Translational mini-review series on immunology of vascular disease: accelerated atherosclerosis in vasculitis.

Premature atherosclerosis has been observed during the course of different systemic inflammatory diseases such as rheumatoid arthritis and sytemic lupus erythematosus. Remarkably, relatively few studies have been published on the occurrence of accelerated atherosclerosis in patients with vasculitis. In giant cell arteritis (GCA), mortality because of ischaemic heart disease is not increased. In addition, intima media thickness (IMT) is lower in patients with GCA than in age-matched controls. In contrast, IMT is increased significantly in Takayasu arteritis, another form of large vessel vasculitis occurring in younger patients. In Takayasu arteritis and in Kawasaki disease, a form of medium-sized vessel vasculitis, accelerated atherosclerosis has been well documented. In small vessel vasculitis because of anti-neutrophil cytoplasmic autoantibodies-associated vasculitis, cardiovascular diseases are a major cause of mortality. IMT measurements reveal conflicting results. During active disease these patients experience acceleration of the atherosclerotic process. However, when inflammation is controlled, these patients have atherosclerotic development as in healthy subjects. Several risk factors, such as diabetes and hypertension, are present more often in patients with vasculitis compared with healthy controls. In addition, steroids may be pro-atherogenic. Most importantly, many patients have impaired renal function, persistent proteinuria and increased levels of C-reactive protein, well-known risk factors for acceleration of atherosclerosis. Enhanced oxidation processes, persistently activated T cells and reduced numbers of regulatory T cells are among the many pathophysiological factors that play a role during acceleration of atherogenesis. Finally, autoantibodies that may be relevant for acceleration of atherosclerosis are found frequently in elevated titres in patients with vasculitis. Because patients have an increased risk for cardiovascular events, vasculitis should be treated with as much care as possible. In addition, treatment should be considered with angiotensin-converting-enzyme inhibitors and/or angiotensin receptor-1 blockers, statins and acetylsalicyl acid. Finally, classical risk factors for cardiovascular disease should be monitored and treated as much as possible.

Deoxyspergualin in relapsing and refractory Wegener's granulomatosis.

OBJECTIVES: Conventional therapy of Wegener's granulomatosis with cyclophosphamide and corticosteroids is limited by incomplete remissions and a high relapse rate. The efficacy and safety of an alternative immunosuppressive drug, deoxyspergualin, was evaluated in patients with relapsing or refractory disease. METHODS: A prospective, international, multicentre, single-limb, open-label study. Entry required active Wegener's granulomatosis with a Birmingham vasculitis activity score (BVAS) > or =4 and previous therapy with cyclophosphamide or methotrexate. Immunosuppressive drugs were withdrawn at entry and prednisolone doses adjusted according to clinical status. Deoxyspergualin, 0.5 mg/kg per day, was self-administered by subcutaneous injection in six cycles of 21 days with a 7-day washout between cycles. Cycles were stopped early for white blood count less than 4000 cells/mm(3). The primary endpoint was complete remission (BVAS 0 for at least 2 months) or partial remission (BVAS <50% of entry score). After the sixth cycle azathioprine was commenced and follow-up continued for 6 months. RESULTS: 42/44 patients (95%) achieved at least partial remission and 20/44 (45%) achieved complete remission. BVAS fell from 12 (4-25), median (range) at baseline to 2 (0-14) at the end of the study (p<0.001). Prednisolone doses were reduced from 20 to 8 mg/day (p<0.001). Relapses occurred in 18 (43%) patients after a median of 170 (44-316) days after achieving remission. Severe or life-threatening (> or = grade 3) treatment-related adverse events occurred in 24 (53%) patients mostly due to leucopaenias. CONCLUSIONS: Deoxyspergualin achieved a high rate of disease remission and permitted prednisolone reduction in refractory or relapsing Wegener's granulomatosis. Adverse events were common but rarely led to treatment discontinuation.

A novel enzyme-linked immunosorbent assay using a mixture of human native and recombinant proteinase-3 significantly improves the diagnostic potential for antineutrophil cytoplasmic antibody-associated vasculitis.

BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCA) with a C-ANCA or P-ANCA pattern are detected in ANCA-associated vasculitis (AAV). While in most patients with AAV a C-ANCA pattern is due to reactivity with proteinase-3 (PR3)-ANCA, some C-ANCA-positive sera do not react with PR3. OBJECTIVE: The development and evaluation of a direct enzyme-linked immunosorbent assay (ELISA) for PR3-ANCA with increased sensitivity. METHODS: A mixture of human native (hn) and human recombinant (hr) PR3 was used as antigen coating. The resulting ELISA (anti-PR3-hn-hr) was compared with ELISAs using directly coated hn-PR3 or hr-PR3, as well as with a hn-PR3 capture ELISA. Assay characteristics were determined in patients with AAV (n = 248), with special attention for those patients with C-ANCA (n = 132), as well as disease controls (n = 585) and healthy controls (n = 429). Additionally, for prediction of relapses serial samples of 46 patients with PR3-AAV were analysed. RESULTS: At a predefined specificity of 99% both ELISAs containing hr-PR3 revealed a substantial increase in sensitivity. For the prediction of relapses by rises in PR3-ANCA titres the capture ELISA was most optimal (odds ratio 12.5). With an odds ratio of 8.9 the novel anti-PR3-hn-hr ELISA was second best. CONCLUSIONS: Owing to the very high sensitivity of the novel anti-PR3-hn-hr ELISA for the detection of PR3-ANCA in C-ANCA-positive samples of patients with AAV this assay has an excellent diagnostic performance. This feature is combined with a good predictability of clinical relapses in patients with PR3-AAV. These characteristics challenge the dogma that, for detection of PR3-ANCA, capture ELISAs are superior for diagnosis and follow-up.

Anti-oxLDL antibody isotype levels, as potential markers for progressive atherosclerosis in APOE and APOECD40L mice.

In humans and animal models of atherosclerosis, antibodies against oxidized LDL have been associated with atherosclerotic lesion development. It has been suggested that IgM anti-oxLDL antibodies are anti-atherogenic, whereas IgG anti-oxLDL antibodies are pro-atherogenic. In this study, we examined the relation between IgM and IgG antibody levels and atherosclerosis severity in APOE(-/-)CD40L(-/-) mice, which are deficient for IgG and develop moderate advanced atherosclerosis, and compared results with mice developing severe (APOE(-/-)) or no atherosclerosis (C57Bl/6). Mice were followed in time for anti-oxLDL antibodies while on high-fat diet or normal chow. Anti-oxLDL antibody levels were determined by ELISA. Results revealed that 24-week-old APOE(-/-)CD40L(-/-) mice had enhanced IgM anti-oxLDL antibody levels when compared with wild-type mice, but similar levels to those of APOE(-/-) mice. As expected, IgG anti-oxLDL antibody levels were almost absent in APOE(-/-)CD40L(-/-) mice. The transition from early to advanced lesions in APOE(-/-) mice was reflected by elevated IgM anti-oxLDL antibody levels. IgM anti-oxLDL levels did not further increase during progression to more advanced lesions. No relation was found between IgG anti-oxLDL levels and atherosclerosis severity. In conclusion, the severity of advanced atherosclerosis in mice is not reflected by IgM and/or IgG anti-oxLDL antibody levels. Furthermore, less advanced atherosclerotic lesion development in APOE(-/-)CD40L(-/-) mice does not seem to be the result of higher levels of protective IgM anti-oxLDL antibodies. Therefore, our study does not support the idea that the previously observed inconsistency in the relation between anti-oxLDL and atherosclerosis severity is due to differences in antibody isotypes.

[ANCA(antineutrophil cytoplasmic antibodies)-associated vasculitis in a man with extreme fatigue, fever and progressive renal dysfunction]. / Vasculitis geassocieerd met antineutrofielencytoplasmatische antistoffen (ANCA) bij een man met ernstige vermoeidheid, koorts en een progressieve nierfunctiestoornis.

A 55-year-old man, with no previous history, presented with extreme fatigue and fever and was admitted to hospital. He had progressive renal dysfunction and his serum anti-neutrophil cytoplasmic antibodies (ANCA) were markedly elevated. Renal histology was consistent with ANCA-associated vasculitis. The patient was successfully treated with cyclophosphamide and prednisolone. The classification and management of the ANCA-associated vasculitides are described. The classification was guided by the clinical presentation, serology and results of tissue biopsies. The ANCA inflammation had affected the middle sized and small vessels of especially the upper and lower airways, and the kidneys. The antibodies were directed at proteinase-3 (PR3) or myeloperoxidase (MPO). PR3-ANCA is predominantly found in Wegener's granulomatosis, while MPO-ANCA is related to microscopic polyangiitis. Tissue studies showed granulomatous inflammation of the airways which is typical of Wegener's disease. This type of inflammation is absent in microscopic polyangiitis. The initial treatment schedule consists of prednisone 1 mg/kg daily and oral cyclophosphamide 2 mg/kg daily. In the remission phase, the cyclophosphamide is replaced by azathioprine. It is not yet known how long maintenance treatment should be continued and which parameters have prognostic value.

