ANCA - Nephropathology Working Group

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

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)

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

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

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)

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)

Sensitivity and specificity of ANCA (IIF + ELISA for PR3, MPO) from different studies in the literature Disease Sensitivity of ANCA ___________________________________________________ Limited Wegener's granulomatosis 50-66 % Generalized Wegener's granulomatosis 80-98 % Microscopic polyangiitis 82-90 % Pauci-immune necrotizing extracap GN 90-95 % Churg-Strauss syndrome 60-70 % Control group Specificity of ANCA ____________________________________________________ Patients with various other diseases 76-91 % Healthy subjects 94-99 %

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)

59.614.2

60.717.0

40.117.5

53.718.2

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

Age Female/male

IBD = inflammatory bowel diseases, AHBD = autoimmune hepato-biliary disorders

Clinico-pathologic diagnosis in relation to ANCA antigen specificity in 423 ANCA positive patients (Institute of Pathology, Faculty of Medicine, Ljubljana) No of pts

PR3

MPO

PR3+ MPO

Other ags

56 54 28

45 3 2

6 45 24

2 2 0

3 4 0

Skin vasculitis

2 8

0 1

1 3

0 0

1 4

Goodpasture sy + ANCA

5

0

5

0

0

59 211

12 20

26 76

6 22

15 93

Diagnosis Wegener’s granulomatosis Microscopic polyangiitis Pauci-immune necr GN Churg-Strauss syndrome

Suspected vasculitis IBD, AHBD, other diseases

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

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

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

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.