Glomerular autoimmune multicomponents of human lupus nephritis in vivo: α-enolase and annexin AI

Abstract

Renal targets of autoimmunity in human lupus nephritis (LN) are unknown. We sought to identify autoantibodies and glomerular target antigens in renal biopsy samples from patients with LN and determine whether the same autoantibodies can be detected in circulation. Glomeruli were microdissected from biopsy samples of 20 patients with LN and characterized by proteomic techniques. Serum samples from large cohorts of patients with systemic lupus erythematosus (SLE) with and without LN and other glomerulonephritides were tested. Glomerular IgGs recognized 11 podocyte antigens, with reactivity varying by LN pathology. Notably, IgG2 autoantibodies against α-enolase and annexin AI were detected in 11 and 10 of the biopsy samples, respectively, and predominated over other autoantibodies. Immunohistochemistry revealed colocalization of α-enolase or annexin AI with IgG2 in glomeruli. High levels of serum anti-α-enolase (>15 mg/L) IgG2 and/or anti-annexin AI (>2.7 mg/L) IgG2 were detected in most patients with LN but not patients with other glomerulonephritides, and they identified two cohorts: patients with high anti-α-enolase/low anti-annexin AI IgG2 and patients with low anti-α-enolase/high anti-annexin AI IgG2. Serum levels of both autoantibodies decreased significantly after 12 months of therapy for LN. Anti-α-enolase IgG2 recognized specific epitopes of α-enolase and did not cross-react with dsDNA. Furthermore, nephritogenic monoclonal IgG2 (clone H147) derived from lupus-prone MRL-lpr/lpr mice recognized human α-enolase, suggesting homology between animal models and human LN. These data show a multiantibody composition in LN, where IgG2 autoantibodies against α-enolase and annexin AI predominate in the glomerulus and can be detected in serum.

Keywords: GN; immunology and pathology; lupus nephritis.

Copyright © 2014 by the American Society of Nephrology.

Figures

Figure 1.

Characterization of autoantibodies eluted from glomeruli of patients with LN and in corresponding serum. (A and E) Representative two-dimensional electrophoresis of podocyte cell extracts stained by colloidal Coomassie. The same cell extract was incubated with antibodies eluted from microdissected glomeruli obtained from patients with different classes of LN: (B) class III, (C) class IV, and (D) class V. Microeluted antibodies from normal kidneys did not react with any protein. Several spots were recognized by LN eluates and identified as 11 different proteins (A–K). One was characterized by LC-MS (spot I; α-enolase), and the remaining spots were characterized by MALDI-MS (Table 2). Their identity as predicted by MS is reported in Table 2. The same podocyte cell extracts separated by two-dimensional electrophoresis were incubated with normal sera (not shown) and (F) pooled LN sera (classes III–V) and then developed with anti-IgG (total) antibodies. Several proteins were detected, and most corresponded to proteins recognized in glomerular eluates (A–C and G–I); three proteins were recognized only in serum or a few glomerular eluates (K and L). All spots were characterized by LC-MS or MALDI-MS as above (Table 2, Supplemental Table 1). Their identity is reported in Table 2.

Figure 2.

Glomerular α-enolase and annexin AI: isotypes and levels in single biopsies. The following studies were done to better characterized (A) anti–α-enolase and (B) anti-annexin AI antibodies: study 1, definition of isotype with dot blot analysis; study 2, single biopsy analysis; and study 3, competition experiment using the same glomerular eluates as above and increasing amounts of α-enolase/annexin AI from 5 to 15 ng to saturate antibodies. Results show complete inhibition and confirm the presence of anti–α-enolase/anti-annexin AI IgG2 in glomerular eluates. (C) Hierarchical cluster analysis heat map for a single antibody in each renal biopsy; antibody intensities (black, high; gray, medium; white, low) are reported in lines and refer to single patient biopsies that are reported at the bottom of the figure. Results are given for 20 LN patients of our study cohort and four normal kidneys.

Figure 3.

