Kf Cheung

Anti-dsDNA antibodies bind to mesangial annexin II in lupus nephritis

Production of anti-dsDNA antibodies is a hallmark of lupus nephritis, but how these antibodies deposit in organs and elicit inflammatory damage remains unknown. In this study, we sought to identify antigens on the surface of human mesangial cells (HMC) that mediate the binding of human anti-dsDNA antibodies and the subsequent pathogenic processes. We isolated anti-dsDNA antibodies from patients with lupus nephritis by affinity chromatography. We used multiple methods to identify and characterize antigens from the plasma membrane fraction of mesangial cells that crossreacted with the anti-dsDNA antibodies. We found that annexin II mediated the binding of anti-dsDNA antibodies to HMC. After binding to the mesangial cell surface, anti-dsDNA antibodies were internalized into the cytoplasm and nucleus. This also led to induction of IL-6 secretion and annexin II synthesis, mediated through activation of p38 MAPK, JNK, and AKT. Binding of anti-dsDNA antibodies to annexin II correlated with disease activity in human lupus nephritis. Glomerular expression of annexin II correlated with the severity of nephritis, and annexin II colocalized with IgG and C3 deposits in both human and murine lupus nephritis. Gene silencing of annexin II in HMC reduced binding of anti-dsDNA antibody and partially decreased IL-6 secretion. In summary, our data demonstrate that annexin II mediates the binding of anti-dsDNA antibodies to mesangial cells, contributing to the pathogenesis of lupus nephritis. This interaction provides a potential target for therapeutic intervention.

Mediators of Inflammation and Their Effect on Resident Renal Cells: Implications in Lupus Nephritis

Lupus nephritis affects up to 70% of patients with systemic lupus erythematosus and is a major cause of morbidity and mortality. It is characterized by a breakdown of immune tolerance, production of autoantibodies, and deposition of immune complexes within the kidney parenchyma, resulting in local inflammation and subsequent organ damage. To date, numerous mediators of inflammation have been implicated in the development and progression of lupus nephritis, and these include cytokines, chemokines, and glycosaminoglycans. Of these, type I interferons (IFNs) can increase both gene and protein expression of cytokines and chemokines associated with lupus susceptibility, and interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and hyaluronan have been shown to elicit both pro- and anti-inflammatory effects on infiltrating and resident renal cells depending on the status of their microenvironment. Expression of IL-6, TNF-α, type I IFNs, and hyaluronan are increased in the kidneys of patients and mice with active lupus nephritis and have been shown to contribute to disease pathogenesis. There is also evidence that despite clinical remission, ongoing inflammatory processes may occur within the glomerular and tubulointerstitial compartments of the kidney, which further promote kidney injury. In this review, we provide an overview of the synthesis and putative roles of IL-6, TNF-α, IFN-α, and hyaluronan in the pathogenesis of lupus nephritis focusing on their effects on human mesangial cells and proximal renal tubular epithelial cells.

Anti-dsDNA antibody induces soluble fibronectin secretion by proximal renal tubular epithelial cells and downstream increase of TGF-β1 and collagen synthesis

The level of anti-dsDNA antibodies correlates with disease activity in lupus nephritis, but their role in pathogenic mechanisms remains to be defined. We investigated the effect of anti-dsDNA antibodies isolated from lupus nephritis patients on fibronectin synthesis and downstream fibrogenesis in proximal renal tubular epithelial cells (PTEC). Kidney biopsies were obtained from patients with active severe proliferative lupus nephritis. In vitro studies with cultured PTEC were performed to investigate the effect of human polyclonal IgG anti-dsDNA antibodies and mycophenolic acid (MPA). The role of IL-6, IL-8, MCP-1, TNF-α, TGF-β1, and MAPK and PKC signaling pathways on soluble and cell-associated fibronectin synthesis was investigated using neutralizing antibodies or specific inhibitors. The effect of exogenous endotoxin-free soluble fibronectin on downstream fibrotic processes was also examined. Fibronectin expression was markedly increased in the tubulo-interstitium of lupus nephritis renal biopsies and it co-localized with IgG deposition. Anti-dsDNA antibodies significantly increased both secreted and cell-associated fibronectin, through prior activation of ERK, p38 MAPK, JNK, PKC-α and PKC-βII. There was downstream induction of IL-6, IL-8, MCP-1, TNF-α and TGF-β1. MPA inhibited the induction of inflammatory and fibrotic processes by anti-dsDNA antibody. Exogenous soluble fibronectin induced TGF-β1 secretion and type I collagen synthesis in PTEC in a dose-dependent manner. Our data demonstrate that anti-dsDNA antibody contributes to tubulo-interstitial fibrosis in lupus nephritis through its action on PTEC. Anti-dsDNA antibody induces both cell-associated and soluble fibronectin secretion in PTEC, the former adds to extracellular matrix deposition while the latter amplifies the fibrotic process through induction of TGF-β1 and collagen type I. The pro-fibrotic effects of anti-dsDNA antibody are ameliorated by MPA.

