmune disease, whereas deletion of the inhibitory Fcgamma receptors increased autoantibody production and susceptibility to immune complex-induced inflammation. Additionally, functional polymorphisms in Fcgamma receptors were shown to be associated with development of human systemic lupus erythematosus. This review focuses on the role of Fcgamma receptors in the initiation of autoantibody production, inflammatory handling of immune complexes, and disease development in systemic lupus erythematosus.     

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.     

Characterization of human complement receptor type 2 (CR2/CD21) as a receptor for IFN-alpha: a potential role in systemic lupus erythematosus

Human complement receptor type 2 (CR2/CD21) is a B lymphocyte membrane glycoprotein that plays a central role in the immune responses to foreign Ags as well as the development of autoimmunity to nuclear Ags in systemic lupus erythematosus. In addition to these three well-characterized ligands, C3d/iC3b, EBV-gp350, and CD23, a previous study has identified CR2 as a potential receptor for IFN-alpha. IFN-alpha, a multifunctional cytokine important in the innate immune system, has recently been proposed to play a major pathogenic role in the development of systemic lupus erythematosus in humans and mice. In this study, we have shown using surface plasmon resonance and ELISA approaches that CR2 will bind IFN-alpha in the same affinity range as the other three well-characterized ligands studied in parallel. In addition, we show that IFN-alpha interacts with short consensus repeat domains 1 and 2 in a region that serves as the ligand binding site for C3d/iC3b, EBV-gp350, and CD23. Finally, we show that treatment of purified human peripheral blood B cells with the inhibitory anti-CR2 mAb 171 diminishes the induction of IFN-alpha-responsive genes. Thus, IFN-alpha represents a fourth class of extracellular ligands for CR2 and interacts with the same domain as the other three ligands. Defining the role of CR2 as compared with the well-characterized type 1 IFN-alpha receptor 1 and 2 in mediating innate immune and autoimmune roles of this cytokine should provide additional insights into the biologic roles of this interaction.     

Clearance of dying cells and systemic lupus erythematosus: the role of C1q and the complement system

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease of unknown etiology characterized by the presence of pathogenic high-titer autoantibodies to a diverse group of autoantigens. In 88% of patients, autoantibodies are present an average of 3.3 years before diagnosis. Antinuclear, anti-Ro, anti-La, and anti-phospholipid antibodies appear first, followed by anti-DNA, anti-Smith and anti-ribonucleoprotein. These autoantibodies have features of an antigen-driven, T-cell-dependent immune response. Once present, the course of SLE is characterized by disease flares and autoimmune dysregulation. Programmed cell death (PCD), an essential developmental and homeostatic mechanism, is the preferred physiological death processes for cells as well as an important immune response regulator. Appropriate clearance of apoptotic material completes the PCD process, and is essential for regulating of inflammation and maintaining self-tolerance. Early complement proteins are important in protecting humans against the development of SLE and the protective role of C1q and complement in SLE is mainly related to their role in clearance of dying cells. However, the complement system is also an important ingredient in inflammation, which mediates SLE pathogenesis. Thus, the question remains whether complement factors have either a protective or a destructive role, or a combination of both.     

Autoimmune response to U1 small nuclear ribonucleoprotein (U1 snRNP) associated with cytomegalovirus infection

The induction of autoantibodies to U1 small nuclear ribonucleoprotein (U1 snRNP) complexes is not well understood. We present evidence that healthy individuals with cytomegalovirus (CMV) infection have an increased frequency and quantity of antibodies to ribonucleoprotein, directed primarily against the U1-70k protein. A significant association between the presence of antibodies to CMV and antibodies to the total RNP targeted by the immune response to the spliceosome (to both the Sm and RNP; Sm/RNP) was found for patients with systemic lupus erythematosus (SLE) but not those with mixed connective-tissue disease. CMV thus may play a role in inducing autoimmune responses in a subset of patients with systemic lupus erythematosus.     

Autoimmunity and HCV infection in porphyria cutanea tarda: a controlled study.

