Measuring Circulating Complement Activation Products in Myeloperoxidase- and Proteinase 3-Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

Abstract

Objective: There is accumulating evidence that complement activation is important in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) pathogenesis. This study was undertaken to investigate complement activation in AAV with myeloperoxidase (MPO) positivity and AAV with proteinase 3 (PR3) positivity after determining optimal methods for measuring activated complement factors in circulation.

Methods: Participants included 98 patients with AAV (45 MPO-ANCA positive, 53 PR3-ANCA positive) and 35 healthy controls. Plasma was obtained from blood collected using EDTA tubes, with or without 100 μg/ml Futhan. Levels of Bb, C3a, C5a, soluble C5b-9 (sC5b-9), properdin, and C4d were measured by enzyme-linked immunosorbent assay. Group comparisons were made using Wilcoxon's 2-sample test. Paired data were analyzed using a matched pairs signed rank test.

Results: Compared to healthy controls, certain complement analyte levels were high in patients with active AAV with MPO positivity, including C3a (P < 0.0001), C5a (P = 0.0004), and sC5b-9 (P = 0.0007). During remission, levels of Bb (P = 0.001), C3a (P < 0.0001), and sC5b-9 (P = 0.003) were higher. Compared to healthy controls, C3a (P < 0.0001), C5a (P = 0.002), sC5b-9 (P = 0.0001), and C4d (P = 0.005) levels were higher in patients with active AAV with PR3 positivity; levels of C3a (P < 0.0001) and C4d (P = 0.007) were also higher duriing remission. There were no significant differences in any complement analyte for either ANCA serotype between patients with active disease and those with disease in remission. Among patients with paired samples, sC5-9 levels were significantly lower during disease remission compared to active disease. C5a was significantly lower among patients with disease in long-term remission who were not receiving therapy. For Bb, C5a, and sC5b-9, median levels and individual values were considerably higher in control and patient samples processed without Futhan compared to those processed with Futhan.

Conclusion: Complement activation occurs in both MPO-positive AAV and PR3-positive AAV. The complement activation profile differs according to disease activity and possibly ANCA serotype. Futhan reduces in vitro complement activation and provides a more accurate measurement.

Figure 1A-F

Measuring complement activation with futhan in healthy controls and MPO-ANCA and PR3-ANCA vasculitis. Bb (A), properdin (B), C3a (C), C5a (D), sC5b-9 (E), C4d (F). Horizontal lines represent median values and interquartile ranges. Data were analyzed using the Wilcoxon two-sample test. After Bonferroni correction, a p-value <0.0083 is considered statistically significant. MPO, myeloperoxidase; PR3, proteinase 3.

Figure 2A-F

Changes in complement activation in patients with ANCA vasculitis and paired samples from active and remission disease states. Changes in complement activation in patients with ANCA vasculitis and paired samples from active and remission disease states. Bb (A), properdin (B), C3a (C), C5a (D), sC5b-9 (E), C4d (F). Samples processed with futhan. Data were analyzed using the paired signed-rank test. A p-value <0.05 is considered statistically significant.

Figure 3A-F

Measuring complement activation in patients with ANCA vasculitis in long-term remission off therapy ≥ 2 years (LTROT≥2). Bb (A), properdin (B), C3a (C), C5a (D), sC5b-9 (E), C4d (F). Horizontal lines represent median values and interquartile ranges. Wilcoxon two-sample test was used to compare patients with active disease to those with LTROT status. A p-value <0.05 is considered statistically significant. MPO, myeloperoxidase; PR3, proteinase 3.