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.

Lupus-like nephrotropic autoantibodies in non-autoimmune mice harboring an anti-basement membrane/anti-DNA Ig heavy chain transgene.

Autoantibodies target a diverse group of tissue antigens in human and experimental autoimmune nephritis. The proximal events that generate and regulate these various pathogenic Ab remain obscure. To examine the origins and fate in normal mice of autoantibodies reactive with renal basement membrane antigen, we established mice transgenic for an IgM H chain encoding an unmutated nephrotropic V region, termed LamH, derived from an MRL/lpr mouse and directed against basement membrane laminin. We previously demonstrated that in vitro transfectants combining LamH-Cmu with unmutated L chains generate distinct nephrotropic autoantibodies. Herein we report in vivo reconstruction of diverse pathogenic autoreactivity by association of LamH-Cmu with endogenous L chains. Progeny of one founder, termed M7, express a distinct phenotype characterized by minimal B cell mIgM and spontaneous production of LamH-Cmu autoreactivity. Similar Ab were not recovered from two phenotypically distinct transgenic lines expressing abundant transgene mIgM. The results suggest that lupus-like autoantibodies are readily generated in the normal genetic background by random recombinatorial events in the absence of mutation and that these Ab may contribute to disease if normal regulation is disturbed.

Molecular and structural analysis of nuclear localizing anti-DNA lupus antibodies.

To determine the structure of three nuclear localizing lupus anti-DNA immunoglobulins (Igs) and to search for clues to mechanisms of cellular and/or nuclear access, their H- and L-chain variable region sequences were determined and subjected to three-dimensional modeling. Although the results indicate heterogeneity in their primary structures, the H chains are encoded by 3 members of the J558 VH gene family with a common tertiary conformation that is not shared by a J558-encoded nonnuclear localizing anti-DNA control Ig. Furthermore, at least two of the Igs share a conformational motif in the H-chain CDR3, and all three Igs contain multiple positively charged amino acids in their CDRs, resembling nuclear localization signals that direct protein nuclear import. Notably, each VH and VK gene is also found recurrently among previously described autoantibodies. Molecular analysis further indicates that both germline-encoded and significantly mutated V genes can generate nuclear localizing anti-DNA Ig.

Structural properties of a subset of nephritogenic anti-DNA antibodies.

Structural analysis of lupus autoantibodies is beginning to provide clues to the molecular basis for antigenic specificity and pathogenicity. The present analysis indicates that multiple light and heavy chains contain residues which can facilitate DNA binding, reaffirming the notion that there are multiple ways that different amino acids combine to form an antigen-binding pocket with affinity for dsDNA and ssDNA. Furthermore, this analysis suggests that these conformations and contact residues are intrinsic to germline sequences, although amino acid changes at critical locations (somatically introduced) modulate antigen binding, and appear to influence the capacity of individual immunoglobulin to form immune deposits. Analysis of additional individual immunoglobulins with closely related V-region sequences and differing pathogenic properties will be required to resolve the contribution of specific motifs to pathogenecity.

Chronic salicylism resulting in noncardiogenic pulmonary edema requiring hemodialysis.

Salicylate intoxication is frequently overlooked as a cause of noncardiogenic pulmonary edema and altered mental status in adult patients. We describe a 42-year-old woman who presented with two episodes of recurrent noncardiogenic pulmonary edema requiring intubation. The first admission to hospital triggered an extensive initial workup that did not indicate a cause for the pulmonary edema. At the second presentation, recognition of the clinical syndrome in the emergency department led to the correct diagnosis of salicylate intoxication. The patient was successfully treated with hemodialysis and urinary alkalinization, leading to rapid resolution of pulmonary edema and extubation. Several aspects of the clinical presentation suggest that the patient suffers from chronic salicylism, probably complicated by episodic superimposed acute intoxication, a condition often misdiagnosed or diagnosed late in the course of disease, contributing to substantial morbidity and mortality in these patients. Maintenance of a high index of suspicion and rapid institution of appropriate therapy including hemodialysis once the diagnosis is established is an important determinant of outcome in this serious but underdiagnosed disorder.

Relevance of systemic lupus erythematosus nephritis animal models to human disease.

