Harini Bagavant

Breaking Tolerance to Double Stranded DNA, Nucleosome, and Other Nuclear Antigens Is Not Required for the Pathogenesis of Lupus Glomerulonephritis

In lupus-prone NZM2328 mice, a locus Cgnz1 on chromosome 1 was linked to chronic glomerulonephritis, severe proteinuria, and early mortality in females. A locus Adnz1 on chromosome 4 was linked to antinuclear antibody (ANA) and anti–double stranded DNA (dsDNA) antibody (Ab) production. In this investigation, two congenic strains, NZM2328.C57L/Jc1 (NZM.C57Lc1) and NZM2328.C57L/Jc4 (NZM.C57Lc4), were generated by replacing the respective genetic intervals containing either Cgnz1 or Adnz1 with those from C57L/J, a nonlupus-prone strain. The NZM.C57Lc1 females had markedly reduced incidence of chronic glomerulonephritis and severe proteinuria. NZM.C57Lc4 females had chronic glomerulonephritis and severe proteinuria without circulating ANA, anti-dsDNA, and antinucleosome Ab. These data confirm the linkage analysis. Unexpectedly, NZM.C57Lc1 females had little anti-dsDNA and related Ab, suggesting the presence of a second locus Adnz2 on chromosome 1. The diseased NZM.C57Lc4 kidneys had immune complexes by immunofluorescence and electron microscopy. The eluates from these kidneys did not contain ANA, anti-dsDNA, and antinucleosome Ab, indicative of the presence of non–anti-dsDNA nephritogenic Ab. Thus, breaking tolerance to dsDNA and chromatin is not required for the pathogenesis of lupus nephritis. These results reaffirm that anti-dsDNA and related Ab production and chronic glomerulonephritis are under independent genetic control. These findings have significant implications in the pathogenesis of systemic lupus erythematosus.

Pathogenesis of kidney disease in systemic lupus erythematosus

Purpose of reviewA combination of systemic autoimmunity and tissue response to immune injury underlie renal involvement in lupus erythematosus. In this review, we discuss recent literature investigating pathogenetic mechanisms of lupus glomerulonephritis. Recent findingsIn lupus glomerulonephritis, glomerular immune complexes were believed to be the primary mediators of renal disease. Recent studies make it apparent that autoantibodies of multiple specificities participate in the formation of immune complexes, deposited in the kidneys. Renal infiltration by T cells, macrophages, and dendritic cells have a dominant role in the progression of lupus glomerulonephritis leading to renal failure. Activation of Toll-like receptors modulates autoantibody production and systemic interferon responses. However, glomerular cell responses to immune injury influence disease outcome. In addition, new insights on the genetics of susceptibility to end-organ damage in lupus glomerulonephritis have been discovered. Differential glomerular responses reflected in gene expression profiles during disease progression provide potential markers for diagnosis of lupus glomerulonephritis progression and flares. In addition, studies of end-organ responses provide new targets for therapeutic interventions. SummaryLupus glomerulonephritis is a prototype of immune complex disease mediated by autoantibodies of multiple specificities, one of which is anti-DNA. Murine models of spontaneous systemic lupus erythematosus have been critical for understanding the underlying disease. Recent studies demonstrate that in addition to systemic autoimmunity, end-organ responses, and end-organ resistance to damage are also critical in determining disease outcome. This understanding should influence design of novel therapeutic approaches in systemic lupus erythematosus.

Failure of CD25+ T Cells from Lupus-Prone Mice to Suppress Lupus Glomerulonephritis and Sialoadenitis

The development of organ-specific autoimmune diseases in mice thymectomized on day 3 of life (d3tx mice) can be prevented by transferring CD4+CD25+ T cells from syngeneic, normal adult mice. Using a d3tx model, we asked whether CD4+CD25+ T cell deficiency contributes to glomerulonephritis (GN) in lupus-prone mice. New Zealand Mixed 2328 (NZM2328) mice spontaneously develop autoantibodies to dsDNA and female-dominant, fatal GN. After d3tx, both male and female NZM2328 mice developed 1) accelerated dsDNA autoantibody response, 2) early onset and severe proliferative GN with massive mesangial immune complexes, and 3) autoimmune disease of the thyroid, lacrimal gland, and salivary gland. The d3tx male mice also developed autoimmune prostatitis. The transfer of CD25+ cells from 6-wk-old asymptomatic NZM2328 donors effectively suppressed dsDNA autoantibody and the development of autoimmune diseases, with the exception of proliferative lupus GN and sialoadenitis. This finding indicates that NZM2328 lupus mice have a selective deficiency in T cells that regulates the development of lupus GN and sialoadenitis. After d3tx, the proliferative GN of female mice progressed to fatal GN, but largely regressed in the male, thereby revealing a checkpoint in lupus GN progression that depends on gender.

