Anti-neutrophil cytoplasm antibody IgG subclasses in Wegener's granulomatosis: a possible pathogenic role for the IgG4 subclass

Abstract

A characteristic feature of Wegener's granulomatosis is the presence of antineutrophil cytoplasm antibodies (ANCA) to proteinase 3 (PR3). In vitro, ANCA activate neutrophils by co-ligating PR3 and FcgammaRIIa/IIIb receptors. ANCA are predominantly of the IgG isotype, and IgG1, IgG3 and IgG4 subclasses are particularly represented. To address the pathogenic role of individual ANCA-IgG subclass antibodies, patients' sera were screened using indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and subclass PR3-ELISA to identify patients with high titres of PR3-ANCA within the IgG1, IgG3 or IgG4 subclasses. Unfractionated ANCA-IgG and subclass fractions were isolated by affinity chromatography and compared for their capacities to stimulate superoxide production by primed human neutrophils. Donor neutrophils were analysed for constitutive and induced FcgammaRI expression by flow cytometry. The IgG1, IgG3 and IgG4 subclass fractions, isolated from three different ANCA sera, each stimulated superoxide production from neutrophils derived from multiple donors. Subsequently, IgG4 subclass fractions isolated from a further four ANCA positive sera demonstrated varying abilities to stimulate release of superoxide; unrelated to PR3-ANCA titre, neutrophil donor, or neutrophil FcgammaRI expression. The stimulation of superoxide release by IgG1- and IgG3-ANCA subclass fractions is consistent with the proposed mechanism of co-ligation of PR3 antigen and FcgammaRIIa/IIIb receptors. However, the demonstration of similar activity for the IgG4-ANCA subclass fractions isolated from some sera was unexpected. This activity was independent of neutrophil donor and expression of FcgammaRI, suggesting it was capable of activating neutrophils via constitutively expressed FcgammaRIIa/IIIb or co-ligation of other, unidentified, cell surface molecules.

Fig. 1

Mean superoxide production stimulated by ANCA-IgG and fractionated ANCA-IgG subclasses from patients P2, P8 and P3 in primed neutrophils. Superoxide production was measured for 100 min in response to 100 µg/ml of ANCA-IgG or normal IgG. For ANCA-IgG subclasses, IgG1-ANCA from patient P2, shown in (a), was used at 49, 100 and 200 µg/ml; IgG3-ANCA from P8, shown in (b), was used at 13, 25, 50 and 100 µg/ml and IgG4-ANCA from P3, shown in (c), was used at 22, 50, 100 and 200 µg/ml. Results are expressed as nmol/1 × 10⁵ cells. Data shown are mean ± s.e.m. for experiments performed in triplicate on four neutrophil donors.

Fig. 2

Mean superoxide production stimulated by unfractionated ANCA-IgG and IgG4-ANCA samples from patients P1, P5, P7 and P9 in primed neutrophils. Superoxide production was measured for 100 min in response to 100 µg/ml of unfractionated ANCA-IgG (black bars) or normal-IgG (white bars) and 200 µg/ml IgG4-ANCA (dark grey bars). Results are expressed as nmol/1 × 10⁵ cells. Data shown are mean ± s.e.m. for experiments performed in triplicate on four neutrophil donors.s.e.m.

Fig. 3

(a) FACS analysis histograms of isolated neutrophils to detect PR3 and FcγRI expression. Dark grey histograms represent expression by unprimed, freshly isolated cells and black histograms represent expression after priming of cells for 15 min with 2 U/ml of TNF-α. Light grey bars histograms represent isotype control IgG1 binding. (b) FACS analysis histograms of isolated neutrophils, dual stained to detect annexin V and FcγRI expression. Dark grey histograms represent expression after priming of cells for 15 min with 2 U/ml of TNF-α. Black histograms represent expression after treatment with 400 U/ml of IFN-γ for 18 h while dashed line histograms represent cells cultured without IFN-γ for 18 h. Light grey histograms represent isotype control IgG1 binding.

Fig. 4

Mean superoxide production stimulated by P3 and pooled ANCA-IgG in fresh and IFN-γ-treated neutrophils. Unfractionated ANCA-IgG and IgG4-ANCA were isolated from the serum of patient P3 and from a pool made up of serum from patients P1, P5, P7 and P9. Superoxide production was measured for 100 min in response to unfractionated 100 µg/ml ANCA or normal IgG and 200 µg/ml IgG4-ANCA. Neutrophils were cultured for 18 h in medium supplemented with 400 U/ml IFN-γ or freshly isolated from the same donors. Results are expressed as nmol/l ×10⁵ cells superoxide measured at 100 min. Data shown are mean ± s.e.m. for experiments performed in triplicate on three neutrophil donors.