P-ANCA staining pattern is the result of a redistribution of cationic hydrophilic substances such as MPO, elastase and lysozyme onto the oppositely charged nucleus after permeabilization of cells by ethanol. This ANCA reactivity was found in most patients with MPA and iNCGN, some patients with CSS and a few WG patients. "Atypical" P-ANCA staining patterns with different frequency were found in patients with IBD, autoimmune liver diseases, infectious diseases and connective tissue diseases (CTD) such as SLE and RA. MPO represents the P-ANCA target antigen with the greatest clinical utility because of the frequent association of MPO-ANCA with SVV. All serum samples should be assayed in PR3-ANCA and MPO-ANCA ELISAs, since about 5% of serum samples are positive only by ELISA. However, many sera that produce P-ANCA or "atypical" P-ANCA staining pattern on ethanol-fixed neutrophils do not contain autoantibodies to MPO or PR3 as tested by antigen-specific assays. Recent studies show that a number of these sera contain autoantibodies directed against a multiplicity of neutrophil constituents. In particular, autoantibodies to human leukocyte elastase (HLE), cathepsin G (CG), lactoferrin (LF), lysozyme (LZ), azurodicin (AZ), α-enolase, catalase, actin, tropomyosin, high motility groups of non-histone chromosomal proteins 1 and 2 (HMG1 and HMG2), bactericidal/permeability-increasing protein (BPI), lamin B1, histone H1 and 50 kD nuclear envelope membrane protein (Table 3.) Recently, a new term it has been proposed for this autoantibody population, namely "neutrophil-specific autoantibodies-NSA".

Table 3. Antineutrophil cytoplasmic antibodies (ANCA) staining patterns and associated target antigen in patients with systemic vasculitides and nonvasculitic disorders

WG-Wegener's granulomatosis; MPA-microscopic polyangiitis; CSS-Churg-Strauss syndrome; UC-ulcerative colitis; CD-Crohn's disease; PSC-primary sclerosing cholangitis; SLE-systemic lupus erythematosus; RA-rheumatoid arthritis; AIH-autoimmune hepatitis; HLE-human leukocyte elastase; BPI-bacteridical/permeability-increasing protein; HMG1/2-high mobility group of non-histone chromosomal proteins 1 and 2

Diagnostic significance of both PR3-ANCA and MPO-ANCA for SVV is not questionable (Table 4). The diagnostic relevance of ANCA depends on the clinical ordering guidelines for ANCA testing. The positive predictive value (PPV) of IIF ANCAs for SVV was very low (55-59%), with negative predictive value (NPV) between 84% and 99%, during ANCA testing in a routine clinical setting. Adherence to clinical ordering guidelines for ANCA testing restricted to patients with a reasonably high likelihood of SVV (Table 5) was reported to reduce the number of false-positive tests by 27%, without missing any cases of SVV. The total number of tests performed may thus be reduced by more than 20%.

The most clear-cut association of a disease with ANCA directed against a specific target antigen is the association between WG and PR3-ANCA. Between 80% to 95% of all ANCA in WG is C-ANCA. The use of more sensitive PR3-ANCA methods (capture ELISA) of detection has confirmed that the C-ANCA in WG is almost always associated with anti-PR3. An estimated 5-20% of ANCA in WG may be P-ANCA, which are mostly directed against MPO and only rarely directed against human leukocyte elastase (HLE). The sensitivity of C-ANCA/PR3-ANCA for WG is related to the extent, severity and activity of disease. In a meta analysis of C-ANCA in WG, the pooled sensitivity was 91% for the subset of patients with active disease compared to 63% for those with inactive disease. The specificity and positive predictive value of C-ANCA/PR3-ANCA for WG are very high (Table 4.). Most patients with MPA, iNCGN and CSS are ANCA positive, either with specificity for MPO or PR3. Other target antigens for ANCA in patients with SVV, such as BPI and AZ, may simultaneously occur with PR3-ANCA and MPO-ANCA. Whereas ANCA

Table 4. Disease associations of C-ANCA (PR3-ANCA) and P-ANCA (MPO-ANCA) in primary systemic small vessel vasculitides

PPV-positive predictive value

Table 5. Clinical ordering guidelines for ANCA testing.

as detected by IIF is found frequently in other inflammatory diseases, PR3-ANCA and MPO-ANCA are only rarely detected in disorders other than SVV. MPO-ANCA is also found in patients with anti-GBM disease. Futhermore, false-positive MPO-ANCA may be found occasionally in patients with SLE or RA and in other inflammatory disorders such as autoimmune liver disease and inflammatory bowel disease. These false positive results, however, can be avoided by using a capture ELISA to detect MPO-ANCA. For PR3-ANCA, the capture ELISA system does not seen to differ much from a direct ELISA system with respect to specificity but the capture ELISA seems to be more sensitive. Even though the presence for WG, MPA and/or CSS, a positive ANCA result should always be interpreted with consideration of the clinical setting since the presence of specific clinical patterns plays a major role in determinig the diagnostic probability of vasculitis.

