Detection of masked anti-DNA antibodies in lupus sera by a monoclonal anti-idiotype

We report here the use of a monoclonal anti-idiotype 3I to human anti-DNA antibodies to detect in serum idiotype-positive antigen-binding antibodies lacking DNA-binding activity as measured by conventional antigen binding assays. We studied paired serum samples from 13 patients with systemic lupus obtained at two times in the course of their disease: in each patient, one serum sample has anti-DNA activity and the second serum sample has no anti-dsDNA activity detectable by Millipore filter, ELISA, or Crithidia assay. Reactivity with 3I as detected with a radioimmunoassay (RIA) was present in all 13 sera with anti-dsDNA activity. Six patients showed a decrease in 3I reactivity to normal levels in the second serum sample, in which anti-dsDNA antibodies were not detectable by conventional antigen-binding assays. The other seven patients' second serum sample continued to show elevated 3I reactivity by RIA even though no anti-dsDNA activity was apparent. When the 3I-reactive antibodies from these latter patients' sera were eluted from a 3I affinity column, they revealed DNA-binding activity. Furthermore, dsDNA binding by these sera was apparent when they were displayed on Western blots of isoelectric focusing gels run in 8 M urea and incubated with radiolabeled dsDNA. These results indicate that the 3I anti-idiotype can detect anti-DNA antibodies in some sera of SLE patients that lack anti-DNA activity by ordinary assays. These antibodies may be inhibited in binding dsDNA by excess antigen or autologous anti-idiotype, and their DNA binding activity can be unmasked by procedures promoting immune complex dissociation.     

SLE血清中抗-DNA抗体分离和性质的研究

本文用国产高分子树脂(T)接枝小牛胸腺DNA,通过亲合层析从系统性红斑狼疮SLE患者血清中纯化出抗-ds DNA抗体和抗-ss DNA抗体。酶联免疫吸附分析(ELISA)的研究表明:SLE抗-DNA抗体和DNA结合的差异性很大,是高度非均一性的。抗-ss DNA抗体不仅组成成分比抗-ds DNA抗体复杂,ss DNA/抗-ssDNA亲合能力也明显高于ds DNA/抗-ds DNA。纯化的抗-DNA抗体以IgG类抗体占主导,同时也有其它类型抗体存在(例如IgM等)。抗-ds DNA抗体有较抗-ss DNA抗体高的IgG含量(两者的IgG/IgM分别是7.0和4.0),说明IgG抗-DNA抗体更倾向于同dsDNA结合。     

Cutting edge: a role for B lymphocyte stimulator in systemic lupus erythematosus

Increased levels of B lymphocyte stimulator (BLyS) are associated with systemic autoimmunity in animal models of spontaneous autoimmune disease, and transgenic animals expressing BLyS develop typical autoimmune disease. Here, we demonstrate significant elevations of BLyS in the patients with systemic lupus erythematosus (SLE). The BLyS isolated from the sera of SLE patients had the same m.w. as the natural soluble form and was able to stimulate B cell activation in vitro. Increased BLyS in SLE patients was partially associated with higher levels of anti-dsDNA Ab of the IgG, IgM, and IgA classes, but not associated with the disease activity. Our results suggest that BLyS may be a useful marker for early activation of an autoimmune diathesis and likely plays a critical role in triggering activation of self-Ag-driven autoimmune B cells in human SLE. BLyS may provide an effective therapeutic target in systemic autoimmunity.     

Quantitative analyses of C3b capture and immune adherence of IgM antibody/dsDNA immune complexes

We isolated the IgM fraction from the plasma of an SLE patient with high titer anti-dsDNA antibodies and prepared soluble IgM/dsDNA immune complexes (IC) that fixed C and captured sufficient C3b to bind to human E via their C3b/C4b receptor, CR1 (immune adherence, IA). We used specific 125I-labeled mAb to IgM, C3b, and IgG to measure the stoichiometries of these C-opsonized IC. They contained 10 to 60 C3b and 10 to 30 IgM per PM2 dsDNA, had no detectable IgG, and the vast majority of the C3b was bound to the IgM, and not to the dsDNA. These stoichiometries are in contrast to those we observed for comparable E-bound IC prepared with IgG anti-dsDNA antibodies (100 to 200 C3b, and 200 to 500 IgG). Our results help explain the greater lability of the IgM IC with respect to IA as evidenced by their plasma-mediated release from human E (presumably due to factor I), and confirm previous predictions of a lower density of "packing" of IgM on dsDNA, compared to IgG. The detailed stoichiometry of C3b capture by the IgM IC (typically 1.5 to 3 C3b per IgM) suggests that individual IgM molecules with multiple C3b facilitate IC binding to clusters of CR1. Finally, comparison of the IgM/dsDNA IC with other IgM IC which have been investigated with respect to C activation, and review of the proposed mechanism by which IgM activates C, suggests that the nature of the Ag plays a fundamental role in determining whether or not an IgM IC can activate C and participate in IA.     

