Department of Autoimmunology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S., Denmark
Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies that are specifically directed to multiple intracellular antigens in neutrophils and monocytes. Such neutrophil-specific autoantibodies have been assumed to exist since the early 1960s, when the indirect immunofluorescence (IIF) technique became popular as a screening method for demonstrating autoantibodies to tissue structures and cells. Intense interest in ANCA serology as a potential tool for diagnosing and monitoring disease activity in patients with Wegener's granulomatosis (WG) and necrotizing and crescentic glomerulonephritis (NCGN) arose in the 1980s. As the classical granular cytoplasmic ANCA (C-ANCA) seemed to be monospecific for the elastinolytic enzyme proteinase 3 (PR3) and the perinuclear ANCA (P-ANCA) appeared to be monospecific for the enzyme myeloperoxidase (MPO) in patients with primary small vessel vasculitides (SVV), it was assumed by some researchers that all neutrophil-specific autoantibodies were selectively directed to cytoplasmic granule constituents even in other ANCA-positive conditions, e.g. rheumatoid arthritis, Felty's syndrome and ulcerative colitis. Later studies have shown that this is not true, but the early nomenclature of ANCA, agreed upon at the second international workshop on ANCA, has been preserved. IIF-positive ANCA patterns not fitting the typical descriptions of C-ANCA and P-ANCA have been called atypical ANCA. Sera giving rise to such IIF patterns have been shown to target multiple antigens in neutrophils and the IIF pattern can be considered to be a summation of multiple reactivities to nuclear, cytosolic and various granular autoantigens [1].
Most patients with primary SVV, e.g. WG, microscopic polyangiitis (MPA), ChurgStrauss syndrome (CSS) and limited forms of these diseases, harbour ANCA directed against either PR3 or MPO, which occur in the azurophilic granules of neutrophils (PR3-ANCA and MPO-ANCA). PR3 is a 2930 kDa serine protease consisting of 229 amino acids and has a structure with considerable homology to neutrophil elastase. It has antimicrobial properties which are not solely dependent on its enzymatic activity, but the enzymatic activity can cause both tissue damage and degradation of phagocytosed bacteria and proteinaceous materials. MPO is another enzyme that is important in the bactericidal properties of neutrophils and monocytes by catalysing the formation of intracellular toxic products, e.g. HOCl, H2O2 and oxygen radicals. MPO-derived products can cause tissue damage by the halidation and oxidation of proteins and may pave the way for serine protease-induced proteolysis by inactivating the important protease inhibitor 1-antitrypsin. The MPO 140 kDa heterodimeric enzyme easily breaks down into smaller fragments by reduction and heating. The main epitopes on PR3 and MPO are conformational [2, 3], and only recently have investigators succeeded in producing recombinant PR3 and MPO with enzymatic activity and immunoreactive epitopes that are ideal for ANCA binding.
Most laboratories screen initially for the presence of ANCA using IIF on normal human peripheral blood leucocytes [4]. The classical C-ANCA pattern is strongly linked to the presence of PR3-ANCA, although exceptions exist. Certain sera containing MPO-ANCA give rise to a classical C-ANCA pattern for reasons that are unexplained. In contrast, the P-ANCA pattern can be caused by the binding of antibodies to a number of soluble cationic proteins which, upon ethanol permeabilization of the leucocytes, migrate and attach to the oppositely charged nucleus of the neutrophil or even the nucleus of an adjacent leucocyte. Thus, the diagnostic specificity of P-ANCA is low if the diagnosis of the patient is unknown. It is strongly recommended that the target antigen reactivity of any ANCA detected by IIF should be established. This is mostly done by direct enzyme immunoassay (EIA) using purified PR3 and MPO as the antigenic substrates, attached to the wells of high-binding microplates [5]. When this technique is used in conjunction with negative and positive calibrators and a positive standard serum, the presence and content of PR3-ANCA and MPO-ANCA can then be determined. PR3-ANCA are mostly found to be in the immunoglobulin (Ig) G1, IgG2 and IgG4 subclasses, but in active systemic disease the IgG3 subclass also occurs. Patients presenting with a haemorrhagic renopulmonary syndrome may also have IgM-class ANCA.
