Pathogenic Anti-DNA Antibodies in SLE: Idiotypic Families and Genetic Origins

We have adopted an idiotypic approach to study the double stranded DNA (dsDNA) binding antibodies of systemic lupus erythematosus (SLE). Three anti-idiotypic reagents, 8.12, 31, and F4, identify cross reactive idiotypes that are each expressed on anti-dsDNA antibodies in the sera of many patients with SLE. These idiotypic antibodies are implicated in the pathogenesis of SLE as they are present in immune complex deposits in the kidneys of patients with SLE glomerulonephritis. The autoantibody associated idiotypes are also expressed on antibodies that do not bind DNA. We are investigating the origin of the pathogenic anti-dsDNA antibodies of SLE by comparing the autoantibodies, the antibodies to foreign antigens, and the myeloma proteins that express each SLE associated idiotype. In conjunction with serological analysis of these idiotypic systems, molecular genetic studies indicate that both the 8.12 and the 31 autoantibody associated idiotypes may be germline encoded, while the F4 idiotype is generated by somatic mutation. The data further suggest that the antigenic specificity of the pathogenic anti-DNA antibodies of SLE is acquired through somatic mutation of germline immunoglobulin genes. By studying the regulation of genes capable of encoding pathogenic autoantibodies, in both SLE patients and non-autoimmune individuals, we may be able to elucidate the pathogenesis of autoimmune disease and begin to design more effective therapeutic interventions.     

Lupus anti-DNA antibodies bearing the 8.12 idiotype appear to be somatically mutated

Anti-DNA antibodies in systemic lupus erythematosus (SLE) sera were analyzed using an antiidiotype designated 8.12 which recognizes a determinant on lambda light chains highly expressed in SLE sera. Eight of ten normal individuals had peripheral blood lymphocytes which produced high-titered 8.12-positive antibodies, following transformation with Epstein Barr virus, implying that the 8.12-reactive sequence originates in the germline gene (GLG). Of 58 SLE sera, 32 contained elevated titers of 8.12-reactive antibodies. Twenty-three of these sera had 8.12-reactive anti-DNA antibodies, suggesting a strong correlation between 8.12 idiotype and DNA binding. Moreover, 20 of 26 8.12-reactive IgG antibodies and only 4 of 10 8.12-reactive IgM antibodies bound DNA (P<0.05). These observations strengthen our previous findings in myeloma sera that DNA binding is associated with IgG isotype in the 8.12 idiotype system and suggest that the acquisition of anti-DNA reactivity in antibodies bearing the GLG idiotype 8.12 is achieved by somatic mutation, a feature of an antigen-driven response.     

Shared idiotypes are expressed on mouse and human anti-DNA autoantibodies.

The expression of common idiotypes on human and mouse anti-DNA monoclonal autoantibodies made by hybridomas was examined by their competitive binding to anti-idiotype antibodies. Some murine autoantibodies inhibited the binding of a human anti-DNA autoantibody 16/6 to monoclonal or polyclonal anti-idiotypic antibodies. Another human antibody (134) was not inhibited in its binding to homologous anti-idiotypic antibodies. The expression of the human 16/6 idiotype on mouse antibodies was restricted to those that had a specificity similar to the 16/6 antibody itself, their major properties being that they reacted more strongly with single stranded DNA (ssDNA) than double stranded DNA (dsDNA). One mouse antibody expressing the 16/6 idiotype also bound weakly to RNA. The results imply structural similarities between the binding sites of the antibodies in the two species, and are consistent with evolutionary conservation of V genes coding for primitive ancestral antibodies that react with DNA and become diversified through somatic mutation.     

Antinuclear Autoantibodies in Flaky Skin ( fsn ) Mutant Mice

Mice homozygous for the flaky skin ( fsn ) single gene mutation have a severe hyperproliferative disease resulting complex phenotype, which includes widespread inflammation and autoimmunity. Flaky skin mice have several serological and pathological features that share similarities with the human systemic autoimmune disease systemic lupus erythematosus (SLE). Analyses of the antinuclear and anti-dsDNA autoantibodies in fsn / fsn mice indicate that they are low titer IgG antibodies. These low titer anti-dsDNA autoantibodies can ultimately form immune complex deposition in the glomeruli associated kidney damage. IgE antibodies were identified in the immune complex deposition, however their role in the pathology is not determined. It is hypothesized that the mechanism of autoantibody production and autoimmune disease pathogenesis in mice homozygous for the fsn mutation is initiated by non-specific polyclonal activation of B-lymphocytes resulting in the synthesis of low affinity autoantibodies.     

