anti-DNA Ab Research Articles

Neonatal peptide exposure can prime T cells and, upon subsequent immunization, induce their immune deviation: implications for antibody vs. T cell-mediated autoimmunity.

Neonatal exposure to antigen is believed to result in T cell clonal inactivation or deletion. Here we report that, contrary to this notion, neonatal injection of BALB/c mice with a hen egg lysozyme peptide 106-116 in putative "tolergenic" doses induced a T cell proliferative and an immunoglobulin G (IgG) antibody (Ab) response of both T helper cell 1 (Th1)- (IgG2a, IgG2b, and IgG 3) and Th2-dependent (IgG1) isotopes. Upon subsequent challenge with the peptide in complete Freund's adjuvant in adult life, although this neonatal regimen suppressed proliferation and the production of Th1 cytokines (interleukin[IL]-2 and interferon gamma), Th2 cytokine (IL-5, IL-4, and IL-10) secretion was increased, and the serum levels of Th1- and Th2-dependent isotypes of peptide-specific Ab remained elevated. The in vitro proliferative unresponsiveness in Th1 cells could be reversed by Abs to Th2 cytokines (IL-4 and IL-10). Thus, neonatal treatment with a peptide antigen induces T cell priming including production of IgG Abs of both Th1- and Th2-dependent isotypes. Upon subsequent peptide exposure, the peptide-specific T cell responses undergo an effective class switch in the direction of Th2, resulting in T cell proliferative unresponsiveness. Accordingly, this shift towards increased Ab production to autoantigen could be deleterious in individuals prone to antibody-mediated diseases. Indeed, neonatal treatment with a self-autoantigenic peptide from an anti-DNA monoclonal Ab (A6H 58-69) significantly increased the IgG anti-double-stranded DNA Ab levels in lupus-prone NZB/NZW F1 mice, despite suppressing peptide-specific T cell proliferation. This adverse clinical response is in sharp contrast to the beneficial outcome of neonatal treatment with autoantigens in Th1-mediated autoimmune diseases, such as autoimmune encephalomyelitis, as reported by others. A Th1 to Th2 immune deviation can explain the discordant biological responses after the presumed induction of neonatal tolerance in autoantibody- vs. Th-1 mediated autoimmune diseases.

Light chain contribution to specificity in anti-DNA antibodies.

We studied mice expressing one of two H chain transgenes. Both transgenes expressed the same 3H9 anti-DNA VDJ, but differed in their constant domains. The IgM transgene efficiently induced tolerance and selected for a subset of endogenous L chains that prevented dsDNA binding. In contrast, the IgG2b secreted-only H chain allowed expression of a broad range of L chains, most of which yielded anti-dsDNA Ab. To deduce the features of L chains that affect DNA binding, we derived hybridomas from LPS-stimulated splenic B cells from the two transgene lines and compared the V kappa sequences of Ab they secreted. Identification of L chains with related sequences but different binding to ssDNA, dsDNA, and cardiolipin allowed us to pinpoint L chain residues that correlate with enhanced or reduced binding. Arginines at the junction of V kappa 1 or V kappa 8 regions and J kappa 1, and arginines or asparagines in CDR1 or CDR2 enhanced DNA binding. Negatively charged residues at the same positions were found to interfere with binding. Thus, we predict that appropriate amino acids at these positions may form contacts with DNA. The likely locations of contact residues in the combining site were evaluated by inspection of previously determined Ab structures. Our results indicate that L chains in anti-DNA Ab are able to modulate DNA binding and contribute contact sites for additional determinants on a complex autoantigen.

Constitutive secretion of transgene-encoded IgG2b autoantibodies leads to symptoms of autoimmune disease.