Human and murine anti-DNA antibodies induce the production of anti-idiotypic antibodies with autoantigen-binding properties (epibodies) through immune-network interactions

To examine the potential role of immune-network interactions in the production of lupus autoantibodies, normal NZW rabbit antibody responses were analyzed after immunization with one of the following Ig preparations: human lupus serum anti-dsDNA antibodies, human lupus serum anti-ssDNA antibodies, a mixture of human lupus serum anti-dsDNA and anti-ssDNA antibodies, the MRL-lpr/lpr anti-dsDNA mAb H241, and the MRL-lpr/lpr anti-ssDNA mAb H130. Four of five rabbits produced Ig typical of lupus autoantibodies: individual rabbit Ig cross-reacted with multiple autoantigens including nucleic acids, cardiolipin, SmRNP, glomerular extract, laminin, and exogenous Ag. Rabbit anti-Id against human anti-dsDNA antibodies were highly specific for dsDNA. Notably, in each serum the autoantibody activity was confined to the anti-Id Ig fraction. A similar spontaneously occurring Id-anti-Id interaction was also found between anti-ssDNA and anti-dsDNA antibodies isolated from an individual lupus patient. These results indicate that lupus autoantibodies which share Ag binding properties with pathogenic Ig, including both cross-reactive and anti-dsDNA antibodies, can induce the production of Ig with similar autoantigen binding properties through immune-network interactions. This phenomenon, if unregulated, could lead to the amplification of pathogenic autoantibody production in individuals with systemic lupus.     

Early diagnosis of systemic lupus erythmatosus using ANN models of dsDNA binding antibody sequence data

In this paper a new method based on artificial neural networks (ANN), is introduced for identifying pathogenic antibodies in Systemic Lupus Erythmatosus (SLE). dsDNA binding antibodies have been implicated in the pathogenesis of this autoimmune disease. In order to identify these dsDNA binding antibodies, the protein sequences of 42 dsDNA binding and 608 non-dsDNA binding antibodies were extracted from Kabat database and encoded using a physicochemical property of their amino acids namely Hydrophilicity. Encoded antibodies were used as the training patterns of a general regression neural network (GRNN). Simulation results show that the accuracy of proposed method in recognizing dsDNA binding antibodies is 83.2%. We have also investigated the roles of the light and heavy chains of anti-dsDNA antibodies in binding to DNA. Simulation results concur with the published experimental findings that in binding to DNA, the heavy chain of anti-dsDNA is more important than their light chain.     

VH sequence of a human anti-Sm autoantibody. Evidence that autoantibodies can be unmutated copies of germline genes

The utilization of germline genes for the synthesis of autoantibodies has been suspected for many years based on the presence of cross-reactive idiotypes among patients as well as in some healthy first-degree relatives of patients with several autoimmune diseases including SLE. One such system of idiotypes involves anti-Sm antibodies, which are highly specific for SLE. To definitively establish the utilization of germline genes in the Sm system, we produced human-human B cell hybridomas from a patient with SLE who had circulating anti-Sm antibodies. One stable hybridoma designated 4B4 secretes an IgM-kappa mAb that binds Sm and shares idiotypic determinants with other anti-Sm antibodies. A second anti-Sm antibody (3C3), isolated from the same patient was also studied. Oligo(dT) priming was used to produce cDNA corresponding to full length IgM. Sequence analysis revealed that the VH gene segment (1-96) of 4B4 is identical to a VH sequence previously detected in a fetal liver cDNA library by Schroeder and his co-workers as well as a germline VH recently described by Berman and his associates. The identity of a lupus mAb and sequences derived from unrelated individuals provides strong evidence that this autoantibody is a direct copy of a germline gene.     