Idiotypic heterogeneity of VKIII autoantibodies to red blood cell antigens.

VKIII light (L) chains are commonly expressed by human autoantibodies with diverse binding specificities, including red blood cell antigens. To better understand the physiologic and pathologic expression of these L chain variable region genes, we have created a panel of murine monoclonal anti-idiotypic antibodies by immunization with a human lymphoblastoid B cell line that secretes an IgM VKIII autoantibody specific for the I red blood cell carbohydrate determinant. The binding specificities of these nine murine monoclonal antibodies, termed IV.1-IV.9, were evaluated against a large panel of monoclonal Ig proteins and compared to two previously well-characterized monoclonal anti-idiotypes, 6B6.6 and 17.109; these two anti-idiotypes have been shown to primarily identify VKIII rheumatoid factors derived from the kv328 (VKIIIa) and kv325 (VKIIIb) genes, respectively. In contrast, our anti-idiotypic antibodies identified (public) cross-reactive idiotypes present on many VKIII proteins that included both anti-erythrocyte and rheumatoid factor autoantibodies. Certain anti-idiotypic antibodies (IV.2 and IV.6) were restricted to VKIIIa L chains but differed from the 6B6.6 anti-idiotype by binding to a larger subset of VKIIIa proteins representing the products of at least two VKIIIa genes. One antibody of our panel (IV.5) recognized a private idiotope expressed only by the immunizing antibody. Using the panel of anti-idiotypic antibodies to evaluate erythrocyte autoantibodies with different serologic specificities, we found striking heterogeneity of L chain idiotype expression, even among known VKIII anti-i/I autoantibodies. These findings differ from the recently described structural and idiotypic conservation associated with the H chain of anti-i/I autoantibodies. From correlations of idiotypic reactivity with L chains of known sequence, it is postulated that the observed heterogeneity of L chain idiotype expression is due to differences in the genetic origin and/or somatic diversification of L chain variable region genes. Furthermore, subtle variability of L chain structure may contribute in part to the differences in fine binding specificity among anti-I and anti-i autoantibodies.     

Antinuclear autoantibodies in flaky skin (fsn) mutant mice

Mice homozygous.for the flaky skin (fsn) single gene mutation have a severe hyperproliferative disease resulting complex phenotype, which includes widespread inflammation and autoimmunity. Flaky skin mice have several serological and pathological features that share similarities with the human systemic autoimmune disease systemic lupus erythematosus (SLE). Analyses of the antinuclear and anti-dsDNA autoantibodies in fsn/fsn mice indicate that they are low titer IgG antibodies. These low titer anti-dsDNA autoantibodies can ultimately form immune complex deposition in the glomeruli associated kidney damage. IgE antibodies were identified in the immune complex deposition, however their role in the pathology is not determined. It is hypothesized that the mechanism of autoantibody production and autoimmune disease pathogenesis in mice homozygous for the fsn mutation is initiated by non-specific polyclonal activation of B-lymphocytes resulting in the synthesis of low affinity autoantibodies.     

Parallelism of serum anti-F(ab')2 and anti-cationic IgG reactivities in patients with systemic lupus erythematosus

Cationic anti-DNA antibodies may be related to glomerular injury in murine lupus nephritis or in patients with systemic lupus erythematosus (SLE). Therefore, anti-cationic antibodies in SLE could include antibodies with regulatory function on such pathogenic cationic molecules. Since anti-F(ab')2 antibodies may be involved in the idiotype control of anti-DNA antibodies in some patients with inactive SLE, the present study was aimed to determine if SLE patients with significant serum levels of anti-F(ab')2 produce antibodies reacting with cationic IgG molecules. Three SLE sera with high titers of anti-F(ab')2 antibodies were individually adsorbed by sequential affinity chromatography on three Sepharose columns coupling normal IgG from Cohn Fraction II, pooled cationic IgG myeloma paraproteins displaying idiotypic anti-DNA markers (F4 and 8.12), and F(ab')2 fragment from allogeneic IgG, respectively. Eluates obtained from cationic IgG adsorption showed predominant anti-F(ab')2 reactivity. A similar profile was also detected in a serum from a normal control donor with high levels of anti-F(ab')2. Biotinylation of anti-cationic eluates showed that such antibodies were significantly more reactive with cationic than anionic or neutral IgG, confirming their apparent affinity for positively charged antigens on IgG molecules. Since anti-cationic absorptions were able to remove the anti-F(ab')2 activities in the SLE sera studied, it is possible that anti-cationic antibodies could function as immunoregulatory antibodies in the idiotypic control of some SLE autoreactive phenomena, including glomerular anti-DNA deposition.     