Type II cryoglobulinemia is not associated with hepatitis C infection: the Dutch experience.

Mixed cryoglobulinemia (MC) are cryoprecipitable immunocomplexes. In type II MC, a combination of polyclonal and monoclonal immunoglobulins is found, whereas in type III a combination of polyclonal immunoglobulins is detected. MC is usually associated with hepatitis C (HCV) infection as has been found in studies that have been performed in countries with a high prevalence of HCV. Because HCV has an extremely low prevalence in the Netherlands (<0.1% of the population), we wondered whether HCV is also associated with MC in our regional referral center. To answer this question, we tested consecutive patients with type II MC for HCV antibodies and for HCV-mRNA by polymerase chain reaction (PCR). Between January 2000 and June 2005, 22 patients tested positive for type II MC. Seven patients had essential MC, 2 patients had MC due to a lymphoproliferative disease, 10 patients had MC in the context of a systemic autoimmune disease, and 3 patients had MC without a clear diagnosis. HCV antibodies were not detected in any of the 22 patients. Also, all samples tested negative for HCV-mRNA. During follow-up none of these patients developed an HCV infection. In summary, the estimated occurrence of HCV in 60-90% of patients with MC is not found in our region where MC is only infrequently associated with HCV. In a substantial proportion of our patients a really "essential MC" is observed. A search for yet unknown etiological factors is clearly needed in these patients, who frequently have severe renal involvement warranting aggressive immunosuppressive therapy.

Evaluation of the FIDIS vasculitis multiplex immunoassay for diagnosis and follow-up of ANCA-associated vasculitis and Goodpasture's disease.

We have evaluated a new-multiplex immunoassay (FIDIS Vasculitis) for simultaneous detection and quantification of anti-MPO, -PR3, and -glomerular basement membrane (GBM) antibodies in diagnosis and follow-up of ANCA-associated vasculitides (AAV) and Goodpasture's disease. ANCA were determined in sera of (a) 87 consecutive patients with biopsy-proven pauci-immune NCGN and 72 controls; (b) 9 patients with Goodpasture's disease; and (c) 60 WG patients and 60 controls, previously used in a multicenter comparison of direct and capture ELISA for PR3-ANCA. Finally, for prediction of relapses, PR3-ANCA was measured in samples preceding relapse in 23 PR3-AAV patients and in 23 matched PR3-AAV patients without relapse. The relative sensitivity of the FIDIS Vasculitis assay was 97.4% for MPO-ANCA and 92.3% for PR3-ANCA; specificity was 100% and 97.2%, respectively. Evaluation of the anti-GBM antibody detection revealed a sensitivity of 100% and a specificity of 99.6%. The sensitivity for WG of the PR3-ANCA detection (71.6%) approached the performance of capture ELISA (74%), although at the cost of specificity (96.7% versus 100%). For prediction of relapses a rise of 50% in ANCA level by FIDIS Vasculitis appeared optimal (ROC curve) for prediction of relapses. However, as compared to capture ELISA, both positive (63% versus 76%) and negative (68% versus 72%) predictive values were reduced. In conclusion, simultaneous detection of anti-MPO, -PR3, and -GBM antibodies in the multiplex FIDIS Vasculitis assay has excellent performance in terms of diagnosis of patients with AAV or Goodpasture's disease. However, detection of rises in PR3-ANCA for prediction of relapses gives less optimal results when compared to capture ELISA.

Induction of endothelial cell apoptosis by IgG antibodies from SLE patients with nephropathy: a potential role for anti-endothelial cell antibodies.