Annexin II-binding Immunoglobulins in Patients with Lupus Nephritis and their Correlation with Disease Manifestations

Annexin II on mesangial cell surface mediates the binding of anti-dsDNA antibodies and consequent downstream inflammatory and fibrotic processes. We investigated the clinical relevance of circulating annexin II-binding immunoglobulins (Igs) in patients with severe proliferative lupus nephritis, and renal annexin II expression in relation to progression of nephritis in New Zealand Black and White F1 mice (NZBWF1/J) mice. Annexin II-binding Igs in serum were measured by ELISA. Ultrastructural localization of annexin II was determined by electron microscopy. Seropositivity rates for annexin II-binding IgG and IgM in patients with active lupus nephritis were significantly higher compared with controls (8.9%, 1.3% and 0.9% for annexin II-binding IgG and 11.1%, 4.0% and 1.9% for annexin II-binding IgM for patients with active lupus nephritis, patients with non-lupus renal disease and healthy subjects respectively). In lupus patients, annexin II-binding IgM level was higher at disease flare compared with remission. Annexin II-binding IgG and IgM levels were associated with that of anti-dsDNA and disease activity. Annexin II-binding IgG and IgM levels correlated with histological activity index in lupus nephritis biopsy samples. In NZBWF1/J mice, serum annexin II-binding IgG and IgM levels and glomerular annexin II and p11 expression increased with progression of active nephritis. Annexin II expression was present on mesangial cell surface and in the mesangial matrix, and co-localized with electron-dense deposits along the glomerular basement membrane. Our results show that circulating annexin II-binding IgG and IgM levels are associated with clinical and histological disease activity in proliferative lupus nephritis. The co-localization of annexin II and p11 expression with immune deposition in the kidney suggests pathogenic relevance.

208 Eda+ fibronectin in tubulo-interstitial injury in lupus nephritis

Background and aims Anti-dsDNA antibody plays a critical role in the pathogenesis of lupus nephritis and contributes to inflammatory and fibrotic processes in the kidney. EDA+ spliced variant of fibronectin (EDA+ FN), normally only weakly expressed, is markedly increased in pathological conditions. We investigated the effect of human polyclonal anti-dsDNA antibodies on the expression of EDA+ FN in proximal tubular epithelial cells (PTEC) and the functional consequence. Methods EDA+ FN expression in human renal biopsies of Class III/IV±V lupus nephritis was assessed by cytochemistry. Cultured PTEC were incubated with control IgG or IgG anti-dsDNA antibodies isolated from lupus nephritis patients for 24 hour and the expression of EDA+ FN was investigated. Recombinant human EDA peptide was used to investigate the functional role of EDA+ FN in PTEC. Results The results showed that EDA+ FN was absent from normal kidney tissue but was markedly increased in the tubulo-interstitium in lupus nephritis patients. Cultured PTEC constitutively expressed native FN but not EDA+ FN. Anti-dsDNA antibodies, compared with serum-free-medium and control IgG, increased EDA+ FN expression by 5.8- and 5.6-fold respectively (p<0.05 for both), and the induction was mediated through PI3K and mTOR activation. Exogenous IL-1β and TGF-β1, but not IL-6, IL-8 or MCP-1, induced EDA+ FN by 1.8- and 2.3-fold respectively. Recombinant EDA peptide increased native FN, collagen I, laminin and SNAIL expression, but decreased E-cadherin expression, in PTEC. Conclusions Our data demonstrated a role of EDA+ FN in the pathogenesis of tubulo-interstitial disease in lupus nephritis.

205 Annexin ii-binding immunoglobulin g level correlates with clinical and renal histological disease activity in lupus nephritis

Background and aims Annexin II mediates anti-dsDNA antibody binding to mesangial cells and downstream inflammatory and fibrotic processes. We investigated the relationship between annexin II-binding IgG and clinical or histological activity in lupus nephritis. Methods Serial serum samples from 28 patients with Class III/IV±V lupus nephritis were studied. Annexin II-binding IgG level was measured with an in-house ELISA. Glomeruli were isolated from NZBWF1 mice, gene and protein expression of annexin II and its binding protein p11 were investigated by real-time PCR and cytochemical staining respectively. Ultrastructural localization of annexin II was determined by electron microscopy and immunogold staining. Results Annexin II-binding IgG level was associated with anti-dsDNA level and disease activity in 42% of lupus nephritis patients. Annexin II-binding IgG level correlated with Activity Index (r=0.44, p=0.04), leukocyte infiltration score (r=0.52, p=0.02), and karyorrhexis/fibrinoid necrosis score (r=0.66, p=0.002) in renal biopsies, and also with the amount of mesangial electron-dense deposit scored semi-quantitatively (r=0.63, p=0.009). Glomerular annexin II and p11 expression increased with disease progression in NZBWF1 mice, and annexin II was found on the surface of mesangial cells and in the mesangial matrix, co-localising with electron-dense deposits. Conclusions Our data demonstrated an association between annexin II-binding IgG level and clinical/histological disease activity in proliferative lupus nephritis. Co-localization of annexin II with electron-dense deposits suggests a pathogenic role for annexin II.