Pathogenic anti-DNA idiotype-reactive IgG in intravenous immunoglobulin preparations.

This study was addressed to explore the reactivity of natural anti-idiotypes from commercial lots of immunoglobulins to several idiotypes (Ids), usually expressed by anti-DNA molecules in lupus nephritis. Eleven intravenous immunoglobulin (IVIG) preparations and nine (three polyvalent and six hyper-immune) intramuscular IgG were investigated for specific content of anti-DNA, anti-F(ab')2 and antibodies reacting with several anti-DNA IgG Ids. Two samples (nos 6 and 11) showed high reactivity with allogeneic F(ab')2 and with F(ab')2 of myeloma proteins bearing the anti-DNA Id 3I+ and the 8.12+. Since both 3I and 8.12 Id markers are known to characterize pathogenic anti-DNA IgG in systemic lupus erythematosus (SLE), anti-Id antibodies to these markers were obtained by absorbing the IVIG samples nos 6 and 11 to Sepharose columns coupled with pooled F(ab')2 fragments of 3I(+)-F4(+)-8.12(+)-myeloma proteins. Inhibition experiments showed that anti-8.12 Id-eluted IgG induced a selective suppression of the DNA-reactive antibodies derived from patients with active lupus nephritis to their substrate, suggesting the involvement of 8.12+ molecules in the SLE glomerular damage. Since 8.12+ anti-DNA are nephritogenic antibodies, the occurrence of anti-8.12+ Id in commercial IVIG may be of potential therapeutic relevance in modulating the pathogenic SLE Id network. Previous variable results of IVIG treatment in SLE, such as resolution of proteinuria or worsening nephritis, could be related to variable enrichment of different lots of IVIG in suppressive anti-pathogenic Id antibodies.     

Expression of an interspecies idiotype in sera of SLE patients and their first-degree relatives.

Sera from 29 SLE patients and 81 first-degree healthy family members were tested for quantitative expression of a cross-reactive idiotype present on a murine monoclonal anti-Sm autoantibody (Y2). Forty-one percent of SLE patients and 27% of all relatives showed increased serum levels of the Y2 idiotype compared to 6% in a normal, unrelated control group. In addition, female relatives of SLE patients showed slightly increased levels of anti-Sm antibodies compared to male relatives (15% vs 3%). In one of the 28 families and three unrelated SLE patients studied, there was a significant correlation between the Y2 idiotype expression and expression of another idiotype present on anti-DNA antibodies (1341d). Affinity column absorption studies showed that these two idiotypes were present on different antibody molecules. This study demonstrates: (1) a genetic predisposition for an anti-Sm antibody idiotype expression in humans; and (2) that two different idiotypes may be under parallel or coordinate regulation.     