Stable expression of a recombinant human antinucleosome antibody to investigate relationships between antibody sequence,binding properties,and pathogenicity

When purified under rigorous conditions, some murine anti-double-stranded-DNA (anti-dsDNA) antibodies actually bind chromatin rather than dsDNA. This suggests that they may actually be antinucleosome antibodies that only appear to bind dsDNA when they are incompletely dissociated from nucleosomes. Experiments in murine models suggest that antibody–nucleosome complexes may play a crucial role in the pathogenesis of glomerulonephritis in systemic lupus erythematosus. Some human monoclonal anti-DNA antibodies are pathogenic when administered to mice with severe combined immunodeficiency (SCID). Our objective was to achieve stable expression of sequence-altered variants of one such antibody, B3, in Chinese hamster ovary (CHO) cells. Purified antibodies secreted by these cells were tested to investigate whether B3 is actually an antinucleosome antibody. The pathogenic effects of the antibodies were tested by implanting CHO cells secreting them into SCID mice. Purified B3 does not bind to dsDNA unless supernatant from cultured cells is added, but does bind to nucleosomes. The strength of binding to dsDNA and nucleosomes is dependent on the sequence of the light chain. Mice that received CHO cells secreting wild-type B3 developed more proteinuria and died earlier than control mice that received nonsecreting CHO cells or mice that received B3 with a single light chain mutation. However, none of the mice had histological changes or deposition of human immunoglobulin G in the kidneys. Sequence changes may alter the pathogenicity of B3, but further studies using different techniques are needed to investigate this possibility.     

Ophiopogonin D attenuates the progression of murine systemic lupus erythematosus by reducing B cell numbers

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by immune abnormalities leading to multi-organ damage. The activation of autoreactive B cell differentiation will lead to the production of pathogenic autoantibodies, contributing to the development of SLE. However, the effects of Ophiopogonin D (OP-D) on B cell activation and autoantibody production as well as renal injury in the pathogenesis of SLE remain unclear. MRL/lpr mice, one of the most commonly used animal models of SLE, were intragastrically administered with 5 mg/kg/d OP-D at 17 weeks of age for 3 weeks. The survival rates of mice in each group were monitored for 6 weeks until 23 weeks of age. Proteinuria and serum creatinine levels were measured. Serum levels of immunoglobulin (Ig)G, IgM, and anti-dsDNA autoantibodies were detected by enzyme-linked immunosorbent assay. Numbers of CD19⁺ B cells in the blood, spleen and bone marrow and numbers of splenic germinal center (GC) B cells were calculated by using flow cytometry. OP-D treatment prolonged survival in MRL/lpr mice. OP-D treatment reduced proteinuria and serum creatinine levels as well as mitigated renal pathological alternation in MRL/lpr mice. Furthermore, serum levels of IgG, IgM, and anti-dsDNA autoantibodies were reduced by OP-D treatment. OP-D lessened not only CD19⁺ B cells in the spleen and bone marrow but also plasma cells that secreted anti-dsDNA autoantibodies, IgG and IgM in the spleen and bone marrow. OP-D ameliorated the progression of SLE by inhibiting the secretion of autoantibodies though reducing B cell numbers.     

Anti-dsDNA Antibody Isotypes in Systemic Lupus Erythematosus: IgA in Addition to IgG Anti-dsDNA Help to Identify Glomerulonephritis and Active Disease

Objectives

To evaluate the role of serum IgG, IgM and IgA anti-dsDNA antibody isotypes in the diagnosis of systemic lupus erythematosus (SLE), and their association with clinical features and disease activity, in a large cohort of SLE patients.

Methods

Sera of 200 SLE patients (mean age 34±10.3 years; 26 male and 174 female; median duration of disease 115 months, range 7–378), and of 206 controls, including 19 Sjögren''s syndrome, 27 rheumatoid arthritis, 26 psoriatic arthritis, 15 idiopathic inflammatory myopathies (IIM), 13 systemic sclerosis, 49 infectious diseases and 57 healthy subjects, were tested for anti-dsDNA IgG, IgM and IgA isotypes.

Results

Selecting a cutoff corresponding to 95% specificity, the sensitivity of IgG, IgM and IgA anti-dsDNA antibodies in SLE was 55%, 30% and 49%, respectively; 12.5%, 1% and 7.5% of SLE patients had positive IgG, IgM or IgA isotype alone, respectively. SLE patients with glomerulonephritis showed higher levels of IgA anti-dsDNA (p = 0.0002), anti-dsDNA IgG/IgM (p = 0.001) and IgA/IgM (p<0.0001) ratios than patients without renal disease. No significant associations have been found between anti-dsDNA isotypes and other clinical features. IgA anti-dsDNA (p = 0.01) (but not IgG or IgM) and IgG/IgM ratio (p = 0.005) were significantly higher in patients with more active disease (ECLAM score >4).