Anti-DNA Ab are strictly regulated, except in autoimmunity, where they are expressed and may contribute to pathogenicity. To study constitutive anti-DNA Ab secretion in nonautoimmune mice, two anti-dsDNA H/L chain transgene combinations were constructed using an IgG2b C region with secretory but no transmembrane domain exons. One H/L combination, consisting of the VH3H9 H and V kappa 4 L chain transgenes, was chosen to recreate 3H9, an autoantibody that originally arose in an autoimmune MRL/lpr mouse; the other paired a higher affinity variant of VH3H9, 56R, with the same V kappa 4 L chain. Elevated titers of IgG2b along with normal levels of other isotypes were observed in transgene-positive mice, indicating that constitutive transgene-directed Ab secretion was achieved. Sera and hybridoma supernatants from VH3H9 gamma transgene-positive animals exhibited binding to dsDNA, ssDNA, and cardiolipin. Mice expressing the 56R gamma H chain and the V kappa 4 L chain showed enhanced binding. Expression of the transgenes correlated with signs of autoimmune disease, including prolonged plasma clotting in vitro, and reduced litter size. The results suggest that even a single autoreactive H chain that escapes tolerance may suffice to induce features of autoimmune disease.

Preferential recognition of specific DNA motifs by anti‐double‐stranded DNA autoantibodies

Although antibodies (Ab) specific for double-stranded (ds) DNA are thought to be involved in the etiopathogenesis of systemic lupus erythematosus (SLE), the fine structure of their DNA targets remains elusive. We have adapted a polymerase chain reaction (PCR)-assisted immunoprecipitation method to define the binding sites in DNA sequences recognized by high affinity anti-dsDNA Ab of SLE patients. SLE sera were used to bind templates from a pool of double-stranded oligonucleotides (ON). A central part of 20 base-pair random sequence was flanked by restriction endonuclease recognition sites and sequences complementary to predefined PCR primers. Immunoselected ON were precipitated, isolated from the immune complexes and then subjected to a further immunoprecipitation step after amplification by PCR. After five cycles of immunoprecipitation and PCR, the resulting ON were cloned. Sequence analysis revealed that sera from SLE patients and two human monoclonal anti-dsDNA Ab obtained from SLE patients preferentially select sequences expected to form non-B-DNA structures. Inhibition studies of the Farr assay confirmed the increased affinity of the selected epitopes for anti-DNA Ab as compared to random B-DNA.

CD4+ T cells determine the ability of spleen cells from F1 hybrid mice to induce neonatal tolerance to alloantigens and autoimmunity in parental mice

Spleen cells from F1 hybrid mice injected into newborn parental mice induce a state of cytolytic unresponsiveness to the corresponding alloantigens. However, these mice develop a transient autoimmune syndrome characterized by the production of multiple autoantibodies and glomerulonephritis. Previous reports indicated that the depletion of F1 donor T cells, shortly prior the injection into parental mice, does not interfere with any of these events. Here, we have explored whether the continuous absence of T cells in F1 mice influences the ability of their spleen cells to induce neonatal tolerance to alloantigens and the associated autoimmune manifestations. Our results revealed that spleen cells from athymic (BALB/c x C57BL/6) F1 hybrid (CB6F1) nu/nu mice or from euthymic CB6F1 mice depleted from birth of CD4+ T cells, but not of CD8+ T cells, are unable to induce neonatal tolerance to alloantigens and autoimmune manifestations. By contrast, the partial reconstitution of T cells in CB6F1 nu/nu mice, after the neonatal graft of a syngeneic thymus, restored the capacity of spleen cells from these mice to induce tolerance and autoimmunity when injected into newborn BALB/c mice. These results demonstrate that the functional defect of spleen cells from athymic CB6F1 nu/nu mice to induce neonatal tolerance to alloantigens is directly related to the long-term absence of mature CD4+ T cells. Interestingly, a new increase in the titers of anti-DNA Ab was observed when spleen cells from athymic CB6F1 nu/nu mice were injected into adult BALB/c mice that had been tolerized at birth with normal CB6F1 spleen cells. This finding indicates that B cells from CB6F1 nu/nu mice recover their capacity to interact with alloreactive Th2 cells when they are placed into mice having functional CD4+ T cells. These data indicate that the continuous absence of CD4+ T cells causes a reversible functional defect in F1 spleen cells that determines their inability to induce neonatal tolerance and autoimmunity.