Idiotypic spreading promotes the production of pathogenic autoantibodies

We propose that a major immunoregulatory abnormality in murine and human autoantibody-mediated disease is idiotypic spreading. By this mechanism, B cells with the genetic information to produce immunoglobulin (Ig) bearing certain public idiotypes (Ids) are selectively upregulated, probably by Id-recognizing helper T cells. The model in which we are testing the hypothesis is systemic lupus erythematosus (SLE) in humans and NZB/NZW F1 (BW) female mice. Recent experiments have shown that the number of public Ids expressed on the Ig of nephritic BW mice is quite restricted. IdX is the dominant Id on serum Ig; IdGN1 and IdGN2 are also common. All three Ids were initially derived from spontaneous antibodies to DNA. Together the three are present on 85% of the total Ig repertoire. Such restriction suggests idiotypic spreading. In glomerular Ig deposits from nephritic BW mice, IdGN1 and IdGN2 are found on 45% of the total Ig: IdX is present in minute amounts. Furthermore, suppression of IdGNs by administration of anti-IdGN1 to BW mice resulted in significant delay in the onset of nephritis, but the IdGNs escaped from control and eventually caused a fatal nephritis. Finally, studies of glomerular Ig deposits in renal biopsies of patients with SLE have shown that IdGN2 dominates such Ig, being present in 76% of renal biopsies from SLE patients and in 6% from patients with non-lupus immune nephritis. Therefore, we have concluded that IdGN1 and IdGN2 are markers of nephritogenic subsets of autoantibodies and are probably the products of idiotypic spreading most likely to cause disease. Finally, after a review of recent experiments suggesting the dominance of autoreactive, Mossman Type 2 T helper cells in nephritic BW mice, it is hypothesized that autoreactive, IdGN-recognizing helper T cells may be central to the sustained upregulation of pathogenic autoantibodies in murine and human SLE.     

Anti-alpha-actinin antibodies: a new marker of lupus nephritis

The exact role of anti-ds (double stranded) DNA antibodies in the pathogenesis of kidney injury in lupus nephritis remains a focus of continuing investigation. One theory explaining the pathogenicity of anti-dsDNA antibodies in lupus nephritis is direct cross-reactivity with renal antigens. Several years ago, alpha-actinin was identified as a major cross-reactive target for pathogenic anti-dsDNA antibodies in murine SLE. Indeed, binding of a nephritogenic murine anti-dsDNA antibody was stronger to the alpha-actinin derived from a lupus prone mouse mesangial cell line as compared to alpha-actinin in a non-autoimmune mouse mesangial cell line. Furthermore, we recently showed that immunization of non-autoimmune mice with alpha-actinin induces anti-chromatin antibodies, glomerular IgG deposition and proteinuria. In humans, anti-alpha-actinin autoantibodies (Ab) were associated with anti-dsDNA Ab in SLE. In those patients, anti-alpha-actinin rather than anti-dsDNA Ab were significantly associated with glomerulonephritis and disease activity. The anti-alpha-actinin reactivity was associated with high avidity anti-dsDNA Ab. Moreover, the anti-alpha-actinin response was related to the actin-binding site of alpha-actinin. Taken together, these studies indicate that detection of anti-alpha-actinin Ab, in association with anti-dsDNA Ab, may constitute a new marker in lupus nephritis.     

DNA Antibody Idiotypes: An Analysis of Their Clinical Connections and Origins

Approximately thirty common DNA antibody idiotypes have been described on hybridoma derived or affinity purified DNA-binding antibodies. There are associations between some idiotypes and the clinical manifestations of systemic lupus erythematosus although none are sufficiently firm to be clinically useful in identifying subsets of SLE or in assessing disease activity in individual patients. The expression of these idiotypes is not confined to DNA antibodies in SLE. They may be found in the serum from patients with a range of autoimmune rheumatic disorders, infectious diseases and blood dyscrasias. In most cases the antigen binding specificity of the antibody bearing the idiotype is unknown. The precise relationship between the various idiotypes is becoming better understood with increasing availability of genetic and structural data. DNA antibody idiotype manipulation may provide a potential new therapeutic modality in SLE.     

Immunization with peptides derived from the idiotypic region of lupus-associated autoantibodies delays the development of lupus nephritis in the (SWR x NZB)F1 murine model

Systemic lupus erythematosus (SLE) is a multiorgan autoimmune disease affecting 40-50/100,000 Americans. Although most of the research on pathogenic antibodies focuses on antigenic specificity, there is increasing evidence that specific immunoglobulin idiotypes may mediate lupus nephritis independent of autoantigen specificity. In previous work, our laboratory characterized a set of nephritogenic monoclonal antibodies with substantial idiotypic cross-reactivity, produced by the spontaneous SLE model (SWR x NZB)F(1) (SNF(1)), termed Id(LN)F(1). Peptides derived from one of these antibodies, Id540, was previously shown to stimulate pathogenic T-cells from prenephritic SNF(1) mice, similar to what has been seen for pathogenic A6.1 antibody produced by the (NZB x NZW)F(1) model. In this study, we immunized pre-nephritic SNF(1) mice with p62-73, a peptide derived from the variable region of Id540 and, in separate experiments, with p58-69, a peptide derived from the variable region of A6.1. In both cases, immunization resulted in increased survival and delayed nephritis; however, while both peptides affected levels of anti-DNA antibodies, immunization with p62-73 only affected levels of Id(LN)F(1) antibodies. These findings confirm the roles of pathogenic idiotypes in the pathogenesis of lupus nephritis and suggest that therapies that target specific idiotypes might be a potential tool in the management of SLE.     