Conclusions

The detection of IgA anti-dsDNA autoantibodies seems to improve our ability to diagnose SLE and to define lupus nephritis phenotype and active disease. By contrast, IgM anti-dsDNA antibodies might be protective for renal involvement. These data support the hypothesis that anti-dsDNA antibody class clustering may help to refine SLE diagnosis and prognosis.     

Characterization of warm-reactive IgG anti-lymphocyte antibodies in systemic lupus erythematosus. Relative specificity for mitogen-activated T cells and their soluble products

In addition to previously described cold-reactive IgM anti-lymphocyte antibodies maximally cytotoxic for resting cells at 15 degrees C, sera from patients with systemic lupus erythematosus (SLE) were found to contain a new type of antibody preferentially reactive at physiologic temperatures with mitogen-activated lymphocytes. This antibody lacked specificity for unstimulated lymphocytes, and was shown to be of the IgG class both by indirect immunofluorescence and in immunochemical experiments. Certain SLE sera also contained IgG antibodies with the capacity to develop plaques with mitogen-activated T lymphocyte preparations used in a reverse hemolytic plaque assay, indicating reactivity with products released by activated cells. The elimination of the ability of SLE sera to develop plaques after absorption with viable mitogen-stimulated lymphocytes, but not with resting cells, suggested that these antibodies were directed toward activation "neoantigen(s)" shed from the cell surface membrane. Surface membrane phenotype analyses performed by using a variety of monoclonal antibody reagents indicated that the plaque-forming cells (PFC) detected with SLE sera were activated T lymphocytes not restricted to single OKT4+, OKT8+, or Ia antigen+ subpopulations. Essentially all PFC expressed transferrin receptors. The present data raise the possibility that certain of the interesting effects of anti-lymphocyte antibodies on immunologic function in SLE may be mediated by interactions of these new type(s) of antibodies with activated lymphocytes or their products, rather than through blocking or depletion effects on resting precursor cells.     

Characterization of mouse and human monoclonal antibodies cross-reactive with SLE serum antibodies to guanosine

Two new monoclonal antibodies, one a mouse IgM and the other a human IgM that reacted with guanosine, were compared to human serum antibodies from patients with systemic lupus erythematosus (SLE). The human monoclonal antibody was polyspecific in its binding to the nucleoside bases, whereas the mouse monoclonal antibody was relatively specific for guanosine when compared by using an enzyme-linked immunosorbent assay (ELISA). Neither antibody bound polyguanylic acid or denatured single-stranded (ss) DNA, however. Serum IgG antibodies from seven patients with SLE cross-reacted with the mouse monoclonal antibody and showed considerable specificity for guanosine. In contrast, the human serum IgG antiguanosine antibodies also bound ssDNA but not dsDNA or polyguanylic acid. Serum IgG antibodies to guanosine measured by ELISA from the seven SLE patients had a decreased response when compared to the total serum IgG response to ssDNA, and most of the antibodies that bound guanosine also bound ssDNA. These studies provide new evidence that there are specific IgG antibodies to guanosine in SLE sera that are a small fraction of the antibodies to ssDNA. Further efforts to define the role of these guanosine antibodies in SLE may provide a better understanding of the basic mechanisms responsible for the development of SLE in man.     

Generation and characterization of cloned T helper cell lines for anti-DNA responses in NZB.H-2bm12 mice.