Periocular inflammation in mice with experimental systemic lupus erythematosus. A new experimental blepharitis and its modulation.

Experimental systemic lupus erythematosus (SLE) can be induced in mice by immunization with a human monoclonal anti-DNA Ab, bearing a major Id 16/6Id. Immunized mice initially produce Abs to 16/6Id, DNA and nuclear Ags, and subsequently develop various clinical manifestations including leukopenia and renal immune complex disease. MHC class I Ags play a critical role in the induction and progression of experimental SLE. The present study reports that ocular changes also occur in mice with experimental SLE. The ocular disease is characterized by bilateral subacute and chronic inflammation of the eyelids (blepharitis) with immune complex IgG deposition and hypertrophic meibomian glands. The severity of ocular changes was strain dependent: most severe in 129 mice, less intense in BALB/c animals and only minimal in C3H.SW mice. No blepharitis developed in mice deficient in MHC class I expression. Further, the disease was strongly inhibited in BALB/c mice treated with methimazole, an agent that has been shown to repress transcription of MHC class I. In these cases, there was no IgG deposition and a decreased infiltration of inflammatory cells in the eyelids. These observations thus suggest that, similar to the observation with experimental SLE, MHC class I is critical in the onset of this experimental autoimmune blepharitis. The new experimental eye disease described here provides an animal model for chronic blepharitis in humans, a common condition for which such a model has been sought.

Decreased CD4-CD8- TCR-alpha beta + cells in lpr/lpr mice lacking beta 2-microglobulin.

The CD4-CD8- T cells that accumulate in lpr/lpr mice have previously expressed CD8, on the basis of studies of CD8 alpha gene demethylation. The actual requirement for CD8 interaction with class I MHC molecules to promote the appearance of CD4-CD8- T cells in lpr/lpr mice has also been suggested. To examine this point in more detail, the lpr mutation was bred onto a beta 2-microglobulin-deficient background (beta 2-m-/-). C57BL/6 (B6) mice homozygous for both the lpr mutation of the fas gene and inactivation of the beta 2-m gene (beta 2-m-/- lpr/lpr) develop less alpha beta T cell lymphadenopathy than the parental B6 lpr/lpr strain. This is caused by the near absence of CD8+ T cells and a considerable reduction in CD4-CD8- T cells, revealing an important role for positive selection on class I MHC molecules during the ontogeny of lpr CD4-CD8- T cells. Although absolute numbers of peripheral T cells are decreased in beta 2-m-/- lpr/lpr mice, they manifest a B cell lymphadenopathy with age. beta 2-m-/- lpr/lpr mice display only subtle indications of autoimmune disease with age, compared with parental B6 (beta 2-m+/+)lpr/lpr mice. These include limited histopathologic stages of kidney disease and lack of proteinuria, despite the presence of serum anti-DNA Abs. Thus, absence of class I MHC-positive selection of CD8+ and CD4-CD8- TCR-alpha beta + cells limits the autoimmune diathesis observed in beta 2-m+/+ lpr/lpr mice.

DNA mimics a self-protein that may be a target for some anti-DNA antibodies in systemic lupus erythematosus.