Systemic lupus erythematosus (SLE) is characterized by spontaneous B and T cell autoreactivity and multiorgan immune injury including severe glomerulonephritis. This autoimmune syndrome results from a global derangement in immune regulation dependent on the interaction of complex genetic and environmental susceptibility factors. Animal models have provided a powerful tool to study disease mechanisms and novel therapeutic interventions under well-defined conditions, and bypass the barriers inherent in the study of human lupus. Classical models of spontaneous and investigator-induced murine lupus, their mutant variants, and novel transgenic and gene-targeted mutant lineages have been particularly useful. Extensive genome typing in inbred and recombinant lupus-prone strains permits mapping and characterization of multiple lupus susceptibility loci and genes and their contribution to various disease phenotypes. Murine models provide important insight into the identity of targeted self-antigens, the molecules and pathways that maintain tolerance, immune cell and cytokine interactions that promote autoimmunity, and mechanisms of renal localization and injury by immune effectors. These models reveal that multiple and independent mechanisms contribute to disease pathogenesis and provide a better understanding of the remarkable phenotypic and histopathologic heterogeneity that characterizes human SLE.

Lupus nephritis: update on pathogenesis and disease mechanisms.

Immune-mediated nephritis is a common complication of systemic lupus erythematosus (SLE). It is now clear that multiple and independent mechanisms contribute to disease onset and pathogenesis, which may explain the remarkable phenotypic and histopathological heterogeneity observed in human SLE. Identification and characterization of disease-relevant autoantibodies, cellular effectors, and soluble immune elements have provided crucial insight into the immunologic interactions that promote renal immune injury. It is now clear that nephritogenic autoantibodies of diverse specificity localize to the kidney by a variety of mechanisms. They are accompanied by activated macrophages and T cells recruited in part through enhanced and abnormal production of macrophage growth factors and cytokines. These pathways provide novel targets for therapeutic intervention to prevent or ameliorate the aggressive autoimmune nephritis that characterizes SLE.

Anti-laminin reactivity and glomerular immune deposition by in vitro recombinant antibodies.

Growing evidence suggests that recombinatorial events prior to antigen contact can generate pathogenic autoantibodies in the nonautoimmune individual, thus providing potential disease mediators if conditions arise that permit bypass of tolerance and activation of autoreactive lymphocytes. To examine the disease potential of selected germline antibody genes, Ig were created de novo by in vitro recombination of Ig H and L chains. H chain loss variant (i.e., L-chain only) cell lines were transfected with a DNA construct encoding the variable region and regulatory sequences (LamH) of a nephrotropic murine lupus anti-laminin Ig, and the resultant Ig were examined for in vitro antigen reactivity and in vivo glomerular immune deposition. The results indicate that two light chains, LamL (Vk8, Jk5) and 238L (Vk4, Jk5), expressing unrelated germline V1 genes, combine with LamH to generate Ig that bind basement membrane laminin in vitro, diverge in their capacity to bind ssDNA, and produce two distinct patterns of glomerular immune deposits in vivo: dense mesangial matrix (LamH/LamL) and dramatic linear glomerular basement membrane (LamH/238L) deposits. The Ig genes used by both LamH and 238L are present in nonautoimmune mice as well as in lupus-prone strains. We conclude that certain unmutated Ig genes can contribute to multiple distinct disease associated specificities, including binding to intrinsic kidney antigens, and that mutation is not essential to generate these Ig. Collectively, these observations suggest that pathogenic autoantibodies can be generated in the normal preimmune repertoire by random recombinatorial and somatic events in the absence of mutation.

Variable region sequence analysis of anti-DNA antibodies: evidence for a family of closely related germ-line VH genes encoding lupus autoantibodies.

Cloning and sequencing of the V regions of the anti-DNA monoclonal antibodies (mAbs), H438 and H130, indicate that H438 is encoded by a J558 VH gene, a single D region nucleotide, and unmutated JH1, V kappa-1C and J kappa 1 genes, and the H130 L chain is encoded by a V kappa-21 subgroup gene J kappa 1 gene. Identification of VH438, which shared VH hybridization pattern with 6% of a panel of 352 MRL/lpr hybridomas, suggests that the frequency of J558 use among spontaneously activated B cells in MRL/lpr mice is greater than previously reported. The VHH438 J558 family gene is identical to VHPAR, which encodes the independently derived MRL/lpr autoantibody, MRP-2, and is highly homologous to the previously reported VHH130, which is identical to a BALB/c germ-line VH gene. Comparison of consensus sequences of homologous autoantibodies and previously reported restriction mapping suggest that a minimum of three highly related J558 germ-line genes encode lupus autoantibodies.