Autoimmunity and high rates of autoantibodies have been implicated in the pathogenesis of porphyria cutanea tarda. These abnormalities could be in part virus-induced, since porphyria cutanea tarda in most geographical regions is highly associated with hepatitis C virus infection. We analyzed the link of autoantibodies, autoimmune hepatitis and systemic lupus erythematosus in 111 patients with porphyria cutanea tarda and sex- and age-matched controls (mean age 58+/-13 years) in Germany, a region with a low prevalence of hepatitis C virus infection. Patients with porphyria cutanea tarda displayed lower rates of anti-nuclear antibodies (16/111, 14% vs 28/111, 25%, p<0,05) and of antibodies against smooth muscle (25/111, 23% vs 48/111, 43%, p<0,01), than controls. The percentage of patients with porphyria cutanea tarda with positive anti-HCV was low but significantly higher than in our controls (9/111, 8% vs 0/111, 0%, respectively), (p<0,05). Two patients with porphyria cutanea tarda (2/111, 2%) fulfilled the criteria for systemic lupus erythematosus and not one of 65 patients was found to have clinical autoimmune hepatitis. In the first controlled study of a large cohort of patients with porphyria cutanea tarda no increased prevalence of selected autoantibodies and autoimmune hepatitis was found. However, a higher prevalence of HCV infection and systemic lupus erythematosus in patients with porphyria cutanea tarda was confirmed.     

Detection of autoantibodies against phenylalanyl-, tyrosyl-, and tryptophanyl-tRNA-synthetase and anti-idiotypic antibodies to it in serum from patients with autoimmune diseases]

Sera of patients bearing autoimmune diseases (rheumatoid arthritis and systemic lupus erythematosus) and sera of clinically healthy donors were examined by ELISA for the presence of autoantibodies against tryptophanyl-, tyrosyl- and phenylalanyl-tRNA synthetases. Pure bovine synthetases served as antigens. It was shown that in patients with both autoimmune diseases all three enzyme autoantibodies were revealed at serum dilution 1/1600-1/3200. Moreover, by means of monoclonal antibodies against the same enzymes used for immunoaffinity sorption, antiidiotypic antibodies of IgG type against autoantibodies were detected. A conclusion has been made that autoimmune diseases are characterized by autoimmune response for many aminoacyl-tRNA synthetases irrespectively of their quaternary structure, intracellular location etc both at the level of primary and secondary antibodies.     

Relationship between anti-dsDNA, anti-nucleosome and anti-alpha-actinin antibodies and markers of renal disease in patients with lupus nephritis: a prospective longitudinal study

Introduction  

Glomerulonephritis is a major cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). Deposition of autoantibodies in the glomeruli plays a key role in the development of lupus nephritis (LN). Different groups have proposed that either anti-nucleosome antibodies or antibodies that bind the intrinsic renal antigen, α-actinin, are central to the pathogenesis of LN. These theories have been based mainly on cross-sectional studies in patients and on experiments in animal models. No previous longitudinal studies have compared the relationships between levels of these antibodies and markers of renal function. We assessed how well anti-α-actinin, anti-nucleosome and anti-double-stranded DNA (anti-dsDNA) antibodies reflected renal outcome measures in patients with new-onset LN followed for up to 2 years.     

IgG autoantibodies against deposited C3 inhibit macrophage-mediated apoptotic cell engulfment in systemic autoimmunity