Recent studies suggest that some anti-DNA Abs in systemic lupus erythematosus may actually be Abs to specific proteins and that binding to dsDNA is a nonspecific cross-reactive event. To identify such proteins that bind to anti-DNA Abs, a cDNA expression library from human placenta was screened with mAb 3E10, a pathogenic anti-dsDNA Ab. MAb 3E10 was shown to bind to a 44-amino-acid fragment of HP8, a newly identified protein with amino acid sequence homology to the family of SPARC extracellular matrix proteins. To determine if Ab binding to both dsDNA and HP8 protein occurs through a common binding site, and therefore represents molecular mimicry, the Ab binding domains for protein and DNA were mapped. Chain recombinations between mAb 3E10 and a non-anti-DNA mAb showed that both the heavy and the light chains of mAb 3E10 were essential for anti-dsDNA and anti-HP8 reactivity. Mutagenesis experiments demonstrated that dsDNA and HP8 shared several critical binding residues located in all three complementarity-determining regions of mAb 3E10 VH. Moreover, Abs to HP8 were demonstrated in the sera of a subset of lupus patients. These results indicate that DNA mimics the HP8 protein in binding a lupus Ab, and that this protein may be a target for a subpopulation of anti-dsDNA Abs in systemic lupus erythematosus.

The mechanism of autoantibody production in an autoimmune MRL/lpr mouse.

Rheumatoid factors (RF) and anti-DNA Abs from MRL/lpr mice have features similar to Abs directed toward foreign Ags, indicating a role of specific activation by Ags during disease. But our previous studies and analogous studies from others concentrated on a limited subset of hybridomas selected on the basis of Ag binding to well characterized target autoantigens. Thus, it has been unclear to what extent clonal expansion is restricted to identifiable autospecificities. To obtain a more complete picture of disease-associated autoantibody production, we designed the following experiment. A large number of B cell hybridomas were generated from the spleen of an MRL/lpr mouse and then analyzed for self-specificity, sequence, and clonal relationship. Surprisingly, we found that clonal expansion was limited to only a few autospecificities, implying a unique property of this response. In addition, we used Southern blotting with heavy and L chain constant region probes to screen both RF and non-RF hybridomas for membership in clones, one of which was first identified among RF hybridomas. We found no non-RF members of this clone. The size and number of mutations of this clone were sufficient for us to conclude that nonspecific (i.e., non-RF) mutant members are rapidly lost. Had an Ag other than IgG2a been driving clonal expansion, we should have seen mutants that retained spectificity for that Ag but that lost specificity for IgG2a. This observation, along with the restriction of clonal expansion to a few autospecificities, provides strong evidence that normal autoantigens themselves drive autoantibody clonal expansion.

Usage of a novel class of germ-line Ig variable region gene for cationic anti-DNA autoantibodies in human lupus nephritis and its role for the development of the disease.

It has been shown that cationic anti-DNA autoantibodies have nephritogenic potential in murine models of lupus nephritis. We have recently reported the close relationship between the presence of cationic anti-DNA Abs and the development of lupus nephritis in humans. To investigate underlining mechanisms responsible for the production of pathogenic autoantibodies, we have isolated a cDNA clone (SC17) encoding cationic anti-DNA Ab of human systemic lupus erythematosus with severe nephritis that was present at the onset of disease but disappeared after disease remission with corticosteroids. We have also cloned a counterpart Ig VL germ-line gene (SG3) from purified neutrophils of the patient and found the presence of replacement mutations only in the CDR of SC17. Surprisingly, predicted isoelectric point (pI) of deduced protein encoded by SG3 was the most cationic one among those encoded by previously reported human V kappa germ-line genes in the DNA database. These results raise the possibility that the use of specific germ-line genes may confer a cationic charge on the anti-DNA Ab, whereas somatic mutations induce affinity maturation of anti-DNA Ab in human lupus nephritis. Anti-DNA Ab-secreting B cells, but not DNA nonbinding B cells of the same patient, constitutively expressed SC17 mRNA. This mRNA is also expressed by B cells from a vast majority of patients at the onset of disease or exacerbation of lupus nephritis. However, the mRNA is absent in B cells from patients with lupus nephritis during disease remission, systemic lupus erythematosus patients without renal involvements, and normal individuals. It is suggested that the SC17 mRNA expression of B cells is rather restricted to systemic lupus erythematosus patients with active renal involvements.

IL-12 stimulates the development of acute graft-versus-host disease in mice that normally would develop chronic, autoimmune graft-versus-host disease.

