Mary H. Foster

Treatment with a Laminin-Derived Peptide Suppresses Lupus Nephritis

The role of DNA as the target for pathogenic lupus autoantibodies in systemic lupus erythematosus is equivocal and renal damage may be due to cross-reactivity of lupus Abs with glomerular components. We have previously shown that lupus autoantibodies bind to the laminin component of the extracellular matrix. In the present work, we have analyzed the fine specificity of the interaction of pathogenic murine lupus autoantibodies with this molecule and the effect of inhibiting their binding to laminin during the course of the disease. We have found that pathogenic murine lupus autoantibodies react with a 21-mer peptide located in the globular part of the α-chain of laminin. Immunization of young lupus-prone mice with this peptide accelerated renal disease. Analysis of transgenic, congenic, and RAG-1−/− mice confirmed the importance of this epitope in the pathogenesis of lupus renal disease. We have synthesized a panel of peptides that cross-react with the anti-laminin Abs and have found that the binding of lupus autoantibodies to the extracellular matrix could be inhibited in vitro by some of these competitive peptides. Treatment of MRL/lpr/lpr mice with these peptides prevented Ab deposition in the kidneys, ameliorated renal disease, and prolonged survival of the peptide-treated mice. We suggest that laminin components can serve as the target for lupus Abs. The interaction with these Ags can explain both the tissue distribution and the immunopathological findings in lupus. Moreover, inhibition of autoantibody binding to the extracellular matrix can lead to suppression of disease.

Glomerular type 1 angiotensin receptors augment kidney injury and inflammation in murine autoimmune nephritis

Studies in humans and animal models indicate a key contribution of angiotensin II to the pathogenesis of glomerular diseases. To examine the role of type 1 angiotensin (AT1) receptors in glomerular inflammation associated with autoimmune disease, we generated MRL-Faslpr/lpr (lpr) mice lacking the major murine type 1 angiotensin receptor (AT1A); lpr mice develop a generalized autoimmune disease with glomerulonephritis that resembles SLE. Surprisingly, AT1A deficiency was not protective against disease but instead substantially accelerated mortality, proteinuria, and kidney pathology. Increased disease severity was not a direct effect of immune cells, since transplantation of AT1A-deficient bone marrow did not affect survival. Moreover, autoimmune injury in extrarenal tissues, including skin, heart, and joints, was unaffected by AT1A deficiency. In murine systems, there is a second type 1 angiotensin receptor isoform, AT1B, and its expression is especially prominent in the renal glomerulus within podocytes. Further, expression of renin was enhanced in kidneys of AT1A-deficient lpr mice, and they showed evidence of exaggerated AT1B receptor activation, including substantially increased podocyte injury and expression of inflammatory mediators. Administration of losartan, which blocks all type 1 angiotensin receptors, reduced markers of kidney disease, including proteinuria, glomerular pathology, and cytokine mRNA expression. Since AT1A-deficient lpr mice had low blood pressure, these findings suggest that activation of type 1 angiotensin receptors in the glomerulus is sufficient to accelerate renal injury and inflammation in the absence of hypertension.

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.

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.

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.

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 αENaC. RA6.3 specifically recognized an amiloride binding domain within the α-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 α-subunit of ENaC. A synthetic peptide spanning this domain inhibited the binding of RA6.3 to αENaC. 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 αENaC.

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.

Central Tolerance Regulates B Cells Reactive with Goodpasture Antigen α3(IV)NC1 Collagen

Patients and rodents with Goodpasture’s syndrome (GPS) develop severe autoimmune crescentic glomerulonephritis, kidney failure, and lung hemorrhage due to binding of pathogenic autoantibodies to the NC1 domain of the α3 chain of type IV collagen. Target epitopes are cryptic, normally hidden from circulating Abs by protein-protein interactions and the highly tissue-restricted expression of the α3(IV) collagen chain. Based on this limited Ag exposure, it has been suggested that target epitopes are not available as B cell tolerogens. To determine how pathogenic anti-GPS autoantibody responses are regulated, we generated an Ig transgenic (Tg) mouse model that expresses an Ig that binds α3(IV)NC1 collagen epitopes recognized by serum IgG of patients with GPS. Phenotypic analysis reveals B cell depletion and L chain editing in Tg mice. To determine the default tolerance phenotype in the absence of receptor editing and endogenous lymphocyte populations, we crossed Tg mice two generations with mice deficient in Rag. Resulting Tg Rag-deficient mice have central B cell deletion. Thus, development of Tg anti-α3(IV)NC1 collagen B cells is halted in the bone marrow, at which point the cells are deleted unless rescued by a Rag enzyme-dependent process, such as editing. The central tolerance phenotype implies that tolerizing self-Ag is expressed in bone marrow.

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

Abstract 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-Cμ. We previously reported recovery of two distinct phenotypes among LamH-Cμ-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-Cμ-endogenous L-chain combinations, we determined in vitro and in vivo antigen reactivity and L-chain V-region sequences of 17 LamH-Cμ-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-Cμ 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.

Humoral autoimmunity to basement membrane antigens is regulated in C57BL/6 and MRL/MpJ mice transgenic for anti-laminin Ig receptors.

Basement membrane proteins are targeted in organ-limited and systemic autoimmune nephritis, yet little is known about the origin or regulation of immunity to these complex extracellular matrices. We used mice transgenic for a nephrotropic systemic lupus erythematosus (SLE) Ig H chain to test the hypothesis that humoral immunity to basement membrane is actively regulated. The LamH-Cμ Ig H chain transgene combines with diverse L chains to produce nephrotropic Ig reactive with murine laminin α1. To determine the fate of transgene-bearing B cells in vivo, transgenic mice were outcrossed onto nonautoimmune B6 and SLE-prone MRL backgrounds and exposed to potent mitogen or Ag in adjuvant. In this work we demonstrate that transgenic autoantibodies are absent in serum from M6 and M29 lineage transgenic mice and transgenic B cells hypoproliferate and fail to increase Ig production upon exposure to endotoxin or when subjected to B cell receptor cross-linking. Administration of LPS or immunization with autologous or heterologous laminin, maneuvers that induce nonoverlapping endogenous anti-laminin IgG responses, fails to induce a transgenic anti-laminin response. The marked reduction in splenic B cell number suggests that selected LamH-Cμ H chain and endogenous L chain combinations generate autospecificities that lead to B cell deletion. It thus appears that SLE-like anti-laminin B cells have access to and engage a tolerizing self-Ag in vivo. Failure to induce autoimmunity by global perturbations in immune regulation introduced by the MRL autoimmune background and exposure to potent environmental challenge suggests that humoral immunity to nephritogenic basement membrane epitopes targeted in systemic autoimmunity is tightly regulated.