Anti-idiotypic monoclonal Ig specific for an anti-laminin Ig heavy chain transgene variable region.

Techniques currently available to obtain anti-idiotypic reagents reactive with a single chain of a lymphocyte antigen receptor rely on immunization with intact soluble or cell-bound Ig or T-cell receptors. Ready recovery of single-chain-specific monoclonal antibodies (MAbs) depends on the presence of an immunodominant epitope on the desired chain and chance recovery of the responding clone. Here we present a method to maximize recovery of an Ig heavy-chain-specific anti-idiotypic Ig, using sequential immunization with MAbs expressing the H chain V region in combination with different H chain isotypes and with different light chains. The latter was produced by in vitro transfection of an H-chain-loss variant myeloma cell line with a transgene construct expressing the Ig H chain V region of interest. Sequential immunization may be a useful strategy to enhance selection of anti-Id reagents reactive with single chain-specific epitopes.

The nephritogenic immune response.

Recent insights into the etiopathogenesis of nephritogenic immune responses are derived primarily from experimental models of systemic and organ-specific autoimmunity. Genetic analyses and immune-related gene ablation studies indicate that multiple independent mechanisms determine disease susceptibility. However, full characterization of proximal immunologic events in many diseases awaits identification of the renal antigens recognized by nephritogenic lymphocytes. Advances in characterization of effector mechanisms include epitope mapping of several putative pathogenic glomerular antigens and identification of novel pathways of immune-mediated tissue injury, including those involved in glomerular-tubulointerstitial communication and tubulointerstitial fibrosis. Finally, successful interruption of signal transduction pathways and transforming growth factor-beta 1 blockade by gene therapy suggest novel approaches to therapeutic intervention in immunologic renal injury.

Diverse endogenous light chains contribute to basement membrane reactivity in nonautoimmune mice transgenic for an anti-laminin Ig heavy chain.

Basement membrane proteins are targeted in a variety of pathologic autoimmune responses, yet little is known regarding the origins and regulation of this subset of pathogenic lymphocytes. To examine the generation and fate of B cells reactive with a matrix autoantigen, nonautoimmune C57BL/6 mice were rendered transgenic for a nephrotropic lupus anti-laminin immunoglobulin (Ig) H chain, termed LamH-Cmu. We previously reported recovery of two distinct phenotypes among LamH-Cmu-transgenic mice: progeny of founders M6 and M29 contained abundant transgene-expressing B cells but little anti-laminin Ig, whereas spontaneous autoreactivity was readily recovered from the M7 lineage that expressed minimal B-cell mIgM. To explore the spectrum of autoreactivity generated in vivo by different LamH-Cmu-endogenous L-chain combinations, we determined in vitro and in vivo antigen reactivity and L-chain V-region sequences of 17 LamH-Cmu-transgenic anti-laminin Igs. The results reveal a heterogeneous population of anti-laminin Igs with different fine specificities encoded by diverse endogenous L chains, encompassing nine different Vk gene families, 11 Vk genes, and three Jk genes. Many of the L chains are identical to known or putative unmutated germline Vk genes used to encode Igs reactive with self and foreign antigens in nonautoimmune and genetically autoimmune-prone mouse strains. These observations confirm that the LamH-Cmu H chain plays a dominant role in determining anti-laminin reactivity, and indicate that nonautoimmune B6 mice are fully capable of generating a diverse pool of basement-membrane-reactive B cells using unmutated Ig genes. When interpreted in the context of the divergent M6/M29 and M7 transgenic mouse phenotypes, our findings further suggest that these matrix-reactive lymphocytes are not spontaneously activated in vivo under normal circumstances.

Prolonged survival with a remnant kidney.