The injection of DBA/2 (D2) spleen cells into (C57BL/6 x DBA/2)F1 mice (BDF1) induces a chronic, autoimmune graft-vs-host disease (GVHD) that is characterized by: increased production of Th2-associated cytokines; increased levels of serum Ig, including IgE; increased production of IgG anti-DNA Abs; and no detectable antihost CTL activity. Experiments were performed to determine if treatment with the cytokine IL-12, which stimulates the production of Th1-associated cytokines and inhibits Th2-associated cytokine production, would inhibit humoral autoimmunity in this system. Treatment of mice with 100 ng IL-12 per day for 5 days, starting on the day of cell transfer, resulted in: 1) near complete suppression of autoantibody production; 2) decreased serum Ig levels; 3) detectable donor antihost CTL activity; and 4) greatly reduced numbers of host splenic B and T cells. Treatment of mice with a neutralizing anti-IFN-gamma mAb did not reverse these effects of IL-12. Thirty nanograms per day resulted in reduced numbers of host B cells and reduced serum anti-DNA levels, but no detectable antihost CTL activity. IL-12 treatment initiated 7 days after cell transfer had little effect on the development of autoimmune GVHD. These observations suggest the following: 1) IL-12 inhibits humoral autoimmunity in a murine parent-->F1 GVHD model by inducing the activation of host-reactive CTLs that reject the host immune system. 2) This effect is IFN-gamma-independent. 3) IL-12 needs to be present during the initial differentiation of T cells in this system to have this effect.

Lack of allelic exclusion permits autoreactive B cells to escape deletion.

The R4A-gamma 2b transgenic mouse harbors the gene for the gamma 2b heavy chain of an anti-dsDNA Ab. Approximately 80% of B cells expressing the transgene display allelic exclusion. Although the transgenic mice have little to no detectable serum anti-DNA activity, splenic B cells can be stimulated in vitro with LPS to secrete anti-DNA Ab. Hybridomas derived from LPS-stimulated splenic B cells were analyzed for expression of the transgene and for DNA binding. All nine transgene-encoded anti-DNA-producing lines were found to express an endogenous IgM heavy chain. Of 19 randomly selected lines producing a transgene-encoded non-DNA binding Ab, none expressed a second heavy chain. The tight correlation between lack of allelic exclusion and anti-dsDNA specificity provides strong support for the hypothesis that a major function of allelic exclusion is to prevent the maintenance of a pool of potentially activatable autoreactive cells.

In Vitro Role of IL-1 in Heightened IgG, Anti-DNA, and Nephritogenic Idiotype Production by B Cells Derived from the Murine MRL/ lpr Lupus Model

The MRL/ lpr murine model resembles human lupus both in its serologic and immunopathologic features, and is characterized by high-level IgG and autoantibody production. The precise mechanisms for this B cell hyperactivity are poorly understood. This study explored the role of IL-1 in determining high-level IgG and autoantibody production in the MRL/ lpr murine lupus model by blocking IL-1 activity with a recombinant IL-1 receptor antagonist (IL-1Ra). IgG and autoantibody production (anti-DNA ab and Id-H130 activity) by B cells derived from MRL/ lpr mice was significantly suppressed by treating B cell cultures with IL-1Ra. In contrast, IgG and autoantibody production by B cells derived from young MRL/ lpr, MLR/++, or normal C3H/HeJ mice showed virtually no suppression with IL-1Ra. Collectively, these findings indicate that IL-1 may be an important factor in determining the heightened production of IgG, anti-DNA, and id-H130 antibody production in lupus-prone MRL/ lpr mice. Furthermore, heightened IL-1 activity appears to be influenced by both age and the presence of the lpr mutation.