Systemic lupus erythematosus (SLE) is a prototype of an autoimmune disease with vasculopathy as demonstrated by the presence of vascular immune-complex deposition, inflammation, and thrombosis. A pivotal role in the initiation of vasculopathy is ascribed to vascular endothelium. In this respect, anti-endothelial cell antibodies (AECA), which are highly associated with SLE, are putative candidates for the initiation of SLE vasculopathy. In addition to the potency of AECA to induce a proinflammatory endothelial cell phenotype, AECA have also been described to trigger endothelial cell apoptosis. However, in SLE data are not uniform on the potentials of AECA to induce endothelial cell apoptosis in vitro. We have addressed this question in a cohort of SLE patients with nephropathy. AECA levels, and the apoptosis-inducing potentials of serum IgG were measured at the time of renal complication and biopsy. Also serum antibody reactivity with various SLE-related autoantigens including HSP60 was determined in patients. The results show that the SLE patient group has increased AECA levels as well as increased levels of induction of endothelial cell apoptosis by serum IgG. AECA and apoptosis values largely varied among the patients. Our data show that antibodies other than anti-HSP60 are also involved in apoptosis induction. The results are discussed in the context of recent findings on the role of AECA in endothelial cell apoptosis and renal vasculopathy in SLE.

Mechanisms of vasculitis: how pauci-immune is ANCA-associated renal vasculitis?

Both the innate and the acquired immune system are involved in the pathophysiology of renal vasculitis. However, anti-neutrophil cytoplasmic antibody (ANCA)-associated renal vasculitis is characterized by a 'pauci-immune' pattern of immunofluorescence during kidney biopsy, indicating the relative lack of immunoglobulin and complement deposition within the kidney. On the other hand, evidence is accumulating that ANCA, autoantibodies against constituents of primary granules of neutrophils and the lysosomes of monocytes, play a pathogenic role in renal vasculitis. In this review we will discuss both in vitro and in vivo experimental data providing compelling evidence that ANCA are a primary pathogenic factor in renal vasculitis, mainly by augmenting leukocyte-endothelial interactions. We will also address novel data, pointing at the role of, in addition to ANCA, non-specific proinflammatory signals. Finally, we propose a working hypothesis of the pathogenesis of ANCA-associated renal vasculitis.

From ANA to ENA: how to proceed?

Anti-nuclear antibodies (ANA), as detected by indirect immuno-fluorescence, are hallmarks of autoimmune connective tissue diseases. Identification of the specificity for extractable nuclear antigens (ENA) is warranted because this may further differentiate between the distinct types of autoimmune connective tissue diseases. In recent years several different ENA, as recognized by ANA, have been identified and the knowledge of the molecular structure has been expanded. Together with technical developments this has enabled the introduction of several new anti-ENA antibody detection systems. In this review we will discuss the main logistic aspects of anti-ENA antibody testing that have to be solved in order to come to a consensus in order to deal with new developments in this field. We conclude that: 1. a positive ANA test should, depending on the titre and pattern, be followed by an anti-ENA antibody assay, 2: to fully appreciate the value of the new anti-ENA antibody detection systems a large, multicenter clinical evaluation is required, and 3: proper interpretation of reported test results requires that the clinician is aware of the way anti-ENA antibodies are detected and reported.

Leukocyte CD40L deficiency affects the CD25(+) CD4 T cell population but does not affect atherosclerosis.

Inhibition of CD40-CD40L interactions results in a reduction of innate regulatory T cells (Tregs) in CD40(-/-) mice and induces a stable plaque phenotype in atherosclerosis-prone mouse strains. Here we investigated the effects of leukocyte CD40L on the Treg population and on atherosclerosis. LDLR(-/-) mice were reconstituted with wild-type or CD40L(-/-) bone marrow (BM). These BM chimeras were analysed by flow cytometry for the presence of innate Tregs (CD45RB(low) CD25(+) CD4) in lymphoid organs and peripheral blood. As in CD40(-/-) mice, the CD45RB(high):CD45RB(low) CD4 T cell ratio significantly increased and the CD25(+) CD4(+) subpopulation significantly decreased in LDLR(-/-) mice receiving CD40L(-/-) BM compared to LDLR(-/-) mice receiving wild-type BM. However, atherosclerotic plaque progression and plaque phenotype did not change in LDLR(-/-) mice reconstituted with CD40L(-/-) BM. In conclusion, the present study shows that CD40-CD40L interactions on leukocytes are essential for the size of the CD45RB(low) CD25(+) CD4 Treg subpopulation. Nevertheless, CD40L deficiency on hemopoietic cells did not affect atherosclerosis, implying that CD40L expressing leukocytes alone are not responsible for the stable plaque phenotype observed after total CD40L blockade.

