ab2006

Antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis after immunisation with bacterial proteins

J. Savige, L. Nassis, T. Cooper, B. Paspaliaris¹, P. Martinello², D. MacGregor²

University of Melbourne Department of Medicine and ²Department of Anatomical Pathology, Austin and Repatriation Medical Centre, Heidelberg, Victoria; ¹Department of Biochemistry, St Vincent's Hospital, Fitzroy, Victoria, Australia.

^{ABSTRACT

Objective}There is circumstantial evidence for a role for infections in the development of the small vessel vasculitides associated with antineutrophil cytoplasmic antibodies (ANCA). The aim of this study was to determine whether the immunisation of rats with bacterial proteins could result in circulating ANCA, T cells with specificity for ANCA antigens, and a systemic vasculitis.

Methods
Adult male Wistar rats were immunised with pasteurised sonicated S. aureus (n=7), E. coli (n=8), purified protein derivative (PPD, n=5), myeloperoxidase (MPO, n=5) or phosphate-buffered saline (PBS, n=5), in complete and in incomplete FreundÕs adjuvant. ANCA were assayed by indirect immunofluorescent (IIF) examination of normal rat neutrophils, and in ELISAs using human proteinase 3 (PR3), MPO and bactericidal/permeability-increasing protein (BPI). The T cell response to PR3, MPO and BPI was assessed by a whole blood T cell proliferative assay in vitro, and by a delayed type hypersensitivity (DTH) response in vivo. Kidney and bowel were examined histologically for evidence of vasculitis and colitis.

Results
One rat from each group immunised with S. aureus or E. coli developed pauciimmune segmental glomerular sclerosis. The rat immunised with E. coli had additionally an arteritis affecting renal interlobular and gut vessels. This rat had circulating C-ANCA, that produced granular cytoplasmic neutrophil fluorescence with central accentuation, but the target antigen could not be determined in ELISAs using human PR3, MPO or BPI. In animals immunised with S. aureus or E. coli, there was no significant T cell proliferative or DTH response specific for human PR3, MPO or BPI.

Conclusion
The development of ANCA and vasculitis in a rat immunised with bacterial proteins indicates that the relationship between infections and ANCA should be investigated further.

Key words
ANCA, cross-reactivity, epitopes, DTH, vasculitis.

Abbreviations:
BPI: bactericidal/permeability increasing protein; C-ANCA and P-ANCA: cytoplasmic and perinuclear antineutrophil cytoplasmic antibodies;
CFA and IFA: complete and incomplete FreundÕs adjuvant;
DTH: delayed type hypersensitivity;
HSA: human serum albumin;
IIF: indirect immunofluorescence;
MPO: myeloperoxidase;
PBS: phosphate-buffered saline;
PPD: purified protein derivative;
PR3: proteinase 3.

Please address correspondence to: A/Prof J. Savige, Department of Medicine, Austin and Repatriation Medical Centre, Heidelberg, VIC 3084, Australia.
E-mail: jsavige@austin.unimelb.edu.au