Colocalization of IgG2 with α-enolase and annexin AI. In this series, the colocalization of each endogenous antigen (α-enolase and annexin AI) with IgG2 within renal biopsy samples is reported. For each antigen/antibody, confocal images of two renal biopsy specimens are shown relative to patients with LN classes III and V. Double immunofluorescence staining was evaluated for each antigen (red) and IgG2 (green). Merged images are reported in yellow. RNase treatment of the same renal tissue did not modify colocalization of anti–α-enolase and IgG2 (Supplemental Figure 2). Magnification of this figure is in Supplemental Material. Original magnification, ×630. Scale bars, 20 µm.

Figure 4.

Serum anti–α-enolase isotype and levels. (A) Characterization of serum anti–α-enolase isotype with dot blot analysis. (B) The same technique was used to determine serum anti–α-enolase IgG1-IgG2-IgG3-IgG4 in patients with lupus erythematosus with (LN; n=104) and without (SLE; n=112) nephritis and several other control populations, including rheumatoid arthritis (RA; n=50), membranous nephropathy (MN; n=186), IgA nephropathy (IgA; n=60), FSGS (n=32), and normal controls (n=135). (C) Levels of anti–α-enolase IgG2 were very high in most patients with LN, whereas (D) other isotypes (IgG1, IgG3, and IgG4) were undetectable. (D) Patients with LN were evaluated at T0, and after 6 and 12 months of therapy, results showed a decrease of antibody levels after therapy. Dot blot analysis using recombinant α-enolase linked to nitrocellulose as antigen (Supplemental Figure 4); results (evaluated as the signal intensity of chemiluminescence detected by VersaDoc and computed with QuantyOne software; Bio-Rad) were transformed (milligrams per liter) using a standard curve of chemioluminescent IgG2. The horizontal line is set at the 95th percentile of levels titrated in normal controls. Receiver operating characterisitc curves for anti–α-enolase IgG2 were significantly greater in LN patients compared with other patient series: (E) normal subjects, (F) SLE, (G) RA, and (H–J) other nephropathies.

Figure 4.

Serum anti–α-enolase isotype and levels. (A) Characterization of serum anti–α-enolase isotype with dot blot analysis. (B) The same technique was used to determine serum anti–α-enolase IgG1-IgG2-IgG3-IgG4 in patients with lupus erythematosus with (LN; n=104) and without (SLE; n=112) nephritis and several other control populations, including rheumatoid arthritis (RA; n=50), membranous nephropathy (MN; n=186), IgA nephropathy (IgA; n=60), FSGS (n=32), and normal controls (n=135). (C) Levels of anti–α-enolase IgG2 were very high in most patients with LN, whereas (D) other isotypes (IgG1, IgG3, and IgG4) were undetectable. (D) Patients with LN were evaluated at T0, and after 6 and 12 months of therapy, results showed a decrease of antibody levels after therapy. Dot blot analysis using recombinant α-enolase linked to nitrocellulose as antigen (Supplemental Figure 4); results (evaluated as the signal intensity of chemiluminescence detected by VersaDoc and computed with QuantyOne software; Bio-Rad) were transformed (milligrams per liter) using a standard curve of chemioluminescent IgG2. The horizontal line is set at the 95th percentile of levels titrated in normal controls. Receiver operating characterisitc curves for anti–α-enolase IgG2 were significantly greater in LN patients compared with other patient series: (E) normal subjects, (F) SLE, (G) RA, and (H–J) other nephropathies.

Figure 5.

Serum anti-annexin AI isotype and levels. (A) Characterization of serum anti-annexin AI isotype with dot blot analysis. (B) Serum levels of anti-annexin AI IgG2 were determined with a self-made ELISA in the same cohort of patients described in Figure 4; the technique is described in Supplemental Material. (C) Patients with LN were evaluated at T0, and after 6 and 12 months of therapy, results showed a decrease of antibody levels after therapy. (D) Double positivity for both anti–α-enolase and anti-annexin AI IgG2 in the different cohorts of patients with LN was analyzed with hierarchical cluster analysis. Results are presented as a heat map, in which color intensity (from red [high] to black [medium] to green [low]) indicates levels in separate patients. Two clusters could be observed: one cluster had high annexin AI and low anti–α-enolase IgG2 (bottom group; red), and one cluster had low annexin AI and high anti–α-enolase IgG2 (top group; green).

Figure 6.