Cross-reactivity of IgM and IgG anticardiolipin antibodies with oxidized-low density lipoproteins.

Anticardiolipin antibodies (aCLAs) and antibodies to oxidized-low density lipoproteins (oxLDL) are associated with two distinct diseases: the antiphospholipid syndrome and atherosclerosis. Because both diseases may be apparent in patients with systemic lupus erythematosus (SLE), it is important to establish the relationship between these two types of antibodies. In the present study, we examined whether sera containing IgM and/or IgG aCLAs also react with LDL that has been oxidized by conjugation with malondialdehyde (MDA-LDL) or by incubation with copper ions (Cu-LDL). Results revealed a clear correlation between IgM aCLAs and IgM anti-MDA-LDL antibodies, and a weak correlation between IgG aCLAs and IgG anti-Cu-LDL antibodies. Cross-reactivity between both antibodies seemed to be limited. Because aCLAs are heterogeneous, only a minor subset of these antibodies may cross-react with oxLDL. Therefore, identification of both antibodies may be relevant for determination of the prognosis of accelerated atherosclerosis in SLE patients.

Evaluation of a novel line-blot immunoassay for the detection of antibodies to extractable nuclear antigens.

We have evaluated the performance of a novel line-blot immunoassay (LIA; Mikrogen) and compared results with those obtained by CIE (in-house), ELISA (Pharmacia Diagnostics), and FEIA (Pharmacia Diagnostics). Sera from systemic lupus erythematosus (SLE) patients (n = 123), systemic sclerosis patients (n = 25), and healthy controls (n = 40) were analyzed for the presence of antibodies to RNP, Sm, SSA, SSB, CENP-B, Scl-70, and Jo-1. Reading of LIA results, as compared with a cutoff control, was performed by automatic analysis of the test strips. Because LIA enables recognition of separate subunits of RNP (68, A, and C), Sm (B and D), and SSA (52 and 60), at least two of the RNP antigens and either one of the Sm or SSA antigens should be detected for considering the test RNP, Sm, or SSA-positive, respectively. LIA had the highest sensitivity in patients with autoimmune connective tissue diseases: 131 specificities (not PO, PCNA, or histones), as compared with ELISA (121), FEIA (119), and CIE (80). However, LIA revealed three positive reactions in healthy controls; other assays were completely negative. LIA is better than CIE, but similar to ELISA and FEIA, in terms of detecting systemic sclerosis-associated antibodies (CENP-B and Scl-70). Furthermore, LIA had the highest sensitivity (17.9%) for the SLE-specific anti-Sm antibodies, as compared with ELISA (11.4%), CIE (8.1%), and FEIA (5.7%). Finally, anti-SSA antibodies were far more prevalent by LIA in the systemic sclerosis samples because of anti-SSA52 reactivity. The clinical relevance of the latter finding remains to be determined. In conclusion, LIA is suitable for routine evaluation of autoantibodies to extractable nuclear antigens.

Dapsone hypersensitivity syndrome not related to G6PD deficiency.

Dapsone hypersensitivity syndrome (DHS) is a rare, but potentially life-threatening reaction to dapsone. We describe a 55-year-old Caucasian woman with normal glucose-6-phosphate dehydrogenase levels presenting with an extensive skin eruption, high-grade fever, pneumonitis and hepatitis, which occurred within 3 weeks after initiation of dapsone. In addition to supportive care, the patient was successfully treated with high-dose corticosteroids and antibiotics. The combination of high-grade fever, skin rash, lung and liver involvement made a dapsone hypersensitivity syndrome very likely.

Levamisole-contaminated cocaine: a hairy affair.

Levamisole-contaminated cocaine can induce severe systemic vasculitis. The diagnosis can be challenging, especially when substance abuse is uncertain. We present the case of a 42-year-old woman suffering from vasculitis due to levamisole-contaminated cocaine, who persistently denied substance abuse. Symptoms included ulcerating skin lesions, arthralgia and myalgia, and the occurrence of an ileal intussusception. The definitive diagnosis was made using hair testing for toxins. She recovered through cocaine abstinence, but re-exposure resulted in a severe relapse with glomerulonephritis. Importantly, at time of the relapse, the patient became positive for both myeloperoxidase-antineutrophil cytoplasmic antibody (ANCA) and proteinase 3-ANCA. Cocaine-levamisole-induced vasculitis poses a great clinical challenge. The proper diagnostic strategy and therapy is still controversial. We highlight our diagnostic and therapeutic considerations, including hair testing for definitive proof of exposure.

