1. ANCA disease: immunoserology and pathogenesis Alenka Vizjak Institute of Pathology · Faculty of Medicine University of Ljubljana, Ljubljana, Slovenia

2. Antineutrophil cytoplasmic antibodies (ANCA) Davies DJ et al. Necrotizing glomerulonephritis with ANCA. Med J 1982; 285:606 Van der Woude FJ et al. Autoantibodies against neutrophils and monocytes: Tool for diagnosis and marker of disease activity in Wegener's granulomatosis. Lancet 1985; 1:425 Jennette JC et al. ANCA-associated GN and vasculitis. Am J Pathol 1989; 135: 921 Discovery of ANCA – of key importance for the classification of small vessel vasculitides (in the kidney necrotizing crescentic GN) and understanding of their pathogenesis (1. immune complex, 2. anti-GBM, 3. ANCA)

3. ANCA disease Pathogenetically determined group of small- vessel vasculitides including: Wegener’s granulomatosis Microscopic polyangiitis Pauci-immune necrotizing crescentic GN = kidney-limited microscopic poliangiitis Churg-Strauss syndrome

4. ANCA antigens ANCA specific for various proteins, mostly enzymes, localized in the cytoplasmic lyzosomes of neutrophils and monocytes. Major target antigens for ANCA in patients with pauci-immune small vessel vasculitides : Myeloperoxidase (MPO) – epitope expressed by 130 kDa native molecule in the azurophilic granules Proteinase 3 (PR3) – 28 kDa serine proteinase, colocalized with MPO in the azurophilic granules

5. ANCA antigens Other ANCA antigens: lactoferrin, elastase, lysozyme, cathepsin G, azurocidin, bactericidal permeability increasing protein (BPI), alpha-enolase, defensin, unknown human lysosomal-associated membrane protein 2 (h-lamp-2) - homologous to bacterial protein FimH (Kain R et al. Nat Med 2008)

6. ANCA testing Indirect immunofluorescence (IIF) C-ANCA (  PR3) P-ANCA, with nuclear extension (  MPO) P-ANCA, without nuclear extension (  mostly unknown antigens) Atypical C-ANCA (  PR3 after treatment or BPI, MPO, other, often multiple antigens) Atypical ANCA (  other, often multiple antigens) Enzyme-linked immunosorbent assay (ELISA) Antigen specificity, quantitative value (relative)

9. Sensitivity and specificity of ANCA (IIF + ELISA for PR3, MPO) from different studies in the literature DiseaseSensitivity of ANCA ___________________________________________________ Limited Wegener's granulomatosis 50-66 % Generalized Wegener's granulomatosis80-98 % Microscopic polyangiitis 82-90 % Pauci-immune necrotizing extracap GN90-95 % Churg-Strauss syndrome 60-70 % Control groupSpecificity of ANCA ____________________________________________________ Patients with various other diseases 76-91 % Healthy subjects 94-99 %

10. Selected demographic features and serologic findings in 423 ANCA positive patients grouped according to their clinico-pathologic settings (Institute of Pathology, Faculty of Medicine, Ljubljana) FeaturesPauci-immune vasculitis (n=153) Suspected vasculitis (n=59) IBD, AHBD (n=83) Other diseases (n=128) Age 59.6  14.260.7  17.040.1  17.553.7  18.2 Female/male93/6038/2152/2180/48 PR3-ANCA 229  51.051.1  7.824.1  1.933.8  4.4 MPO-ANCA 570.8  71.365.8  9.931.5  4.434.5  2.5 IBD = inflammatory bowel diseases, AHBD = autoimmune hepato-biliary disorders

11. Clinico-pathologic diagnosis in relation to ANCA antigen specificity in 423 ANCA positive patients (Institute of Pathology, Faculty of Medicine, Ljubljana) Diagnosis No of pts PR3MPOPR3+ MPO Other ags Wegener’s granulomatosis 5645623 Microscopic polyangiitis 5434524 Pauci-immune necr GN 2822400 Churg-Strauss syndrome 20101 Skin vasculitis 81304 Goodpasture sy + ANCA 50500 Suspected vasculitis 591226615 IBD, AHBD, other diseases 211207676293

12. Patogenetic role of ANCA Clinical evidence Correlation between ANCA values and activity of vasculitis, as well as relapses Drug-induced ANCA vasculitis In vitro studies Activation of neutrophils by binding of ANCAs  release of destructive enzymes and toxic reactive oxygen radicals, as well as neutrophil extracellular traps; factors released by activated neutrophils activate the alternative complement pathway; ANCAs bind also to ANCA antigens adsorbed to anionic endothelium and GBM, enhancing complement dependent cytotoxicity; ANCAs disregulate neutrophil apoptosis  necrosis; specific T lymphocytes for PR3 Several animal models of ANCA disease MPO-ANCA, PR3-ANCA, anti-LAMP-2 antibodies

13. Falk RJ, Jennette JC. J Am Soc Nephrol 2010, 21: 745-752

14. De Lind van Wijngaarden RAF et al, Clin JASN, 2008, 3: 237-252

15. Conclusions PR3- and MPO-ANCA are highly specific diagnostic marker for pauci-immune small vessel vasculitides determined as ANCA disease. Rarely, ANCA specific for other antigens are involved in ANCA disease. ANCA can be not rarely positive in various other diseases, mostly with an atypical IIF pattern, in low values, and specificities for rare and unknown ANCA antigens. In view of clinical serologic correlations, as well as in vitro studies and in vivo animal models ANCA evidently have a key role in the pathogenesis of glomerular and vascular lesions in ANCA disease.