Histone mediates glomerular deposition of small size DNA anti-DNA complex

Histone can mediate the binding of free DNA to the glomerular capillary wall. We tested whether histone could mediate the deposition of preformed DNA-anti-DNA immune complex (IC). IC were generated using monoclonal anti-DNA Ab and excess of small size 125I-DNA; after further digestion with DNase the IC, containing 5 micrograms DNA (now 20 to 60 bp), was injected into the left kidney of rats. When given alone, only about 0.2% of the IC bound in glomeruli. Prior injection of 200 micrograms of core histones (H2A,H2B,H3,H4) resulted in high glomerular binding of the IC; 18.1% of the injected dose (measured as 125I-DNA) was bound at 15 minutes. Mouse immunoglobulin, representing the IC, could be seen in a capillary pattern. C3 was also present in a similar pattern, showing that complement had been activated. Discrete electron-dense deposits were seen in a subendothelial and subepithelial localization at 15 minutes. Although about 1 microgram of DNA was deposited in the glomeruli, it could not be detected by indirect immunofluorescence or intercalating dyes. These studies provide direct evidence that histones can mediate the binding of particular circulating DNA-anti-DNA immune complexes to the glomerular capillary wall in vivo. If small size DNA fragments (< 100 bp) are involved in lupus nephritis, our results provide a possible explanation for the frequent failure to detect DNA deposits in renal biopsies from SLE patients.

Molecular analysis of spontaneous nephrotropic anti-laminin antibodies in an autoimmune MRL-lpr/lpr mouse.

To explore the genetic relationship between anti-laminin and anti-DNA autoantibodies (autoAb), VH gene and gene family expression were determined among autoAb derived from an individual 6-mo-old MRL-lpr/lpr mouse. Whereas 85% of the anti-DNA Ig were identified by one of two VH family probes, 7183 and VHJ558, none of the anti-laminin antibodies (Ab) examined were recognized by these probes. Subsequent V region sequence analysis of three of the anti-laminin Ab revealed that they in fact utilized a J558 VH gene (VH50). Furthermore, FR2 and CDR2 oligonucleotide probes complementary to VH50 recognized multiple anti-laminin Ab by Northern blot analysis; the FR2 probe recognized two control anti-DNA Ab, but neither probe recognized anti-DNA Ab from the same mouse. Polymerase chain reaction amplification of MRL-lpr/lpr genomic liver DNA using primers generated from VH50 and Vk50 sequences indicated that all three anti-laminin Ig have a single replacement mutation in both their VH and Vk genes. Search of the nucleic acid databases revealed that both germline VH and Vk genes are expressed unmutated by murine lupus anti-dsDNA autoAb, previously sequenced in other laboratories. Sequence comparisons suggest that differences in anti-DNA and anti-laminin reactivity may be dependent upon somatically generated differences in the CDR3 regions of the H and L chains. The results indicate that lupus anti-laminin Ab can arise from distinct B cell populations but express the same unmutated germline V region genes as lupus anti-dsDNA autoAb. They further raise the possibility that these distinct B cell populations may be activated and expanded either: independently, by distinct Ig receptor ligands such as the Ag, laminin and DNA; or simultaneously, by a common ligand such as an anti-Id recognizing a common V region epitope.

Independently derived murine glomerular immune deposit-forming anti-DNA antibodies are encoded by near-identical VH gene sequences.

To examine the influence of variable region sequences on the capacity of individual lupus autoantibodies (autoAb) to form glomerular immune deposits, the complete VH and VL region sequences of three anti-DNA mAb that produced morphologically similar immune deposits after administration to normal mice were determined. The Ig were independently derived from 1-mo-old (H238, IgM), 3-mo-old (H8, IgG2a), and 6-mo-old (H161, IgG3) MRL-lpr/lpr mice, and they all produced subendothelial and mesangial immune deposits after passive transfer to normal mice. In addition, H238 and H161 produced granular deposits in small extraglomerular vessels. The mAb had nearly identical VH gene sequences; H8 differed from H238 and H161 by a single nucleotide in FR1 that resulted in a histidine for glutamine substitution. This VH gene sequence was also > 99% homologous to another anti-DNA Ab (termed H241), that we previously reported to produce glomerular immune deposits in a similar morphologic pattern. H161 and H238 were encoded by DFL16 and JH2 genes, whereas H8 was encoded by a JH4 gene. Different Vk family genes were used to encode the three mAb, however H161 and H238 both used a Jk5 gene. The results indicate that an identical or highly related VH gene is used to encode a subgroup of murine lupus autoAb that share immune deposit forming properties. Furthermore, they raise the possibility that amino acid residues independent from those encoded by VH genes may be influential in immune deposit formation at extraglomerular sites.