Surgical ablation of five-sixths renal mass in Munich-Wistar rats fed a high protein diet leads to focal sclerosis in the remnant kidney and progressive renal failure. Experimental data suggest that this injury results from intraglomerular hypertension and/or chronic glomerular hyperfiltration. Data in humans largely are limited to patients with unilateral renal agenesis or uninephrectomy, either for unilateral renal disease or for kidney transplant donation. Isolated case reports have documented focal sclerosis and progressive renal failure in two patients with a remnant kidney. To obtain data in humans with a remnant kidney, we surveyed more than 800 urologists and nephrologists in the United States and abroad. Criteria for inclusion in the study were (1) surgical resection (in one or more operations) resulting in the presence of a remnant kidney; and (2) an adequate period of follow-up, defined as 5 years or greater. A total of 13 patients were identified (from 13 different centers). Twelve patients had renal cancer and one had tuberculosis. Six patients were observed for 10 or more years postoperatively and all have stable serum creatinine levels of less than 270 mumol/L (3.0 mg/dL); two of these six patients are now more than 25 and 30 years postoperation. The other seven patients, observed for 5 to 7 years, have serum creatinine levels less than 270 mumol/L (3 mg/dL), while one has an increasing serum creatinine level. The two longest surviving patients both have undergone successful pregnancy with no overall change in serum creatinine. These observations demonstrate that it is possible for humans to survive more than 30 years with a stable serum creatinine, despite the presence of only a remnant kidney.

Membranoproliferative glomerulonephritis type I, mixed cryoglobulinemia and lymphoma in the absence of hepatitis C infection.

Chronic hepatitis C virus infection has been linked to cryoglobulinemia, membranoproliferative glomerulonephritis, and malignant B-cell lymphoproliferation, suggesting a possible pathogenetic link between these disorders. We report a patient with the latter clinical triad in the absence of hepatitis C infection. We postulate that the persistent and dysregulated immunologic activity associated with chronic antigen stimulation, inflammation and/or B-cell malignancy induces nephritogenic autoantibodies, including cryoglobulins, that produce a similar clinical syndrome in genetically susceptible individuals.

VH gene analysis of spontaneously activated B cells in adult MRL-lpr/lpr mice. J558 bias is not limited to classic lupus autoantibodies.

To determine the genetic origins of lupus auto-antibodies, we analyzed the relationship between VH gene usage and auto-Ag-binding properties of 352 B cell hybridomas derived from MRL-lpr/lpr mice. The hybridomas were derived from neonatal, 1-month-old, 3-month-old, and 6-month-old mice. The experimental strategy provided that the hybridomas were monoclonal at initial evaluation, so the Ag binding and V gene frequencies of the entire population could be determined. Initially, 1032 Ig-producing hybridomas were evaluated for binding to six Ag; VH gene family use was determined in 119 anti-DNA and anti-rabbit thymus extract (RTE) antibodies (autoantibodies) and in 233 age-matched Ig that did not bind to any of the six Ag (nonbinders). Neonatal B cells, including cross-reactive IgM autoantibodies and nonbinder IgM, used relatively 3' VH genes. The majority of B cells in adult mice used VH genes of the J558 family. Although J558 use was significantly higher among the autoantibodies (anti-DNA and anti-RTE) than among the nonbinder Ig, this difference was due to a higher frequency of J558 use by 1-month-old mice. At 3 months, J558 use by the nonbinder Ig increased to the same frequency of J558 use as in the autoantibody population. J558 use in both groups of antibodies exceeded a previously reported estimation of J558 expression in the functional B cell repertoire of young adult MRL-lpr/lpr mice. Several subgroups of antibodies that share properties with pathogenic Ig, including IgG, cross-reactive Ig, and anti-dsDNA autoantibodies, demonstrated a marked preferential expression of the J558 family. These results suggest that there is an age-related bias in the activation of B cells using J558 VH genes in MRL-lpr/lpr mice that is under the influence of a selective force distinct from, or in addition to, an ssDNA or RTE auto-Ag-driven response.

In vitro and in vivo expression of a nephritogenic Ig heavy chain determinant: pathogenic autoreactivity requires permissive light chains.