Activation of innate immune responses through Toll-like receptor 3 causes a rapid loss of salivary gland function

BACKGROUND Recent studies have demonstrated the expression of Toll-like receptor 3 (TLR3) in salivary glands and epithelial cell lines derived from Sjögrens syndrome (SS) patients. As viral infections are considered to be a trigger for SS, in this study we investigated whether in vivo engagement of TLR3 affects salivary gland function. METHODS Female New Zealand Black/WF1 mice were repeatedly injected with polyinosinic:polycytidylic acid [poly(I:C)]. TLR3 expression within submandibular glands was studied using immunohistochemistry. RNA levels of inflammatory cytokines in the submandibular glands were determined by real time polymerase chain reaction. Pilocarpine induced saliva volume was used as an index of glandular function. RESULTS Immunohistochemical analysis of submandibular glands showed TLR3 expression in epithelium of serous and mucous acini, granular convoluted tubules, and ducts. Poly(I:C) treatment rapidly up-regulated the mRNA levels of type I interferon (IFN) and inflammatory cytokines in the submandibular glands. One week after treatment, the saliva volumes in poly(I:C) treated mice were significantly reduced in comparison with the phosphate-buffered saline (PBS) treated mice. Hematoxylin and eosin staining showed that salivary gland histology was normal and lymphocytic foci were not detected. Glandular function recovered after poly(I:C) treatment was stopped. CONCLUSIONS Our results demonstrate that engagement of TLR3 within the salivary glands results in a rapid loss of glandular function. This phenomenon is associated with the production of type I IFN and inflammatory cytokines in the salivary glands. Restoration of glandular function suggests that for viral etiology of SS, a chronic infection of salivary glands might be necessary.

Role for Nephritogenic T Cells in Lupus Glomerulonephritis: Progression to Renal Failure Is Accompanied by T Cell Activation and Expansion in Regional Lymph Nodes

Autoreactive T cells are critical in the initiation and maintenance of autoantibody responses that are a hallmark of systemic lupus erythematosus. However, the direct contribution of T cells in end-organ disease like lupus glomerulonephritis (GN) is poorly understood. In this study, we investigated the role of T cells in progression of lupus GN in NZM2328 mice, a murine model of spontaneous systemic lupus erythematosus. At 26 wk of age, NZM2328 female mice showed glomerular immune complex deposits and acute proliferative GN. This was associated with up-regulation of MHC class II and the detection of T cells and CD11c+ dendritic cells in the glomeruli. The regional lymph nodes (LN) showed preferential activation of T cells and an oligoclonal T cell response with skewed expansion of certain Vβ families. This suggests an Ag-driven response occurring in the regional LN of nephritic mice during acute GN. In contrast, male NZM2328 mice developed glomerular immune complexes and acute GN, but rarely progressed to fatal chronic GN. Significantly, male kidneys at 40 wk of age did not have detectable dendritic cells and T cells in the glomeruli. Thus, glomerular immune complex deposition initiates an immune response against renal Ags in the regional LN, leading to T cell recruitment into the kidney during acute proliferative GN. This T cell activation and infiltration are influenced by gender-dependent end-organ factors and may determine the progression of acute GN to chronic GN and renal failure.

T cell epitope mimicry between Sjögren's syndrome Antigen A (SSA)/Ro60 and oral, gut, skin and vaginal bacteria

This study was undertaken to test the hypothesis that Sjogrens syndrome Antigen A (SSA)/Ro60-reactive T cells are activated by peptides originating from oral and gut bacteria. T cell hybridomas generated from HLA-DR3 transgenic mice recognized 3 regions on Ro60, with core epitopes mapped to amino acids 228-238, 246-256 and 371-381. BLAST analysis identified several mimicry peptides, originating from human oral, intestinal, skin and vaginal bacteria, as well as environmental bacteria. Amongst these, a peptide from the von Willebrand factor type A domain protein (vWFA) from the oral microbe Capnocytophaga ochracea was the most potent activator. Further, Ro60-reactive T cells were activated by recombinant vWFA protein and whole Escherichia coli expressing this protein. These results demonstrate that peptides derived from normal human microbiota can activate Ro60-reactive T cells. Thus, immune responses to commensal microbiota and opportunistic pathogens should be explored as potential triggers for initiating autoimmunity in SLE and Sjögrens syndrome.