Defective clearance of apoptotic cells has been shown in systemic lupus erythematosus (SLE) and is postulated to enhance autoimmune responses by increasing access to intracellular autoantigens. Until now, research has emphasized inherited rather than acquired impairment of apoptotic cell engulfment in the pathogenesis of SLE. In this study, we confirm previous results that efficient removal of apoptotic cells (efferocytosis) is bolstered in the presence of wild-type mouse serum, through the C3 deposition on the apoptotic cell surface. In contrast, sera from three mouse models of SLE, Mer(KD), MRL(lpr), and New Zealand Black/WF1 did not support and in fact actively inhibited apoptotic cell uptake. IgG autoantibodies were responsible for the inhibition, through the blockade of C3 recognition by macrophages. Consistent with this, IgG removal reversed the inhibitory activity within autoimmune serum, and purified autoimmune IgG blocked both the detection of C3 on apoptotic cells and C3-dependent efferocytosis. Sera from SLE patients demonstrated elevated anti-C3b IgG that blocked detection of C3 on apoptotic cells, activity that was not found in healthy controls or patients with rheumatoid arthritis, nor in mice prior to the onset of autoimmunity. We propose that the suppression of apoptotic cell disposal by Abs against deposited C3 may contribute to increasing severity and/or exacerbations in SLE.     

Human monoclonal multiple-organ-reactive autoantibodies distinguished by mouse monoclonal anti-idiotypic antibodies: expression of idiotopes in humans with and without autoimmune diseases

Previously we reported on the production and characteristics of a number of human monoclonal autoantibodies. All of these autoantibodies were of the IgM class and reacted with antigens in multiple organs. In this study we generated IgG murine monoclonal anti-idiotypic antibodies against five human monoclonal autoantibodies, (i.e., MOR-h2, MOR-h3, MOR-h4, CG1, and CG2). These anti-idiotypic antibodies reacted strongly with the corresponding human monoclonal autoantibody, but minimally or not at all with other human monoclonal autoantibodies. By using these anti-idiotypic antibodies as probes, we screened sera obtained from normal individuals and patients with insulin-dependent diabetes mellitus, Hashimoto's thyroiditis, and systemic lupus erythematosus for the expression of idiotopes. Our study showed that the idiotopes recognized by three of the anti-idiotypic antibodies, i.e., anti-CG1, anti-CG2, and anti-MOR-h2, were not expressed, but the idiotopes recognized by two of the anti-idiotypic antibodies, i.e., anti-MOR-h3 and anti-MOR-h4, were expressed in normal individuals. In patients with autoimmune disorders, there was no increase in the expression of the CG1, CG2, and MOR-h2 idiotopes, but 45 and 23% of the patients with systemic lupus erythematosus showed a significant increase in the expression of the MOR-h3 and MOR-h4 idiotopes respectively. These findings show that there is widespread expression in the B cell repertoire of certain autoantibody-associated idiotopes.     

Complement component C3 is not required for full expression of immune complex glomerulonephritis in MRL/lpr mice

Complement activation and tissue deposition of complement fragments occur during disease progression in lupus nephritis. Genetic deficiency of some complement components (e.g., Factor B) and infusion of complement inhibitors (e.g., Crry, anti-C5 Ab) protect against inflammatory renal disease. Paradoxically, genetic deficiencies of early components of the classical complement pathway (e.g., C1q, C4, and C2) are associated with an increased incidence of lupus in humans and lupus-like disease in murine knockout strains. Complement protein C3 is the converging point for activation of all three complement pathways and thus plays a critical role in biologic processes mediated by complement activation. To define the role of C3 in lupus nephritis, mice rendered C3 deficient by targeted deletion were backcrossed for eight generations to MRL/lpr mice, a mouse strain that spontaneously develops lupus-like disease. We derived homozygous knockout (C3(-/-)), heterozygous (C3(+/-)), and C3 wild-type (C3(+/+)) MRL/lpr mice. Serum levels of autoantibodies and circulating immune complexes were similar among the three groups. However, there was earlier and significantly greater albuminuria in the C3(-/-) mice compared with the other two groups. Glomerular IgG deposition was also significantly greater in the C3(-/-) mice than in the other two groups, although overall pathologic renal scores were similar. These results indicate that C3 and/or activation of C3 is not required for full expression of immune complex renal disease in MRL/lpr mice and may in fact play a beneficial role via clearance of immune complexes.     