The conditions used for determining PR3-ANCA and MPO-ANCA by direct EIA were explored in a European multicentre standardization study carried out between 1991 and 1994. It was shown that PR3 preparations purified by three different methods from normal neutrophils or from purulent sputum were equally suitable as antigens when coated and used as described in detail by the group that produced the purified protein [5]. It took many rounds of studies to reach reasonable alignment of results between centres, and satisfactory results were not obtained until PR3 preparations had been coated on the microplates centrally and all centres used identical conditions for incubation, washing etc., and the same conjugate. After these preparatory studies had been done, coefficients of variation for PR3-ANCA between centres were brought down to <20%. One MPO preparation was used by all centres employing identical test conditions and conjugate, but in this instance the instability of MPO during mailing from the central laboratory to the participating laboratories made it necessary to coat the MPO on microplates locally. The results showed an interlaboratory coefficient of variation of around 30%, showing that this EIA could also be standardized reasonably well. Six of the participating laboratories had no previous experience of ANCA testing when these studies were performed.
In the final phase of the study, the standard IIF technique and the newly standardized EIA methods were used to reveal ANCA in sera from patients with different forms of primary SVV and in a number of disease control groups; these consisted of sera from patients with various connective tissue diseases with and without vasculitis, and healthy control sera that had been gathered from all 14 participating centres in Europe [6]. Altogether, sera from 159 new SVV patients and 103 previously diagnosed SVV patients were compared with sera from 184 inflammatory disease controls and 740 healthy controls. The use of IIF or EIA results alone showed unsatisfactory diagnostic specificity compared with in disease control sera. However, when the IIF and EIA results were combined, the presence of C-ANCA with PR3-ANCA showed 99% specificity for the diagnosis of primary SVV and so did the combination of P-ANCA with MPO-ANCA. It was agreed that both the IIF and the corresponding EIA had to be positive before a serum could be designated ANCA-positive in order to support a diagnosis of primary SVV. Using this definition, the nosological sensitivity of ANCA was found to be 73% in total for newly diagnosed WG and 67% for newly diagnosed MPA patients. Sensitivities in previously diagnosed patients were slightly lower (62 and 42% respectively). The main reason why ANCA were found less frequently than in most other publications describing patients with primary SVV is undoubtedly that the cut-off values for PR3-ANCA and MPO-ANCA positivity in our study were set high enough to give a diagnostic specificity of 9095% by the use of EIA alone compared with inflammatory disease controls. The improvement in diagnostic specificity when using disease controls rather than healthy controls was conspicuous. The last conclusion of the study was that practically no serum from any single patient with primary SVV harbours ANCA towards both PR3 and MPO.
A major obstacle in the past has been the nomenclature and classification of the various forms of vasculitis. The Chapel Hill proposals contained a number of terms and a few sentences defining each term, and was a step towards an easy-to-use terminology with as little possibility of overlap as was attainable at the time [7]. The definitions relating to this terminology do not include ANCA, but it is stated that their presence is limited in practice to WG, MPA, CSS and more limited forms of these diseases, such as renal-limited NCGN, subglottic stenosis and nose- and sinus-limited WG. These diseases have in common the predominant involvement of capillaries and venules, sometimes also of arterioles, but also the early invasion of the vessel walls by neutrophils, which may be responsible for the necrosis of the vascular wall. The involvement of larger vessels is seen in some patients, but by definition polyarteritis nodosa patients cannot simultaneously have small-vessel involvement. In accordance with this concept, polyarteritis nodosa patients do not produce ANCA. The selective presence of ANCA directed to one of two enzymes in the azurophilic granules probably reflects particular pathological events at the level of small vesselsevents that differ from those taking place outside the vessels in other neutrophil-dominated inflammatory conditions.
