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

2. Antineutrophil cytoplasmic antibodies (ANCA)
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)
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

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
Disease Sensitivity of ANCA
___________________________________________________
Limited Wegener's granulomatosis %
Generalized Wegener's granulomatosis %
Microscopic polyangiitis %
Pauci-immune necrotizing extracap GN %
Churg-Strauss syndrome %
Control group Specificity of ANCA
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Patients with various other diseases %
Healthy subjects %

10. Pauci-immune vasculitis (n=153) Suspected vasculitis (n=59)
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)
Features
Pauci-immune vasculitis (n=153)
Suspected vasculitis (n=59)
IBD, AHBD (n=83)
Other diseases (n=128)
Age
59.614.2
60.717.0
40.117.5
53.718.2
Female/male
93/60
38/21
52/21
80/48
PR3-ANCA
22951.0
51.17.8
24.11.9
33.84.4
MPO-ANCA
570.871.3
65.89.9
31.54.4
34.52.5
IBD = inflammatory bowel diseases, AHBD = autoimmune hepato-biliary disorders
Vizjak A et al. Wien Klin Wochenschr 2000, 112:

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
PR3
MPO
PR3+
Other ags
Wegener’s granulomatosis 56 45 6 2 3
Microscopic polyangiitis 54 4
Pauci-immune necr GN 28 24
Churg-Strauss syndrome 1
Skin vasculitis 8
Goodpasture sy + ANCA 5
Suspected vasculitis 59 12 26 15
IBD, AHBD, other diseases
211 20 76 22 93
Vizjak A et al. Wien Klin Wochenschr 2000, 112:

12. Pathogenetic 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.