Anti-DNA antibodies form immune deposits at distinct glomerular and vascular sites

To investigate the capacity of lupus autoAb to produce glomerular immune deposits (ID) and nephritis, 24 murine monoclonal (m) anti-DNA antibodies (Ab), derived from either MRL-lpr/lpr, SNF1 or NZB lupus-prone mice and selected based on properties shared with nephritogenic Ig, were administered i.p. (as hybridomas) and i.v. (as purified Ig) to normal mice; at least four mice/mAb were evaluated. Three general patterns of immune deposit formation (IDF) were observed: extracellular ID within glomeruli (+/- blood vessels, N = 8); intranuclear ID (N = 5); or minimal or no ID (N = 11). The four MRL m anti-DNA Ab that produced significant extracellular ID demonstrated different disease profiles including: (a) mesangial and subendothelial ID with anti-basement membrane staining, associated with proliferative glomerulonephritis, PMN infiltration, and proteinuria; (b) diffuse fine granular mesangial and extraglomerular vascular ID, associated with proliferative glomerulonephritis and proteinuria; (c) dense intramembranous ID and intraluminal ID, associated with capillary wall thickening, mesangial interposition and expansion, aneurysmal dilatation and intraluminal occlusion of glomerular capillary loops, and heavy proteinuria; and (d) mesangial and extraglomerular vascular ID, associated with mild segmental mesangial expansion, without proteinuria. These MRL mAb were derived from four different mice, and they had variable pIs and isotypes. They all cross reacted with multiple autoantigens (autoAg), however, their autoAg binding profiles were distinguishable. Among the SNF1 derived mAb, four produced histologically and clinically indistinguishable disease characterized by diffuse mesangial and capillary wall ID, associated with cellular proliferation/infiltration and proteinuria. Three of the four mAb were derived from the same mouse and were clonally related; they were: IgG2b with SWR allotype, relatively cationic, highly cross reactive with similar Ag binding patterns, idiotypically related and encoded by identical VH and nearly identical VL sequences. We conclude that both the capacity of lupus autoAb to form ID and the location of IDF are dependent on properties unique to individual Ig. The results also indicate that the Ag binding region of the autoAb is influential in this process, and they suggest that multiple Ab-Ag interactions contribute to IDF in individuals with lupus nephritis. Furthermore, these observations raise the possibility that the pathologic and clinical abnormalities resulting from these interactions are influenced by the location of IDF, and that the dominant interaction, in a given individual, may be highly influential in the phenotypic expression of nephritis.

Murine monoclonal anti-DNA antibodies penetrate cells, bind to nuclei, and induce glomerular proliferation and proteinuria in vivo.

The production of relatively high quantities of autoantibodies (autoAb) that react with DNA and other intranuclear antigens is characteristic of individuals with systemic lupus erythematosus and other autoimmune diseases. However, the capacity of these Ab to penetrate cells and induce functional perturbations in vivo is not well appreciated. To address this issue, monoclonal (m) anti-DNA Ab (mAb), derived from MRL-lpr/lpr and (NZB x SWR)F1 mice, were administered to normal mice, and the animals were examined for morphologic and functional abnormalities. A subset of five mAb produced intranuclear immunoglobulin deposits in multiple organs. Intranuclear immunoglobulin deposits were also observed after cross-linking the tissue before direct immunofluorescence and after i.v. injection of F(ab')2 fragments of one anti-DNA Ab. This phenomenon was reproducible and was only associated with this subset of autoAb. Furthermore, intranuclear deposits of anti-DNA Ab within glomeruli were associated with morphologic and functional abnormalities including: hypercellularity, epithelial foot process fusion, new fiber bundle formation within the mesangium suggestive of new collagen synthesis, and proteinuria. These results indicate that a subset of autoAb may penetrate cells in vivo to influence normal cellular and nuclear function and to contribute to functional and pathologic abnormalities in individuals with systemic lupus.