Lymphocyte antigen receptors are promising targets for immune intervention strategies in disorders marked by repertoire skewing or expansion of lymphocyte subsets. Appropriate application of immune receptor modulation is predicated on understanding the role of a particular receptor in pathogenesis and disease regulation. The VHB/W16 gene, restricted to mice carrying the j haplotype for the J558 family, is overexpressed by murine lupus anti-DNA Ig. This gene is also expressed recurrently among nephritogenic anti-DNA Ig recovered from several autoimmune strains, suggesting that cells expressing this pathogenic receptor are positively selected during disease progression. To explore the extent and mechanisms by which Ig H chains expressing this gene contribute to autoimmunity, an Ig H chain gene was engineered for in vitro and in vivo recombination studies. Site-directed mutagenesis generated unique restriction sites to link PCR-amplified V region (VDJ) cDNA to previously isolated genomic fragments containing Ig regulatory and signal sequences. The new 3 kb VDJ gene was then ligated to a 9 kb fragment encoding the IgM constant region. Transfection of H chain loss variant myeloma with the complete 12 kb construct, termed 238H-Cmicro, resulted in secretion of intact Ig pairing 238H-Cmicro, with a lambda L chain; however, transfectant Ig lacked autoreactivity and pathogenicity. Introduction of the 238H-Cmicro H chain as a transgene onto the non-autoimmune C57BL/6 background resulted in abundant B cell surface expression of 238H-Cmicro, however, four transgenic Ig recovered by fusion of LPS-stimulated splenocytes and formed by combination of 238H-Cmicro, with endogenous kappa chains do not bind DNA or laminin. These results indicate that the antigen binding sites encoded by this disease-associated gene and/or H chain must associate with permissive L chains to specify autoimmunity. The 238H-Cmicro, transgenic model should prove useful in dissecting the in vivo fate of 238H-Cmicro, L combinations that produce pathogenic autoreactive receptors and in evaluating receptor-targeted interventions.

Antiidiotypic antibody recognizes an amiloride binding domain within the alpha subunit of the epithelial Na+ channel.

We previously raised an antibody (RA6.3) by an antiidiotypic approach which was designed to be directed against an amiloride binding domain on the epithelial Na+ channel (ENaC). This antibody mimicked amiloride in that it inhibited transepithelial Na+ transport across A6 cell monolayers. RA6.3 recognized a 72-kDa polypeptide in A6 epithelia treated with tunicamycin, consistent with the size of nonglycosylated Xenopus laevis alphaENaC. RA6.3 specifically recognized an amiloride binding domain within the alpha-subunit of mouse and bovine ENaC. The deduced amino acid sequence of RA6.3 was used to generate a three-dimensional model structure of the antibody. The combining site of RA6.3 was epitope mapped using a novel computer-based strategy. Organic residues that potentially interact with the RA6.3 combining site were identified by data base screening using the program LUDI. Selected residues docked to the antibody in a manner corresponding to the ordered linear array of amino acid residues within an amiloride binding domain on the alpha-subunit of ENaC. A synthetic peptide spanning this domain inhibited the binding of RA6.3 to alphaENaC. This analysis provided a novel approach to develop models of antibody-antigen interaction as well as a molecular perspective of RA6.3 binding to an amiloride binding domain within alphaENaC.

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.

A subgroup of murine monoclonal anti-deoxyribonucleic acid antibodies traverse the cytoplasm and enter the nucleus in a time-and temperature- dependent manner.

BACKGROUND: The capacity of lupus autoantibodies to enter living cells and bind to molecules for which they have intrinsic affinity is not well appreciated. In previous studies, we identified a subgroup of three murine monoclonal IgG anti-DNA antibodies, derived from lupus-prone MRL-lpr/lpr mice, that localized within nuclei of cells in multiple organs and induced functional perturbations, in vivo, after passive transfer to normal mice. To examine the mechanisms of this phenomenon, we now extend these observations, using the same monoclonal anti-DNA antibodies and cultured cell lines. EXPERIMENTAL DESIGN: Multiple experimental approaches were utilized to track nuclear localization of anti-DNA antibodies, including direct immunofluorescence, confocal microscopy and immunoelectron microscopy. The requirements for nuclear localization were further evaluated quantitatively, in nuclei isolated from co-cultures of cells and 125I-Ig, under varying experimental conditions. RESULTS: Nuclear localization was observed with the same subset of anti-DNA antibodies that localized within nuclei in vivo; it was dependent on the antigen-binding region of the molecule; and it was not found with other anti-DNA antibodies. At progressive intervals, the Ig were observed: at the cell surface, within the cytoplasm, clustered at the nuclear pore, and within the nucleus. Nuclear localization of Ig was found to be a time- and temperature- dependent process, specific for a subset of anti-DNA antibodies and dependent on the antigen binding region of the Ig. CONCLUSIONS: This is the first demonstration that monoclonal autoantibodies can traverse both the cell and nuclear membranes to localize within the nuclei of cultured cells. Furthermore, nuclear localization of Ig was regulated in a manner analogous to that of other large cytoplasmic proteins that enter the nucleus. This confirms and extends our results using the same antibodies in whole animals, and it provides the basis to further examine the underlying mechanisms and consequences of this phenomenon.