Azurocidin is a novel antigen for anti-neutrophil cytoplasmic autoantibodies (ANCA) in systemic vasculitis

Abstract

SUMMARYWe isolated a 27-kD protein using cation exchange chromatography from an acid extract of neutrophil granules. N-terminal amino acid sequence analysis of the first 10 residues showed that this protein is azurocidin, a member of the family of neutral serine proteinase found in the neutrophil, which shares amino acid sequence homology with the three other neutral serine proteinases, elastase, proteinase 3 (PR3) and cathepsin G, but unlike them is without proteolytic activity. To test whether, in addition to these proteases, azurocidin might be a target for the humoral autoimmune responses associated with human vasculitis, 185 indirect immunofluorescence (1IF)-positive ANCA sera, made up of four groups of sera with specificities for PR3 (n = 37), myeloperoxidase (MPO; n = 50), bactericidal/permeability-increasing protein (BPI; n = 41) and sera that recognized none of them (triple negative, n = 57), and 46 normal sera were screened for IgG anti-azurocidin antibodies using an ELISA incorporating purified azurocidin. Twenty of the 185 IIF-positive sera and 2/46 normal sera displayed reactivity with azurocidin. Positive sera could blot the 27-kD band by Western blot analysis. Further study of the 20 positive sera revealed that: (i) 10 also had autoreactivity for MPO, of which six additionally recognized lactoferrin; (ii) two had reactivity with BPI; (iii) the remaining eight sera were positive only for azurocidin. All 20 sera were from patients with systemic vasculitis, and four of the six sera with triple reactivity (for azurocidin, MPO and lactoferrin) were from patients with hydralazine-induced vasculitis. We concluded that: (i) azurocidin is a novel ANCA antigen; (ii) anti-azurocidin antibodies from a subgroup of patients might represent the consequence of a drug-induced multi-clone activation.

The target antigens of antineutrophil cytoplasmic antibodies (ANCA) induced by propylthiouracil

“…The seven known target ANCA antigens, including MPO, PR3, HLE, lactoferrin, cathepsin G, azurocidin and BPI were prepared as our previous reports [9][10][11][12]. Purified target antigens were used as solid phase ligands and were diluted to 1-2 μg/ml in coating buffer (0.05 mmol/L bicarbonate buffer pH 9.6, BPI in 0.01 mmol/L PBS) and coated onto the wells of a Costar microtitre plate (Data Packaging Corporation, MA, USA).…”

Clinical Implications of Excessive Neutrophil Extracellular Trap Formation in Renal Autoimmune Diseases

“…In addition, co-localization of NETs (determined as extracellular histones) with MPO and PR3 was demonstrated in kidney biopsies of AAV patients 34, 39. Autoantigens for atypical ANCAs, including azurocidin,50, 92 cathepsin G, 53 elastase,51, 53 lactoferrin,51, 53 lysosomal membrane protein-2, 58 and lysozym C 51 were demonstrated in NETs (Table 1). These atypical ANCAs are sometimes present in AAV patients, 92 but are also commonly associated with other systemic inflammatory diseases 92…”

“…The current data on NET-associated molecules, as identified by proteomics or immunofluorescence microscopy studies, that are known autoantigens in AAV and SLE are listed in Table 1 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91Table 1NET-associated molecules that are known autoantigens in AAV and/or SLENET- moleculesMethod of detectionNeutrophil localizationAuto-agRole in autoimmune diseaseRef.AzurocidinProteomics of PMA-induced NETs 49 Azurophilic granulesAAVAutoantibodies (atypical) present in AAV 50 Cathepsin GProteomics of PMA-induced NETs 49 Azurophilic granulesAAVSLEAutoantibodies (atypical) present in AAVAutoantibodies are present in SLE51, 52, 53, 54Neutrophil ElastaseProteomics of PMA-induced NETs 49 Azurophilic granulesAAVSLEAnti-elastase antibodies present in AAVAnti-elastase antibodies are present in SLE.51, 52, 55LactoferrinProteomics of PMA-induced NETs 49 Secondary granulesAAVSLEAtypical ANCA in AAVAutoantibodies are present in SLE…”

Antineutrophil Cytoplasmic Autoantibodies: How Should the Biologist Manage Them?

“…Ten percent of ANCA are anti-azurocidin antibodies. Usually, these antibodies are multispecifc with more frequently either anti-MPO or anti-lactoferrin specificities and could be associated with drug-induced vasculitis, as described for example with hydralazyn [43].…”