A role for the Cr2 gene in modifying autoantibody production in systemic lupus erythematosus

Systemic lupus erythematosus is an autoimmune disease characterized by autoantibody production against nuclear Ags. Recent studies suggest that the Cr2 gene, which encodes for complement receptor (CR)1 and CR2, is important in disease susceptibility. Because the precise disease phenotype related to this gene, in isolation or in relation to other genetic loci, is not known, we analyzed C57BL/6 mice with a targeted mutation in Cr2 (C57BL/6.Cr2(-/-)) with or without a concomitant mutation in Fas (C57BL/6.lpr Cr2(-/-)). The Cr2(null) mutation in a C57BL/6.lpr background markedly increases the serum concentrations of IgG1 and IgG2b and the levels of antinuclear and anti-dsDNA Abs as compared with C57BL/6.lpr controls. There is also a trend for higher concentrations of IgG2a and IgG3. In contrast, isolated deficiencies in either these CRs or Fas have a limited effect in the production of anti-dsDNA Abs. Moreover, the Cr2(null) mutation does not affect other disease manifestations. These findings demonstrate that abnormalities in CR1 and CR2 may be linked to the production of autoantibodies by modifying the effect of other systemic lupus erythematosus susceptibility genes. Phenotypic expression of other disease manifestations need additional Cr2-independent genetic factors.     

Immune Complex Signatures of Patients with Active and Inactive SLE Revealed by Multiplex Protein Binding Analysis on Antigen Microarrays

Systemic lupus erythematosus is characterized by dysfunctional clearance of apoptotic debris and the development of pathogenic autoantibodies. While the complement system is also involved in the disease no attempt has been made to generate a comprehensive view of immune complex formation from various autoantigens. We increased the complexity of autoantibody profiles by measuring the binding of two complement proteins, C3 and C4, in addition to two antibody classes, IgG and IgM, to a collection of autoantigens. These complement components covalently bind to those microarray features where antibodies and other serum components induce complement activation. Using this technology, we compared functional serum antibody profiles of control subjects (n = 31) and patients with lupus erythematosus (n = 61) in the active (n = 22) and inactive (n = 39) phase of the disease. Multivariate analysis was applied to identify contributions of binding data on 25 antigens to the discrimination of the study groups. Receiver operating characteristic analysis was used to portray the discriminative property of each measured parameter for each antigen in pairwise group comparisons. Complement C3 and C4 deposition increased on autoantibody targets in spite of the decreased serum complement concentrations, and decreased on other autoantigens, demonstrating the imbalance of complement function in patients with lupus erythematosus. Our observations confirmed previously known markers of disease and showed that C3 and C4 deposition data were at least as powerful as Ig binding data in separating the study groups.     

Analysis of C4 and the C4 binding protein in the MRL/lpr mouse

Systemic lupus erythematosus is a complement-mediated autoimmune disease. While genetic deficiencies of classical pathway components lead to an increased risk of developing systemic lupus erythematosus, end organ damage is associated with complement activation and immune complex deposition. The role of classical pathway regulators in systemic lupus erythematosus is unknown. C4 binding protein (C4bp) is a major negative regulator of the classical pathway. In order to study the role of C4bp deficiency in an established murine model of lupus nephritis, mice with a targeted deletion in the gene encoding C4bp were backcrossed into the MRL/lpr genetic background. Compared with control MRL/lpr mice, C4bp knockout MLR/lpr mice had similar mortality and similar degrees of lymphoproliferation. There were no differences in the extent of proteinuria or renal inflammation. Staining for complement proteins and immunoglobulins in the kidneys of diseased mice revealed no significant strain differences. Moreover, there was no difference in autoantibody production or in levels of circulating immune complexes. In comparison with C57BL/6 mice, MRL/lpr mice had depressed C4 levels as early as 3 weeks of age. The absence of C4bp did not impact serum C4 levels or alter classical pathway hemolytic activity. Given that immune complex renal injury in the MRL/lpr mouse is independent of Fc receptors as well as the major negative regulator of the classical pathway, new mechanisms for immune-complex-mediated renal injury need to be considered.     