Lupus Glomerulonephritis Revisited 2004: Autoimmunity and End-Organ Damage

Histopathology of the kidney and clinical presentation are critical factors in the diagnosis of immune‐mediated glomerulonephritis (GN). The histological manifestations of glomerular injury are shared by multiple underlying mechanisms. Work from our laboratory and from other investigators shows that antinuclear, antihistone or anti‐dsDNA antibodies are neither required nor sufficient for development of lupus GN. In addition, antibody to dsDNA can be generated by mechanisms other than loss of tolerance to chromatin. Genetic analyses demonstrate that although there is some interaction between autoantibody production and renal disease, the phenotypes are regulated by distinct genetic intervals. Furthermore, renal failure is not an essential outcome of the immune‐complex deposition and proliferative lupus GN. These data are also supported by published studies from systemic lupus erythematosus (SLE) patients. The immune regulation of lupus GN is distinct from other organ‐specific diseases and not influenced by CD25+ or NK1.1+ regulatory T cells. Thus, fatal GN may depend upon a kidney‐reactive T‐cell response that, in turn, may be regulated by gender and intrinsic end‐organ factors. The data discussed in this review call for a re‐evaluation of the current paradigms for pathogenesis of SLE. An interactive model separating autoimmunity from end‐organ susceptibility for the pathogenesis of SLE is proposed.

Mesangial pathology in glomerular disease: targets for therapeutic intervention

The glomerulus is the filtration unit of the kidney. Disruption of glomerular function may be caused by primary glomerular pathology or secondary to systemic diseases. The mesangial, endothelial and epithelial cells of the glomerulus are involved in most pathologic processes. Animal models provide an understanding of the molecular basis of glomerular disease. These studies show that mesangial cells are critical players in the initiation and progression of disease. Therefore, modulation of mesangial cell responses offers a novel therapeutic approach. The complex architecture of the kidney, specifically the renal glomerulus, makes targeted drug delivery especially challenging. Targeted delivery of therapeutic agents reduces dose of administration and minimises unwanted side effects caused by toxicity to other tissues. The currently available modalities demonstrating the feasibility of mesangial cell targeting are discussed.

Anti–α8 integrin immunoliposomes in glomeruli of lupus‐susceptible mice: A novel system for delivery of therapeutic agents to the renal glomerulus in systemic lupus erythematosus

OBJECTIVE Glomerular mesangial cells are active participants in the pathogenesis of lupus glomerulonephritis (GN). Thus, targeted delivery of therapeutic agents to mesangial cells would be an attractive approach to treatment. However, lack of known unique mesangial cell surface markers has hampered this process. This study was undertaken in a mouse model of lupus GN to identify mesangial markers and to develop a system for targeted drug delivery to the glomerulus. METHODS Based on previous observations, alpha8 integrin expressed on the surface of glomerular mesangial cells was selected as a target molecule for delivery. Two mouse strains susceptible to lupus GN, NZM2328 and (NZM2328 x NOD)F1, were studied. Glomerular expression of alpha8 integrin in normal and nephritic mice was confirmed by immunofluorescence and quantitative polymerase chain reaction analysis. Liposomes were formulated and conjugated with an anti-alpha8 integrin antibody. These immunoliposomes were loaded with DiI, a red fluorescent dye, to allow tracking in vivo, and injected into the tail vein of female mice at different ages. Specificity of targeting was studied by fluorescence microscopy and flow cytometry. RESULTS Expression of alpha8 integrin was observed in the glomeruli of normal and nephritic mice. Anti-alpha8 integrin immunoliposomes were detected in the glomerulus and glomerular mesangial cells after tail vein injection in normal and nephritic mice. Delivery of DiI by anti-alpha8 integrin immunoliposomes was tissue specific, being observed predominantly in the glomeruli, with some nonspecific uptake by CD11b cells. CONCLUSION These findings are the first demonstration of specific delivery of anti-alpha8 integrin immunoliposomes to the mesangium following tail vein injection in mice. Anti-alpha8 integrin immunoliposomes thus offer a novel approach for targeted drug therapy in lupus and other glomerular diseases.

Activation of innate immunity accelerates sialoadenitis in a mouse model for Sjögren’s syndrome-like disease

OBJECTIVE Sjögrens syndrome is a chronic autoimmune disorder characterized by progressive lymphocytic infiltration within the salivary and lacrimal glands. This study was undertaken to investigate the effects of innate immunity activation on sialoadenitis in a mouse strain genetically susceptible for development of SS-like disease. METHODS Female New Zealand Black X New Zealand White F1 mice were repeatedly treated with toll-like 3 receptor agonist poly(I:C). Submandibular glands were investigated at different time points for sialoadenitis by immunohistochemistry and for gene expression of different chemokines by quantitative PCR. Submandibular gland-infiltrating cells were characterized by flow cytometry. RESULTS Poly(I:C) treatment significantly upregulated the expression of multiple chemokines within the submandibular glands. The severity and incidence of sialoadenitis was considerably higher in poly(I:C)-treated mice. There was a preponderance of dendritic cells and NK cells in the initial inflammatory cell infiltrates, and these were followed by CD4+ T cells. CONCLUSIONS Our data clearly demonstrate that systemic activation of innate immunity accelerates sialoadenitis in a mouse model for SS-like disease. These findings suggest that chronic activation of innate immunity can influence certain features of SS.