A common anti-DNA idiotype and other autoantibodies in sera of offspring of mothers with systemic lupus erythematosus.

Since the immune response in fetuses of mothers with systemic lupus erythematosus (SLE) is unknown, we investigated sera from six mothers and their paired offspring by enzyme-linked immunosorbent assay (ELISA) for the presence of a common anti-DNA idiotype (16/6 Id) and, as control, for the presence of an unrelated public idiotype of antibody to hepatitis B surface antigen (HBsAg). In addition, maternal as well as fetal sera were evaluated for the presence of antibodies to ssDNA, dsDNA, poly(I), poly (dT), RNA, cardiolipin, total histones and the presence of lupus anticoagulant. Clinically active SLE mothers showed in general increased IgG and, to a lesser extent, IgM autoantibody activity. Circulating lupus anticoagulant was detectable in clinically active mothers only. All offspring of clinically active SLE mothers showed increased IgG autoantibodies to a variety of antigens, while IgM antibodies were detected in only one fetus. In contrast, fetuses of clinically inactive mothers showed only minor IgG activity. Common anti-DNA-idiotype (16/6 Id) activity also correlated with disease activity in both maternal and fetal compartments. One clinically active mother was 16/6-negative; her offspring was, however, positive, indicating de novo production of the idiotype by the fetus. In contrast, a control anti-HBsAg idiotype was not detected in either maternal or fetal sera. It therefore appears that offspring of clinically active SLE mothers serologically reflect maternal disease activity. Furthermore, autoantibodies and common idiotype of autoantibodies can be found within the fetal compartment even in the absence of such antibodies in the maternal serum. Discrepancies between mothers and offspring in IgM-autoantibody levels and the presence of new idiotypes in fetuses are indicative of fetal de novo autoantibody production.     

Cloning and functional analysis of anti-double strand DNA IgG autoantibodies using the phage-display method

To examine the relationship between pathophysiological effects and molecular features of anti-double-stranded (ds)DNA autoantibodies (Abs), we isolated anti-dsDNA Ab fragments by using the phage-display method. Fd ? and light-chain DNA were PCR amplified from peripheral blood lymphocytes of a patient with systemic lupus erythematosus (SLE), complicated with lupus nephritis. They were then inserted into a phagemid vector, pComb3-H. We generated eight Fab fragments that specifically bound to solid-phase DNA. The Fab clones stained positively using Crithidia luciliae, indicating that they were anti-dsDNA antibodies. Nucleotide sequences of VH of Fab clones were very similar and appeared to be derived from VH26 germline gene. Differences in the activities of anti-dsDNA Ab of the Fab clones may be ascribed to the diversity of VL. Fab fragments with anti-dsDNA Ab activities exhibited a positive charge on isoelectric focusing, consistent with pathogenic features. Our results indicate that anti-dsDNA Ab Fab fragments obtained by the phage-display method in the present study may possess molecular and functional characteristics of pathogenic anti-dsDNA autoAbs in SLE.     

DNA antibody idiotypes: an analysis of their clinical connections and origins

Approximately thirty common DNA antibody idiotypes have been described on hybridoma derived or affinity purified DNA-binding antibodies. There are associations between some idiotypes and the clinical manifestations of systemic lupus erythematosus although none are sufficiently firm to be clinically useful in identifying subsets of SLE or in assessing disease activity in individual patients. The expression of these idiotypes is not confined to DNA antibodies in SLE. They may be found in the serum from patients with a range of autoimmune rheumatic disorders, infectious disease and blood dyscrasias. In most cases the antigen binding specificity of the antibody bearing the idiotype is unknown. The precise relationship between the various idiotypes is becoming better understood with increasing availability of genetic and structural data. DNA antibody idiotype manipulation may provide a potential new therapeutic modality in SLE.     