Molecular features of anti–α-enolase antibody. (A–C) Anti–α-enolase IgG2 interacts with specific epitope protein. Characterization of α-enolase epitopes targets of anti–α-enolase IgG2 and IgG4; CNBr digests of anti–α-enolase were prepared as described in Concise Methods and then immunoblotted with IgG2 and IgG4 eluted from glomeruli and serum of patients with LN and MN, respectively. Arrows indicate two different peptides that are recognized by IgG2 (1.349 kD; ILPVGAANFREAM) and IgG4 (6.822 kD; DGTENKSKFGANAILGVSLAVCKAGAVEKGVPLYRHIADLAGNSEVILPVPAFNVINGGSHAGNKLAM); the second peptide contains a site for acetylation. The two epitopes above recognized by IgG2 and IgG4 have been localized in different regions of α-enolase. (D–F) Human anti-DNA IgG does not cross-react with α-enolase. Anti-dsDNA antibodies were purified from sera of LN patients by affinity chromatography; (D) they recognized several histones (only H1 and H2b were negative at Western blot) but (F) did not recognize α-enolase. Dot blot analysis was done by increasing the amount of fixed α-enolase up to 15 μg without showing any interaction. (E) The same histones separated by monodimensional electrophoresis were recognized by glomerular eluates. With the same dot blot assay, anti–α-enolase antibodies recognized the antigen at low concentration (5 μg).

Figure 7.

Experimental lupus nephritis. (A) mAbs from nephritogenic hybridomas recognize α-enolase. Nepritogenic monoclonal anti-DNA IgG2 clone (H147) derived from lupus-prone MRL-lpr/lpr mice was furnished by M.M. On dot blot using dilutions of the original hybridoma, a specific reaction with α-enolase was found. Techniques for developing hybridomas have been reported in detail in previous works., (B–F) Anti–α-enolase IgG infusion produced proliferative renal lesions in BALB/c and SCID mice. Twenty-two BALB/c mice were injected intraperitoneally with 1×106 hybridoma cells producing anti–α-enolase mAbs; hybridoma cells producing IgM anti-DNA antibodies were injected into three BALB/c mice as controls. After 10 days, six mice developed proteinuria (100–300 mg%). (B and C) Controls and other mice had proteinuria less than 1 mg/ml. Gross pathology is reported in D, and it shows hematoxylin/eosin (HE; ×100 and ×200) and periodic–acid Schiff (PAS) staining. In E, immunostaining for nonmuscle myosin (αSMA) is shown; in mice infused with anti–α-enolase antibodies, mesangial deposits of αSMA were seen in glomeruli and small vessels (where αSMA is physiologic), whereas no mesangial staining could be seen in mice injected with anti-DNA IgM (×200). (F) Four of six SCID mice similarly injected with the same hybridomas developed proliferative GN with crescents and tubulointerstitial lesions.

Figure 7.

Experimental lupus nephritis. (A) mAbs from nephritogenic hybridomas recognize α-enolase. Nepritogenic monoclonal anti-DNA IgG2 clone (H147) derived from lupus-prone MRL-lpr/lpr mice was furnished by M.M. On dot blot using dilutions of the original hybridoma, a specific reaction with α-enolase was found. Techniques for developing hybridomas have been reported in detail in previous works., (B–F) Anti–α-enolase IgG infusion produced proliferative renal lesions in BALB/c and SCID mice. Twenty-two BALB/c mice were injected intraperitoneally with 1×106 hybridoma cells producing anti–α-enolase mAbs; hybridoma cells producing IgM anti-DNA antibodies were injected into three BALB/c mice as controls. After 10 days, six mice developed proteinuria (100–300 mg%). (B and C) Controls and other mice had proteinuria less than 1 mg/ml. Gross pathology is reported in D, and it shows hematoxylin/eosin (HE; ×100 and ×200) and periodic–acid Schiff (PAS) staining. In E, immunostaining for nonmuscle myosin (αSMA) is shown; in mice infused with anti–α-enolase antibodies, mesangial deposits of αSMA were seen in glomeruli and small vessels (where αSMA is physiologic), whereas no mesangial staining could be seen in mice injected with anti-DNA IgM (×200). (F) Four of six SCID mice similarly injected with the same hybridomas developed proliferative GN with crescents and tubulointerstitial lesions.