Chimeric antibodies to proteinase 3 of IgG1 and IgG3 subclasses induce different magnitudes of functional responses in neutrophils

Abstract

Background: Antineutrophil cytoplasmic antibodies (ANCA) are associated with small-vessel vasculitis and have been implicated in its pathogenesis. The subclass distribution of ANCA IgG deviates from normal patterns, and it has been suggested that the IgG3 subclass may have pathogenic potential over the IgG1 subclass and may be more likely to be associated with active disease and renal involvement. Objective: To deal with potential pathogenicity, chimeric antibodies were constructed of IgG1 and three subclasses with human IgG1 or three constant regions and a murine-derived variable region that binds an epitope within the ANCA antigen proteinase 3 (PR3) that is recognised by human autoantibodies. Methods: The antibodies were characterised for binding to PR3, including affinity and avidity, before being used as tools to explore their ability to activate human neutrophils for superoxide release, cytokine release, degranulation and ability to induce neutrophil adhesion under flow. Results: Both subclass antibodies elicited similar neutrophil responses for superoxide release, degranulation and interleukin (IL) 8 production, although quantitative responses showed that the IgG1 subclass favoured degranulation and the IgG3 subclass favoured IL8 production. Both antibodies were able to convert neutrophils from selectin-dependent rolling adhesion to integrin-dependent stationary adhesion in a flow assay. Conclusions: These findings indicate that humanised antibodies directed against a single epitope of PR3 can recapitulate the effects of polyclonal human ANCA, which recognises multiple PR3 epitopes. Further, PR3-ANCA of both IgG1 and IgG3 subclasses can activate neutrophils, although the more potent IL8 response by IgG3 PR3-ANCA may encourage further neutrophil recruitment and amplify injury.

ANCA-associated vasculitis is linked to carriage of the Z allele of α1 antitrypsin and its polymers

“…Neutrophil degranulation was assessed by measuring the release of MPO, as previously described 29. Experiments were repeated four times with differing neutrophil donor and IgG combinations.…”

Chimeric IgG4 PR3‐ANCA induces selective inflammatory responses from neutrophils through engagement of Fcγ receptors

“…Studies of the distribution of anti‐neutrophil cytoplasmic autoantibody (ANCA)‐mediated small vessel vasculitis (such as Wegener’s granulomatosis) have consistently reported the presence of antibodies of the IgG1 and IgG3 subclasses and it has been suggested that IgG3 titres may correlate with clinical disease activity, possibly because of their potential enhanced capacity to activate neutrophils (reviewed in ref. 1). However, raised IgG1 and IgG4 levels have been observed in patients with ANCA‐positive vasculitis and this is not the result of a general increase in titres of IgG4 as a whole 2 …”

Anti-Neutrophil Cytoplasmic Antibodies Stimulate Release of Neutrophil Microparticles

“…This priming step caused externalization of the ANCA autoantigens MPO and PR3 on the cell surface (assessed using flow cytometry; data not shown) but did not result in full neutrophil activation as assessed by superoxide release (data not shown), in agreement with the findings of others. 46 Primed neutrophils were then treated with either 200 mg/ml polyclonal MPO or PR3-ANCA derived from patients with AAV, control polyclonal IgG 200 mg/ml from healthy adult donors, 12.5 mg/ml chimeric IgG1 or IgG3 PR3-ANCA, 45 or control chimeric IgG1 or IgG3 antibody with specificity for the 4-hydroxyl-3-nitrophenylacetyl hapten for 60 minutes. In addition, a dose-response curve for NMP generation from primed neutrophils stimulated with chimeric IgG3 PR3-ANCA was plotted over the concentration 0-20 mg/ml ( Figure 2C).…”

“…45 These chimeric ANCAs were used at a concentration of 12.5mg/ml as described previously. 45 Both polyclonal IgG ANCAs from patients and chimeric ANCAs were confirmed to activate superoxide release 46 from human neutrophils; in contrast, both control antibodies had no such effect (data not shown).…”