IgA and IgG antineutrophil cytoplasmic antibody engagement of Fc receptor genetic variants influences granulomatosis with polyangiitis

Abstract

Granulomatosis with polyangiitis (Wegener's) is a rare autoimmune neutrophil-mediated vasculitis that can cause renal disease and mucosal manifestations. Antineutrophil cytoplasmic antibodies (ANCA) are present in many patients, vary in level over time, and induce neutrophil activation through engagement with Fc receptors (FcRs). Given roles for FcRs in ANCA-mediated neutrophil activation and IgA antibodies in mucosal immunity, we hypothesized that FcR genetics and previously unappreciated IgA ANCA affect clinical presentation. We assembled a total of 673 patients and 413 controls from two multicenter cohorts, performed ELISA and immunofluorescence assays to determine IgA and IgG ANCA positivity, and used Illumina, TaqMan, or Pyrosequencing to genotype eight haplotype-tagging SNPs in the IgA FcR (FCAR) and to determine NA1/NA2 genotype of FCGR3B, the most prevalent neutrophil IgG FcR. We evaluated neutrophil activation by measuring degranulation marker CD11b with flow cytometry or neutrophil extracellcular trap formation with confocal microscopy. Functional polymorphisms in FCGR3B and FCAR differed between patient groups stratified by renal involvement. IgA ANCA were found in ∼30% of patients and were less common in patients with severe renal disease. Neutrophil stimulation by IgA or IgG ANCA led to degranulation and neutrophil extracellcular trap formation in a FcR allele-specific manner (IgA:FCAR P = 0.008; IgG:FCGR3B P = 0.003). When stimulated with IgA and IgG ANCA together, IgG ANCA induced neutrophil activation was reduced (P = 0.0001). FcR genotypes, IgA ANCA, and IgG ANCA are potential prognostic and therapeutic targets for understanding the pathogenesis and presentation of granulomatosis with polyangiitis (Wegener's).

Fig. 1.

IgA ANCA, IgG ANCA, and their Fc receptors influence neutrophil activity. (A) Human IgG and IgA anti-PR3 containing antibody fractions impact degranulation in neutrophils from healthy donors (n = 4) measured as change in CD11b surface expression by flow cytometry. These differences in degranulation were compared with stimulation with IgG and IgA antibody fractions not containing anti-PR3. (B) When stratifying by FCAR genotype (rs16986050), the homozygous G allele, which results in a proinflammatory response, was associated with a higher percentage of cells with NET formation when stimulated with IgA anti-PR3 antibody fractions (P = 0.008). Stimulating with media or antibody fractions from healthy controls was used as a negative control. Phorbol myrisitate acetate (PMA) was used as a positive control. (C) When stratifying by FCGR3B NA1/NA2 genotype, the more proinflammatory NA1 genotype results in a higher percentage of cells with NET formation when stimulated by IgG anti-PR3 antibodies (P = 0.003). (D) Stimulating with IgA anti-PR3 antibody fractions can reduce the NET formation potential of IgG ANCA stimulation in a genetically dependent manner. Stimuli included antibody fractions from healthy controls, antibody fractions from IgG anti-PR3–positive patients, and a combination of antibody fractions from an IgG anti-PR3–positive patient and an IgA anti-PR3–positive patient. The presence of the A allele of rs16986050 in FCAR significantly reduced the potential of IgG anti-PR3 antibodies to induce NET formation when costimulated with IgA anti-PR3 antibodies (P = 0.0001).

Fig. 2.

Fluorescent microscopy displaying NET formation based on 180-min stimulation with control serum, PMA-positive control, IgA anti-PR3 antibody fractions, and IgG anti-PR3 antibody fractions.