单克隆抗DNA自身抗体及其抗血清独特型交叉反应

由严重存在SLE,并无需外源免疫能自发产生抗DNA自身抗体的小鼠MRL-Lpr/Lpr脾细胞与SP2/0-Ag-14骨髓瘤细胞杂交,获得一系列杂交瘤细胞株,它们分别分泌单克隆抗DNA自身抗体。我们用其中的细胞株H241的单抗免疫家兔,从免疫血清中收获到抗H241独特型扰体。通过放射免疫分析法(RIA),对H241进行了敏感度分析,并研究了H241与抗H211血清反应中,H241与另24个抗DNA单抗独特型交叉反应。结果表明,H241是高敏感度的单抗,6ng非标记H241能使~(125)I-H241与抗H241独特型抗体的结合达50%抑制。实验还证明,24个抗DNA单抗中有15个与H241不同程度地存在独特型交叉反应。实验提示,虽然抗DNA自身抗体独特型往往结构分离,从而表现抗体多样性,但有些抗体由于独特型结构类似,多样性受到限制;全身性红斑狠疮小鼠控制免疫球蛋白合成的胚系基因含有某些抗DNA抗体的相关位点。     

SLE患者血清抗F(ab′)_2片段抗体的测定及其临床意义

用酶解法制备正常人 IgG-F(ab')_2片段及 SLE 患者血清抗 ds-DNA-F(ab')_2片段,ELISA 法检测同一组(46例)SLE 患者血清中抗 F(ab')_2片段抗体,两者检测结果呈高度相关性.同时发现,缓解期 SLE 患者血清中抗 F(ab')_2片段抗体水平明显高于活动期 SLE 患者,而抗 ds-DNA 抗体水平与之相反.提示,正常人 IgG-F(ab')_2片段与 SLE 患者血清抗 ds-DNA-F(ab')_2片段可能有共同的结构表位,其抗 F(ab')_2片段抗体测定可作为临床观察 SLE 活动性的一个指标.     

An analysis of autoimmunity through studies of DNA antibody idiotypes

The existence of idiotypic networks, first postulated over 12 years ago, is now widely recognised. Idiotypic analyses of autoantibodies have been reported among both hybridoma-derived and naturally occurring immunoglobulins. In this review the many studies of idiotypes detected on anti-DNA antibodies, notably one designated 16/6, are analysed to see what clues they offer to our understanding of autoimmunity. The links between infection and autoimmunity are emphasised by this analysis. It is also obvious that idiotypes first identified an autoantibodies are not confined to these immunoglobulins. Thus, the 16/6 idiotype originally described on a hybridoma-derived monoclonal anti-DNA antibody has also been identified on naturally occurring antibodies binding the Klebsiella polysaccharide K30.     

Antiidiotypic suppression of autoantibodies with normal polyspecific immunoglobulins

A mechanism by which therapeutic normal polyspecific immunoglobulins (IVIg) may suppress autoimmune responses in vivo is that of antiidiotypic suppression of autoantibodies mediated by anti-idiotypes present in IVIg. In vitro incubation with IVIg of either the plasma or the IgG fraction from plasma of patients with autoantibodies against procoagulant factor VIII (VIII:C), DNA, thyroglobulin, peripheral nerve and intrinsic factor resulted in dose-dependent inhibition of autoantibody activity. The pattern of inhibition curves showed a prozone phenomenon. Maximal inhibition was achieved at a ratio of patient's IgG to IVIg which was specific for each antibody tested. Inhibition was dependent on idiotypic/antiidiotypic interactions between autoantibodies and IVIg since: 1) F(ab')2 from IVIg inhibited autoantibody activity in F(ab')2 fragments from patients' IgG; 2) IVIg contained no antigen-like activity and no antibodies against the commonest allotypes expressed in F(ab')2 fragments of human IgG; 3) autoantibody activity in F(ab')2 fragments from patients' IgG was specifically retained on affinity columns of Sepharose-bound F(ab')2 fragments from IVIg. The presence of antiidiotypes against autoantibodies in pooled normal IgG supports the concept of a functional idiotypic network regulating autoimmune responses in man.