MAGE-B2 autoantibody: a new biomarker for pediatric systemic lupus erythematosus

Abstract

Objective: Melanoma-associated antigen gene B2 (MAGE-B2) encodes an embryonic antigen normally silenced after birth except in testis and placenta. We identified the MAGE-B2 gene and autoantibodies in pediatric patients with systemic lupus erythematosus (SLE) glomerulonephritis. We investigated the prevalence of MAGE-B2 autoantibodies in association with active SLE, to determine a pathogenetic role of MAGE-B2 protein through its distribution in cells and tissues.

Methods: A cross-sectional study analyzed the frequency of MAGE-B2 autoantibodies in 40 patients with pediatric SLE, 23 adult controls, and 16 patients with pediatric juvenile rheumatoid arthritis (JRA) using Western blots containing recombinant MAGE-B2. SLE Disease Activity Index 2000 (SLEDAI-2K) and British Isles Lupus Assessment Group (BILAG) index measured SLE disease activity. Tissue distribution of MAGE-B2 protein was assessed by immunohistochemistry, immunofluorescence, and Western blots.

Results: Seventeen (43%) of 40 pediatric SLE patients had MAGE-B2 autoantibodies as compared to 0 of 16 JRA patients and 2 of 23 adult controls. SLE disease activity was significantly higher in MAGE-B2 autoantibody-positive versus autoantibody-negative patients (SLEDAI-2K, mean 10.9 vs 5.2, p = 0.013; BILAG, mean 15.3 vs 6.3, p = 0.023). Active nephritis was more prevalent (60% vs 24%) in MAGE-B2 autoantibody-positive than autoantibody-negative SLE patients. MAGE-B2 protein was visualized in SLE kidney proximal convoluted tubules and in tumor epithelial cells, but not in lymphoblastoid cells.

Conclusion: MAGE-B2 autoantibody appears to be a clinically relevant biomarker for pediatric SLE disease activity and nephritis.

Figure 1

A. Scatter plot showing disease activity distribution of SLE patients with and without the MAGE-B2 autoantibody. The horizontal line in each column depicts the mean value. P values were obtained using Wilcoxon/Kruskal Wallis Rank Sum Test. B. Comparison of active nephritis and all nephritis in patients with and without MAGE-B2 autoantibody. The “active nephritis” category was defined as any positive Renal SLEDAI and/or a urine protein to creatinine ratio > 0.5 or proteinuria >0.5 grams per 24 hours. The “all nephritis” category included patients with past or current lupus nephritis, diagnosed clinically or by renal biopsy. P values were obtained via Fisher’s exact test.

Figure 2

Specificity of MAGE-B2 antibodies using PVDF strips loaded with 0.4μg of recombinant MAGE-B2 protein. A. The commercial MAGE-B2 antibody signal was no longer observed after absorption with blocking peptide (+BP). B. The same PVDF membranes as in A were stripped and re-probed with MAGE-B2 antibody at the same dilution. The weaker signal in − BP membrane in section A most likely reflects the fact that the centrifugation step was omitted in section B immunoblotting. C. The commercial MAGE-B2 signal was blocked by full-length recombinant MAGE-B2 protein (+rMB2). D. A SLE patient’s MAGE-B2 autoantibody signal was blocked after absorption with recombinant MAGE-B2 protein (+rMB2).

Figure 3

Immunoblot loaded with cytosolic (C) and nuclear (N) lysates of HEp-2 cells, SLE lymphoblastoid cell line (LCL), and normal LCL. Recombinant MAGE-B2 (rMAGE-B2) is in the last lane. Note MAGE-B2 expression in both subcellular compartments of HEp-2 cells but not in LCLs. Second panel shows beta-actin antibody loading control. Third panel shows the degree of cross contamination of cytosolic fractions with nuclear protein marker, Lamin A&C. Fourth panel shows the same blot for nuclear fraction contamination, using a cytosolic protein marker, HSP-90.

Figure 4

Indirect surface immunofluorescence (IF) of HEp-2 cells shows A. MAGE-B2 stained green with FITC, B. HLA-ABC stained red with TRITC, and C. colocalization of MAGE-B2 and HLA-ABC (yellow) on the cell membrane. D. Goat serum IgG (control for MAGE-B2 antibodies) and E. mouse serum IgG (control for HLA-ABC antibodies) are stained with FITC and TRITC, respectively. F. Overlay of D and E shows no specific staining or colocalization. G. Within HEp-2 cells, MAGE-B2, stained green with FITC, is distributed mainly in the cytoplasm. H. Intracellular IF with goat serum IgG, labeled with FITC, shows minimal staining. Nuclei are stained blue with 4′,6-diamidino-2-phenylindole.

Figure 5

Immunohistochemistry of kidney biopsies from SLE patients with A. WHO Class IV and B. WHO Class V glomerulonephritis (GN), shows strong MAGE-B2 expression (brown staining) in the brush borders of proximal convoluted tubules (arrowheads). C. Biopsy from a normal kidney resection (from a patient with renal cancer) shows minimal staining of the proximal convoluted tubules (arrowheads). For sections A, B, and C: first column, hematoxylin and eosin (H&E) staining; second column, staining with anti-MAGE-B2 antibody (1μg/mL); third column, goat serum IgG control (1μg/mL). Top row: 400× magnification; bottom rows (insets): 1890× manignification. Immunohistochemistry of kidney biopsy from a SLE patient with WHO Class IV nephritis shows D. MAGE-B2 staining in glomerular epithelial nuclear membranes and cytoplasm (arrowheads). E. A normal glomerulus does not show any MAGE-B2 staining. For sections D and E: first column, anti-MAGE-B2 antibody (1μg/mL); second column, goat serum IgG control (1μg/mL). Top row: 630× magnification; bottom row (insets): 1260× magnification.