Most publications describing patients with active WG report that PR3-ANCA is found in about 7080% of patients and MPO-ANCA in only 10%. About 60% of patients with MPA or renal-limited NCGN have MPO-ANCA and about 30% have PR3-ANCA. The few studies on patients with CSS indicate equal frequencies of PR3-ANCA and MPO-ANCAaround 30% for each [8].
Two more recent techniques for quantifying PR3-ANCA may prove to be superior to direct EIA, namely capture EIA and quantitative immunoprecipitation. The former uses as a capturing ligand for PR3 a mouse monoclonal antibody directed to an epitope on PR3 that is rarely targeted by human ANCA [9]; apart from this variation it is a simple EIA technique. Its sensitivity seems to be somewhat higher than that of direct EIA, but the most important advantage seems to be that this method regularly shows rising concentrations of PR3-ANCA in patients with disease relapses. The epitopes on PR3 may be more accessible using this technique, which is also assumed to preserve the conformation of PR3 better. The immunoprecipitation technique uses radioactively labelled diisopropylfluorophosphate ([3H]DFP), which selectively binds with high affinity to PR3 and neutrophil elastase, as a label for these enzymes [10]. The labelled neutrophil extract is then incubated with serum, after which IgG-class antibodies are bound to Sepharose beads coated with protein G or A. After release of the bound IgG and immune complexes, the labelled constituent (PR3 or elastase) is identified by SDSPAGE (sodium dodecyl sulphatepolyacrylamide gel electrophoresis) or quantified directly on a microfilter. ElastaseANCA is not usually a problem in determining PR3-ANCA with this technique since elastaseANCA is extremely rare in primary SVV. Advantages are that whole neutrophil extracts can be used and the conformation of PR3 is well preserved because of minimal handling in vitro. Though the technique seems promising, it needs to be tested in many laboratories and on many SVV patients to judge its value and specificity in ANCA diagnostics.
Patients with drug-induced vasculitides or lupus erythematosus-like syndromes due to long-term intake of drugs such as propylthiouracil and hydralazine often develop ANCA. The characteristics of such ANCA are the strong ANCA (mostly P-ANCA) directed to multiple neutrophil-derived antigens, e.g. MPO, elastase, PR3 and sometimes cathepsin G, lysozyme, azurocidin or lactoferrin. When the drug treatment is discontinued, remission of clinical symptoms and abrogation of ANCA production is the rule [8].
Patients with long-standing infections or less effective protection against bacterial invasion, such as patients with cystic fibrosis, bronchiectases, ulcerative colitis etc., often produce ANCA directed towards the bacterial permeability-increasing protein (BPI), which is a 55 kDa antimicrobial product of azurophilic granules. In such patients the antibodies are commonly both IgG and IgA.
Though neutrophil-specific autoantibodies can be found in a subpopulation of patients with rheumatoid arthritis, ulcerative colitis, sclerosing cholangitis and chronic active hepatitis, the antibodies are found at much lower titres by IIF, often give atypical ANCA patterns and are directed towards a host of neutrophil proteins, e.g. cathepsin G, elastase, MPO, BPI, lysozyme, lactoferrin and less well characterized cytosolic antigens, with no major antigenic specificity. The most important antigens, however, are localized in the heterochromatin and nuclear membrane [11] and hence these antibodies should not be named ANCA.
ANCA directed to PR3 or MPO may be a tool for diagnosis and disease monitoring, but they are not yet criteria for primary SVV conditions. Diagnosis still rests on characteristic clinical manifestations and histopathological findings in biopsies from vasculitis-affected areas. A new classification system for SVV, based on the terms included in the Chapel Hill proposals and actual disease criteria, should be set up to assist in the early recognition of incipient SVV. This will allow treatment decisions to be taken early with the aim of preserving organ functions.
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