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

Rachel Colman ¹, Abdullah Hussain ¹, Margaret Goodall ¹, Steven P Young ¹, Tanya Pankhurst ¹, Xiaomei Lu ¹, Royston Jefferis ¹, Caroline OS Savage ¹ and Julie M Williams ^1*

¹ University of Birmingham, United Kingdom

^* To whom correspondence should be addressed. E-mail: j.m.williams.med{at}bham.ac.uk.

Accepted 10 December 2006

Abstract

Objectives:Anti-neutrophil cytoplasm antibodies (ANCA) are associated with small vessel vasculitis and have been implicated in 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 IgG1 subclass and may be more likely to be associated with active disease and renal involvement. To address potential pathogenicity, chimeric antibodies were constructed of IgG1 and 3 subclasses with human IgG1 or 3 constant regions and a murine-derived variable region that binds an epitope within the ANCA antigen proteinase 3 (PR3) that is recognized by human autoantibodies.

Methods:The antibodies were characterized 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 IL-8 production although quantitative responses showed that IgG1 subclass favored degranulation and IgG3 subclass favored IL-8 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 humanized antibodies directed against a single epitope of PR3 can recapitulate the effects of polyclonal human ANCA, which recognizes multiple PR3 epitopes. Further, both PR3-ANCA of IgG1 and IgG3 subclass can activate neutrophils, although the more potent IL-8 response by IgG3 PR3-ANCA may encourage further neutrophil recruitment and amplify injury.

Keywords: IgG subclasses, anti-neutrophil cytoplasm antibodies, chimeric, neutrophil, vasculitis

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