Inhibitory Anti-Peroxidasin Antibodies in Pulmonary-Renal Syndromes

Abstract

Background: Goodpasture syndrome (GP) is a pulmonary-renal syndrome characterized by autoantibodies directed against the NC1 domains of collagen IV in the glomerular and alveolar basement membranes. Exposure of the cryptic epitope is thought to occur via disruption of sulfilimine crosslinks in the NC1 domain that are formed by peroxidasin-dependent production of hypobromous acid. Peroxidasin, a heme peroxidase, has significant structural overlap with myeloperoxidase (MPO), and MPO-ANCA is present both before and at GP diagnosis in some patients. We determined whether autoantibodies directed against peroxidasin are also detected in GP.

Methods: We used ELISA and competitive binding assays to assess the presence and specificity of autoantibodies in serum from patients with GP and healthy controls. Peroxidasin activity was fluorometrically measured in the presence of partially purified IgG from patients or controls. Clinical disease severity was gauged by Birmingham Vasculitis Activity Score.

Results: We detected anti-peroxidasin autoantibodies in the serum of patients with GP before and at clinical presentation. Enriched anti-peroxidasin antibodies inhibited peroxidasin-mediated hypobromous acid production in vitro. The anti-peroxidasin antibodies recognized peroxidasin but not soluble MPO. However, these antibodies did crossreact with MPO coated on the polystyrene plates used for ELISAs. Finally, peroxidasin-specific antibodies were also found in serum from patients with anti-MPO vasculitis and were associated with significantly more active clinical disease.

Conclusions: Anti-peroxidasin antibodies, which would previously have been mischaracterized, are associated with pulmonary-renal syndromes, both before and during active disease, and may be involved in disease activity and pathogenesis in some patients.

Keywords: ANCA; Goodpasture-s syndrome; anti-GBM disease; extracellular matrix; glomerulonephritis; vasculitis.

Figure 1.

Antibodies recognizing peroxidasin (PXDN) are present in patients with GP before and at the time of diagnosis. (A) ELISA data for specified native recombinant antigens from 1:100 dilutions of either serum or early plasmapheresis fluid at the time of presentation. Positivity threshold was determined from the healthy control mean (n=7) plus 3 SD for each antigen. Dotted reference line represents control mean for the specified antigen, and gray line represents positivity threshold. (B) ELISA data for specified native recombinant antigens from 1:100 dilutions for each individual serum sample; maximal values from each patient and control are plotted, regardless of time of draw. A schematic of sample timing appears in Supplemental Figure 1, and each patient’s specific time-course appears in Supplemental Figures 2 and 3. Positivity thresholds derived from all control samples. Controls plotted with mean. Dotted reference line represents control mean for the specified antigen, and gray line represents positivity threshold (+3 SD from control mean).

Figure 2.

Peroxidasin is inhibited by patient anti-peroxidasin antibodies in vitro. APF-based peroxidase activity assay (500 pM peroxidasin, 218 µM whole patient IgG [437.5-fold molecular excess], 100 µM NaBr, 140 mM NaCl, 10 µM APF) with initial rates measured after the addition of 7.5 µM H₂O₂ in the presence of partially purified IgG from anti–peroxidasin-positive patients or matched controls demonstrating significant inhibition of activity by anti-peroxidasin antibodies (t test used, variances found to not significantly differ). AFU, arbitrary fluorescence units.

Figure 3.

The subset of patients with GP recognizing peroxidasin (PXDN) has anti–peroxidasin-specific antibodies. Peroxidasin-positive, MPO-positive, and matched controls were tested for antigen specificity using competition ELISA. Antigens were coated (2.8 nM) and native, soluble peroxidasin or MPO were preincubated with 1:500 dilutions of patient sera for 12 hours at 4°C before exposure to coated antigens. Error bars represent the SEM for duplicates of each condition. Gray lines indicate controls, black lines indicate patients with peroxidasin as coated antigen, green lines indicate patients with MPO as coated antigen. Refer to key in the top right of the figure for quick reference regarding the type of competition binding being tested. “self” refers to same antigen competition and “cross” refers to the other antigen. Each line/shape combination refers to an individual patient. (A) Binding to coated antigens in the absence of any competing protein. (B) Peroxidasin-positive patients have peroxidasin and MPO recognition inhibited by peroxidasin, but not MPO, indicating the presence of peroxidasin-specific antibodies. Of note, the top right quadrant demonstrates peroxidasin’s ability to compete away MPO recognition. (C) MPO-only positive patients have MPO binding, which is unaffected by soluble peroxidasin, but completely inhibited by soluble MPO, indicating that these antibodies are MPO specific.

Figure 4.

Anti-peroxidasin antibodies are present in anti-MPO ANCA-associated vasculitis and are significantly associated with worse clinical disease. (A) ELISA data for specified ANCA-associated vasculitis from 1:100 dilutions of serum. Positivity threshold was determined from the control mean (n=23) plus 3 SD. Controls plotted with mean±SD. Dotted reference line represents control mean for the specified antigen, and gray line represents positivity threshold (+3 SD). (B) Birmingham Vasculitis Activity Scores grouped by anti-peroxidasin positivity status for anti-MPO ANCA-associated vasculitis patients (n=54; n=7 anti-peroxidasin positive). Median Birmingham Vasculitis Activity Score for the anti–peroxidasin-positive samples was 12 versus two for the peroxidasin-negative samples. Difference in median tested by Mann–Whitney U test.

Figure 5.

The potential role of anti-peroxidasin autoantibodies in pulmonary renal syndromes based on enzyme inhibition and sulfilimine cross-linking (S=N) of the basement membrane.