Co-Positivity for Anti-dsDNA, -Nucleosome and -Histone Antibodies in Lupus Nephritis Is Indicative of High Serum Levels and Severe Nephropathy

ObjectiveTo characterize the significance of correlated autoantibodies in systemic lupus erythematosus (SLE) and its complication lupus nephritis (LN) in a large cohort of patients.MethodsClinical data were statistically analyzed in 1699 SLE patients with or without nephritis who were diagnosed and treated during 2002–2013 in the northeast region of China. Reactivity to a list of 16 autoantibodies was detected by the serum test Euroline ANA profile (IgG). Serum titers of the anti-nucleosome autoantibodies were measured by ELISA assays. Kidney biopsies were examined by pathologists. Immune complex deposition was identified by immunohistochemistry stain.ResultsSimultaneous positivity of anti-dsDNA, -nucleosome and -histone antibodies (3-pos) was prevalent in SLE patients with LN compared to Non-renal SLE patients (41% vs 11%, p< 0.001). Significant correlations were found between any two of the above three anti-nucleosome antibodies in LN patients. In comparison to non-3-pos cohorts, 3-pos patients with LN had significantly higher serum levels of the three antibodies and more active disease; was associated with type IV disease; suffered from more severe renal damages; received more intensive treatment and had worse disease outcome. The serum levels of these three autoantibodies in 3-pos LN patients were significantly decreased when they underwent clinical recovery.ConclusionsSimultaneous reactivity to anti-dsDNA, -nucleosome and -histone antibodies by Euroline ANA profile (IgG) may indicate severe nephropathy in patients with SLE.     

Role of type I interferons in the activation of autoreactive B cells

Type I interferons (IFNs) are a family of cytokines involved in the defense against viral infections that play a key role in the activation of both the innate and adaptive immune system. IFNs both directly and indirectly enhance the capacity of B lymphocytes to respond to viral challenge and produce cytotoxic and neutralizing antibodies. However, prolonged type I IFN exposure is not always beneficial to the host. If not regulated properly IFN can drive autoantibody production as well as other parameters of systemic autoimmune disease. Type I IFNs impact B-cell function through a variety of mechanisms, including effects on receptor engagement, Toll-like receptor expression, cell migration, antigen presentation, cytokine responsiveness, cytokine production, survival, differentiation and class-switch recombination. Type I IFNs are also cytotoxic for a variety of cell types and thereby contribute to the accumulation of cell debris that serves as a potential source for autoantigens. Type I IFN engagement of a variety of accessory cells further promotes B-cell survival and activation, as exemplified by the capacity of type I IFNs to increase the level of B-cell survival factors, such as B lymphocyte stimulator, produced by dendritic cells. Therefore, it is not surprising that the loss of expression of the type I IFN receptor can have dramatic effects on the production of autoantibodies and on the clinical features of systemic autoimmune diseases such as systemic lupus erythematosus.     

Lack of chromatin and nuclear fragmentation in vivo impairs the production of lupus anti-nuclear antibodies

Nuclear autoantigens in systemic lupus erythematosus are thought to derive primarily from apoptotic cells, yet there is no direct evidence that interfering with apoptosis impairs the generation of lupus autoantibodies. Here we use a mouse model that lacks the endonuclease caspase-activated DNase (CAD), resulting in an absence of chromatin and nuclear fragmentation during apoptotic cell death. We show that in this mouse, production and release into circulation of chromatin is impaired after exposure to several apoptotic triggers, but that the absence of CAD does not interfere with upstream steps of apoptosis or immune system function. Finally we show that in CAD-mutant mice, impaired lupus autoimmunity is skewed toward known cytoplasmic components, and autoimmunity toward membrane autoantigens is preserved, while autoimmunity toward chromatin and other lupus nuclear targets is severely impaired or absent. We also show, as control, that the induction of experimental autoimmune encephalomyelitis is not affected by the absence of CAD. Thus, our work in vivo strongly suggests that apoptotic molecular steps during cell death generate nuclear autoantigens to sustain the specific autoimmune response in systemic lupus erythematosus.