We have previously demonstrated that the introduction of the bm12 mutation into NZB mice results in animals that spontaneously produce high titer IgG autoantibodies to dsDNA. The observation that NZB.H-2bm12 develop lupus although NZB.H-2b control mice do not, provides a unique system to study the role of Th cells in the production of antibodies to dsDNA. We have isolated, in the absence of a known stimulating autoantigen, a series of seven autoreactive T cell clones that provide help in vitro for the production of IgG anti-dsDNA antibodies by syngeneic B cells. The data on these seven cloned T cell lines was compared to two cloned T cell lines specific for keyhole limpet hemocyanin. The seven cloned T cell lines, coined clones 19D, 23G, 410F, 410H, C1, C15, and C52 all show significant help in vitro for production of IgM and IgG antibodies to ssDNA and dsDNA; antibody levels increased 7- to 30-fold compared to cultures without T cells. Clones C1, C15, and C52 were furthered studied and were shown to provide help for IgM antihistone and anti-OVA responses but provided significantly less help for IgG antibodies. In contrast, keyhole limpet hemocyanin-specific cloned T cell lines TK2 and TK5 provided help for IgM antibodies to ssDNA, dsDNA, and histone, but failed to significantly increase IgG antibodies to ssDNA, dsDNA, or histone. The cloned T cell lines were restricted to H-2bm12 and proliferated only in response to APC from NZB.H-2bm12 and B6.C-H-2bm12 but not NZB.H-2b or NZB.H-2d mice; their in vitro helper activity was inhibited by antibodies to class II. All cloned T cell lines expressed Thy-1, CD5, and TCR-alpha/beta. Three of the seven clones used TCR-V beta 4. However, the V beta expression of the four remaining autoreactive T cell clones could not be determined. All of the autoreactive cloned T cell lines produce significant IL-4 but no detectable IL-2 or IFN-gamma. We believe that HPLC-purified peptides eluted from I-Abm12 molecules from APC can potentially provide insight on the putative autoantigen.     

Antibodies elicited in response to EBNA-1 may cross-react with dsDNA

Background

Several genetic and environmental factors have been linked to Systemic Lupus Erythematosus (SLE). One environmental trigger that has a strong association with SLE is the Epstein Barr Virus (EBV). Our laboratory previously demonstrated that BALB/c mice expressing the complete EBNA-1 protein can develop antibodies to double stranded DNA (dsDNA). The present study was undertaken to understand why anti-dsDNA antibodies arise during the immune response to EBNA-1.

Methodology/Principal Findings

In this study, we demonstrated that mouse antibodies elicited in response to EBNA-1 cross-react with dsDNA. First, we showed that adsorption of sera reactive with EBNA-1 and dsDNA, on dsDNA cellulose columns, diminished reactivity with EBNA-1. Next, we generated mononclonal antibodies (MAbs) to EBNA-1 and showed, by several methods, that they also reacted with dsDNA. Examination of two cross-reactive MAbs—3D4, generated in this laboratory, and 0211, a commercial MAb—revealed that 3D4 recognizes the carboxyl region of EBNA-1, while 0211 recognizes both the amino and carboxyl regions. In addition, 0211 binds moderately well to the ribonucleoprotein, Sm, which has been reported by others to elicit a cross-reactive response with EBNA-1, while 3D4 binds only weakly to Sm. This suggests that the epitope in the carboxyl region may be more important for cross-reactivity with dsDNA while the epitope in the amino region may be more important for cross-reactivity with Sm.

Conclusions/Significance

In conclusion, our results demonstrate that antibodies to the EBNA-1 protein cross-react with dsDNA. This study is significant because it demonstrates a direct link between the viral antigen and the development of anti-dsDNA antibodies, which are the hallmark of SLE. Furthermore, it illustrates the crucial need to identify the epitopes in EBNA-1 responsible for this cross-reactivity so that therapeutic strategies can be designed to mask these regions from the immune system following EBV exposure.     

17β-Estradiol enhances response of mice spleen B cells elicited by TLR9 agonist

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies against nucleic acid-associated antigens. B cells play cardinal roles in SLE. Many evidences have proved estrogen contribute to the gender bias in SLE and 17β-estradiol (E2) could accelerate the disease by regulating B cells. On the other hand, B cells express TLR9 which recognized dsDNA and played a critical role in SLE. However, the crosstalk between estrogen and TLR9 in B cells remains unknown. So we investigated the E2 effect in the presence of the TLR9 ligand CpG on mice spleen B cells. We found that the up-regulation of cell viability, life-span, co-stimulation molecules (CD40, CD86) expression, IgM secretion, TLR9 and MCM6 expression were more significant than CpG ODN or E2 stimulated alone. It may provide a new way to investigate the mechanism of how E2 modulate the B cells function in lupus.     