ANCA-associated vasculitis has a 1-year survival of at least 80-90%. Treatment consists generally of a combination of prednisolone and cyclophosphamide, or other immunosuppressive drugs. Since all drugs that are used produce toxic side effect, medications are generally tapered and eventually eliminated in most cases. However, during folow-up, up to 80% of the patients in remission experience relapses. Patients with WG relapse more frequently than patients with MPA or renal limited vasculitis. In addition, patients with PR3-ANCA have more frequent relapses than patients with MPO-ANCA. This is also true when patients are subdivided into groups according to their diagnosis. So, patients with either WG and MPA who are PR3-ANCA positive have a higher relapse rate than patients with MPO-ANCA associated with the respective disease type. Thus, ANCA testing is not only a highly sensitive and specific test for making a diagnosis of WG, MPA or CSS, but ANCA antigen specificity has also a prognostic value with respect to the development of relapses during follow-up. Patients with PR3-ANCA may be at higher risk of death and patients with MPO-ANCA may be at higher risk for renal failures. Futhermore, relapses in PR3-ANCA positive patients are much more fulminant than relapses in MPO-ANCA positive patients. The causes of progression of renal failure differ between PR3 and MPO-ANCA positive patients. In patients with PR3-ANCA renal function is stable during remission, but declines with every relapse. In patients with MPO-ANCA, a slowly progressive course is often observed during follow-up without signs of clinically active disease. In these patients, proteinuria is the most important risk factor for renal failure during follow-up. In addition to ANCA specificity, ANCA levels at diagnosis and during follow-up have been shown to be predictive for patients renal and disease-free survival. A high PR3-ANCA level in capture ELISA at diagnosis is a risk factor for poor patient and renal survival and a constantly elevated MPO-ANCA level is a risk factor for poor renal survival. During induction therapy ANCA levels fall and become negative in many patients within the first few months. Persistent or recurring C-ANCA during the first year is significantly related to subsequent relapse. More than 80% of the patients who were ANCA positive at diagnosis and who experience a relapse, are test positive for ANCA at the time of relapse. So, the patients persistently negative for ANCA have a very low risk of develop relapse, although relapses localized to the respiratory tract can occur in these patients.

Relapses have a major impact on disease outcome in patients with SVV. Renal relapses during follow-up have recently been shown to be the most important predictor of long-term renal survival. Therefore, it is extremely important to identify patients at risk of relapse. The usefulness of serially measuring ANCA titers in predicting disease activity is at present still controversial. Several studies, most of them restrospective, have been published in which the relation between rises in ANCA levels as measured by IIF or by ELISA and disease activity of ANCA associated vasculitis was studied (Table 6 and 7).

Table 6. Relationships between increases in ANCA as determined by IIF and relapse of ANCA-associated small vessel vasculitis as reported by different studies

Table 7. Relationships between increases in ANCA as measured by ELISA and relapse of ANCA-associated small vessel vasculitis as reported by different studies

N.R. - not reported

Although direct evidence for the pathogenicity of ANCA in vasculitis is lacking, ample evidence from in vitro and in vivo studies support a potential role of ANCA in the development of vascular lesions.

PR3-ANCA and MPO-ANCA are able to inhibit enzymatic activity and prevent the inactivation of PR3 and MPO by its natural inhibitor alfa-1 antitrypsin and ceruloplasmin, respectively. In vitro, sera or purified IgG from ANCA-positive patients, as well as, monoclonal antibodies directed against MPO or PR3, have been found to induce an oxidative burst and degranulation in healthy human neutrophils pretreated with inflammatory cytokines such as tumor necrosis factor-α, IL-1β or bacterial lipopolysaccharide (LPS). Furthermore, ANCA-activated PMN are capable of damaging cultured endothelial cells. Indeed, freshly isolated and untreated PMNs from patients with ANCA-associated vasculitis are found to produce significantly more superoxide than PMN from normal control subjects. ANCA have been shown to activate monocytes to production of reactive oxygen species, IL-8, a potent attractant for PMN, and monocyte chemoattractant protein 1 (MCP-1), even without prior priming. Immunopathological studies have shown that inflammatory infiltrate is composed mainly of activated T lymphocytes, the majority of which are CD4+ and macrophages. T- lymphocytes isolated from WG patients proliferate in response to a crude neutrophil extract containing PR3 and MPO.

Although all of the aforementioned mechanisms may be operative in vivo in idiopathic vasculitis, conclusive evidence for the pathogenicity of ANCA awaits, however, a convicing animal model of ANCA-induced vasculitis. In a rat model of autoimmunity, the administration of mercuric chloride (HgCl2) to Brown Norway (BN) rats leads to a syndrome characterized by the presence of autoantibodies against a variety of antigens, including DNA, collagen, thyroglobulin, glomerular basement components and MPO. On pathologic examination of the animals, moderate acute tubular necrosis and lymphocytic infiltration in the interstitium and perivascularly can be observed. Autoantibodies against human MPO that cross-react with rat MPO, can be observed in BN rats immunized with human MPO. The autoimmune response alone does not result in clinical lesions. However, upon administration of human MPO and its substrate H202, these rats develop necrotizing glomerulonephritis with interstitial tubulonephritis, pulmonary and gastrointestinal vasculitis. Recently, it has been shown that Wistar-Kyoto rats immunized with purified human MPO in CFA develop alveolar lung haemorrhage and a mild glomerulonephritis. Moreover, recent studies on mouse models provided elegantly prove that MPO-ANCA alone induce pauci-immune glomerulonephritis and vasculitis. MPO deficient mice were immunized with mouse MPO and circulating anti-murine MPO antibodies were developed. Adoptive transfer, either of splenocytes or purified IgG derived from the MPO-immunized MPO-deficient mice, resulted in the development of crescentic glomerulonephritis and systemic vasculitis mimicking the human disease.

Schematic presentation of an integrative view of ANCA-mediated vascular tissue damage is shown in Figure 2. The model is based on four prerequisities for endothelial cell damage by ANCA: 1) the presence of ANCA; 2) expression of target antigens for ANCA on primed neutrophils and monocytes; 3) the necessity of an interaction between primed neutrophils and endothelium by means of β2-integrins and 4) activation of endothelial cells.