Rabbits Produce Sle-Like Anti-RNA Polymerase I and Anti-DNA Autoantibodies in Responses to Immunization with Either Human or Murine SLE Anti-Dna Antibodies

Anti-DNA and anti-DNA polymerase I (RPI) autoantibody responses are symptoms of systemic lupus erythematosus (SLE). To investigate the relationship between these antibodies (Ab), rabbits were immunized with one of the following preparations: human SLE anti-DNA Ab; human SLE anti-DNA IgG; normal human anti-DNA Ab; human Grave's disease anti-DNA Ab; murine SLE anti-DNA Ab or anti-DNA IgG Fab; various normal human, murine, or rabbit IgG preparations; or complete Freund's adjuvant (CFA), alone. All of the animals immunized with anti-DNA Ab (n = 14) generated Ab reactive in radioimmunoassay with: ssDNA, dsDNA, RPI, the soluble fraction of rabbit liver crude nuclear extract, and the immunogen. Induced rabbit anti-DNA Ab in turn induced these responses in a different rabbit: a rabbit immunized with rabbit anti-DNA IgG Ab which had been previously induced by immunization with human anti-DNA Ab, produced Ab reactive with ssDNA, dsDNA, RPI, and the soluble fraction of rabbit liver nuclear extract. Although an individual animal's antisera reacted consistently over the course of immunization with the same individual RPI subunit(s), antisera from different animals reacted with different subunits of the 9-subunit RPI complex in Western blot analyses: 190 kD (n = 6); 120 kD (n = 1); 62 kD (n = 4); 45 kD (n = 2); and, no reactivity (n = 2). In contrast, animals immunized with normal IgG or CFA produced responses only against the immunogen. Together, these data suggest that anti-DNA and anti-RPI responses are connected through an autoimmune network in SLE.

Cross-reactivity of monoclonal anti-DNA antibodies with heparan sulfate is mediated via bound DNA/histone complexes

To study in more detail the cross-reactive binding of anti-DNA antibodies to heparan sulfate (HS) and heparan sulfate proteoglycan (HSPG) purified from glomerular basement membranes (GBM), the binding pattern of 31 murine IgG anti-DNA MoAbs, derived from MRL/lpr, NZB/W and graft-versus-host diseased mice, was analysed. In ELISA we found binding of 10 anti-DNA MoAbs to HS. Seven of the 10 anti-HS positive clones bound to HSPG but not to the HSPG core protein in ELISA and/or on Western blots. However, DNase-I treatment partly reduced this binding, whereas after purification of MoAb by protein-A sepharose chromatography under dissociative conditions, all clones completely lost their binding capacity to HS and HSPG. Culturing of hybridoma cells in the presence of 3H-thymidine revealed DNA bound to the MoAb. Although the binding to HS and HSPG could be reconstituted by the addition of the protein-A column effluent, this was not possible by the addition of DNA alone. Therefore, we performed immunoprecipitation of the effluent with purified MoAb and subsequent SDS-PAGE which showed that the complex also contained histones. However, histones alone were also not able to reconstitute the binding to HS and HSPG. It is concluded that binding of anti-DNA MoAb to HS and GMB-HSPG is mediated via bound complexes containing both DNA and histones. A comparable reaction with polyclonal anti-DNA Ab might play a role in the pathogenesis of SLE nephritis, since histones have a very high affinity for HS, the major glycosaminoglycan of the GBM.