Development of functional B cells in a line of SCID mice with transgenes coding for anti-double-stranded DNA antibody

Deletion or inactivation of anti-self (DNA) B cells has been reported in non-autoimmune mice bearing Ig transgenes that code for Abs with specificity for dsDNA or ssDNA. However, we report a case in which anti-dsDNA B cells appear to escape both deletion and inactivation. We show that B cells (B220+IgM+) can develop in non-autoimmune SCID mice bearing two site-directed transgenes, 3H9(56R) and Vkappa8, that together code for an anti-dsDNA Ab. The B cells appear inactive, because the mice (56RVkappa8 SCID mice) generally lack serum Ig. However, 56RVkappa8 SCID mice are able to produce IgG Ab with specificity for dsDNA when they become "leaky" for T cells or are reconstituted with exogenous T cells from B cell-deficient JH-/- donors. Thus, anti-dsDNA B cells that escape deletion in 56RVkappa8 SCID mice appear fully functional and can differentiate, class switch, and give rise to IgG-producing cells in the presence of T cells and self-Ag.     

A mimic of p21WAF1/CIP1 ameliorates murine lupus

Systemic lupus erythematosus (SLE) is a progressive autoimmune disease characterized by the production of high levels of affinity-matured IgG autoantibodies to dsDNA and, possibly, visceral involvement. Pathogenic autoantibodies result from the activation and proliferation of autoreactive T and B lymphocytes stimulated by epitopes borne by nucleosomal histones. To inhibit the proliferation of autoreactive cells and abrogate the development of SLE, a novel tool, cell cycle inhibiting peptide therapy, was used. Thus, a peptidyl mimic of p21WAF1/CIP1 that inhibits the interaction between cyclin-dependent kinase 4 and type D cyclins abrogated the in vitro proliferative response of T cells to histones and T-independent and T-dependent proliferative responses of B cells. The WAF1/CIP1 mimic also abrogated the in vitro production of total and anti-dsDNA IgG Abs by B cells. Similarly, the p21WAF1/CIP1 construct inhibited the ex vivo T and B cell proliferative responses to histones and decreased the numbers of activated/memory B and T spleen cells. The alterations in the balance of spleen cell subsets resulted from proapoptotic effects of the p21WAF1/CIP1)construct on activated splenocytes. Finally, in vivo, four i.v. injections of the p21WAF1/CIP1 mimic were sufficient to inhibit the progression of the lupus-like syndrome in (NZB x NZW)F1 mice. The levels of anti-dsDNA IgG autoantibodies and the incidence and severity of renal involvement were lower in treated mice than in nontreated mice. Those observations open new avenues for the treatment of SLE and prompt us to evaluate the potential interest of peptidic therapy in human SLE.     

BAFF overexpression promotes anti-dsDNA B-cell maturation and antibody secretion

Overexpression of BAFF is believed to play an important role in systemic lupus erythematosus and elevated levels of serum BAFF have been found in lupus patients. Excess BAFF also leads to overproduction of anti-dsDNA antibodies and a lupus-like syndrome in mice. In the present study, we use mice transgenic for the R4A-Cμ (IgM) heavy chain of an anti-dsDNA antibody, to study the effects of BAFF overexpression on anti-dsDNA B-cell regulation. We observe that overexpression of BAFF promotes anti-dsDNA B-cell maturation and secretion of antibody and enriches for transgenic anti-dsDNA B cells in the marginal zone and follicular splenic compartments. In addition, our data suggests that BAFF rescues a subset of anti-dsDNA B cells from a regulatory checkpoint in the transitional stage of development.     

CD4+ T lymphocytes with constitutive CD40 ligand in preautoimmune (NZB x NZW)F1 lupus-prone mice: phenotype and possible role in autoreactivity

Lupus disease is marked by B lymphocyte hyperactivity and the production of Abs to dsDNA. The production of these anti-dsDNA Abs is T lymphocyte dependent. However, it is not clear how CD4+ T lymphocytes provide help for B lymphocytes to produce IgG anti-dsDNA Abs. One possible mechanism is suggested by studies showing that human patients with systemic lupus erythematosus and lupus mice have increased numbers of CD40 ligand (CD40L)+ T and B lymphocytes. The results described in this study reveal that young, clinically healthy lupus-prone New Zealand Black x New Zealand White F1 (BWF1) mice have naive CD4+ T cells with preformed CD40L. These cells contribute to a brisk response to immunization and to the production of anti-dsDNA Abs. In vitro experiments revealed that CD4+ T cells with preformed CD40L could, upon stimulation, provide antiapoptotic signals for B cells but could not induce proliferation or reduce activation threshold. These results suggest that the direct target cells for the effect of T cells with preformed CD40L in lupus may not be B lymphocytes.     

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.