AANCANCA disease: disease: immunoserology and immunoserology and

pathogenesispathogenesis

Alenka Vizjak

Institute of Pathology · Faculty of Medicine University of Ljubljana, Ljubljana, Slovenia

Antineutrophil cytoplasmic Antineutrophil cytoplasmic antibodies (ANCA)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 diseaseANCA 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

ANCA antigensANCA 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 antigensANCA 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 testingANCA 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 orBPI, 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 (Sensitivity and specificity of ANCA (IIF IIF ++ ELISA for ELISA for PR3, MPOPR3, MPO) from different studies in the literature) 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 Selected demographic features and serologic findings in 423 findings in 423 ANCAANCA positive patients grouped positive patients grouped according to their clinico-pathologic settings according to their clinico-pathologic settings (Institute of Pathology, Faculty of Medicine, Ljubljana)(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 59.614.2 60.717.0 40.117.5 53.718.2

Female/male 93/60 38/21 52/21 80/48

PR3-ANCA 22951.0 51.17.8 24.11.9 33.84.4

MPO-ANCA 570.871.3 65.89.9 31.54.4 34.52.5

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

Clinico-pathologic diagnosis inClinico-pathologic diagnosis in relation to ANCA relation to ANCA antigen specificity in 423 antigen specificity in 423 ANCAANCA positive patients positive patients

(Institute of Pathology, Faculty of Medicine, Ljubljana)(Institute of Pathology, Faculty of Medicine, Ljubljana)

Diagnosis No of pts

PR3 MPO PR3+

MPO

Other ags

Wegener’s granulomatosis 56 45 6 2 3Microscopic polyangiitis 54 3 45 2 4Pauci-immune necr GN 28 2 24 0 0Churg-Strauss syndrome 2 0 1 0 1Skin vasculitis 8 1 3 0 4 Goodpasture sy + ANCA 5 0 5 0 0Suspected vasculitis 59 12 26 6 15IBD, AHBD, other diseases 211 20 76 22 93

Patogenetic role of ANCAPatogenetic role of ANCA • Clinical evidence

Correlation between ANCA values and activity of vasculitis, as well as relapsesDrug-induced ANCA vasculitis

• In vitro studiesActivation 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 diseaseMPO-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

ConclusionsConclusions

• 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.