AECA and ANCA in patients with premature atherosclerosis.

UNLABELLED: Autoimmunity is suggested to play a pathogenetic role in premature atherosclerosis. Since atherosclerosis and vasculitis seem pathogenetically related, we hypothesized that ANCA, an important antibody in vasculitis, plays a role in atherosclerosis as well. We therefore investigated the prevalence of ANCA in patients with premature atherosclerosis and related the presence of these antibodies to levels of AECA and markers of inflammation. METHODS & RESULTS: In a cohort of 286 patients with premature atherosclerosis the prevalence of ANCA was 5.6% (16/286). All had perinuclear ANCA. More females were ANCA-positive (8M/8F vs. 200M/70F, p = 0.03). In a nested case-control study, comparing the 16 ANCA-positive patients with 32 controls, levels of AECA were higher in the first (7.32 +/- 0.91U vs. 5.52 +/- 0.41U, p < 0.05). CONCLUSION: ANCA does not seem to play a major role in premature atherosclerosis. Whether elevated levels of AECA in ANCA-positive patients with premature atherosclerosis reflect more extended vascular disease remains to be determined.

Cardiac involvement of Churg Strauss syndrome demonstrated by magnetic resonance imaging.

Churg Strauss syndrome (CSS) may lead to cardiac involvement in up to 60% of patients. The myocardium, coronary vasculature, valves and pericardium may be affected. This results in significant morbidity and mortality, accounting for 48% of deaths due to CSS. Cardiac magnetic resonance imaging (CMR) is used to evaluate cardiac structure and function, and is able to evaluate myocardial perfusion and delineate scar tissue. We are the first to demonstrate these features in a 53-year-old CSS patient who presented with palpitations and atypical chest pains, and was found to have myocardial perfusion defects and scar tissue, most likely secondary to vasculitis of the small myocardial vasculature and myocardial infiltration.

Increased alpha-linolenic acid intake lowers C-reactive protein, but has no effect on markers of atherosclerosis.

OBJECTIVE: To investigate the effects of increased alpha-linolenic acid (ALA)-intake on intima-media thickness (IMT), oxidized low-density lipoprotein (LDL) antibodies, soluble intercellular adhesion molecule-1 (sICAM-1), C-reactive protein (CRP), and interleukins 6 and 10. DESIGN: Randomized double-blind placebo-controlled trial. SUBJECTS: Moderately hypercholesterolaemic men and women (55 +/- 10 y) with two other cardiovascular risk factors (n = 103). INTERVENTION: Participants were assigned to a margarine enriched with ALA (fatty acid composition 46% LA, 15% ALA) or linoleic acid (LA) (58% LA, 0.3% ALA) for 2 y. RESULTS: Dietary ALA intake was 2.3 en% among ALA users, and 0.4 en% among LA users. The 2-y progression rate of the mean carotid IMT (ALA and LA: +0.05 mm) and femoral IMT (ALA:+0.05 mm; LA:+0.04 mm) was similar, when adjusted for confounding variables. After 1 and 2 y, ALA users had a lower CRP level than LA users (net differences -0.53 and -0.56 mg/l, respectively, P < 0.05). No significant effects were observed in oxidized LDL antibodies, and levels of sICAM-1, interleukins 6 and 10. CONCLUSIONS: A six-fold increased ALA intake lowers CRP, when compared to a control diet high in LA. The present study found no effects on markers for atherosclerosis. SPONSORSHIP: The Dutch 'Praeventiefonds'.

Pathophysiology of ANCA-associated vasculitides: are ANCA really pathogenic?

The strong relation between antineutrophil cytoplasmic autoantibodies (ANCA) and primary vasculitic syndromes suggests a pathophysiological role for ANCA. Experimental evidence for the pathogenic potential of ANCA has been derived from in vitro studies that demonstrate that ANCA can activate tumour necrosis factor alpha primed neutrophils, monocytes and/or endothelial cells. The binding of ANCA to primed neutrophils results in activation of these cells by a process that is largely dependent on engagement of beta-2 integrins and on the interaction of the Fc portion of ANCA. An Fc-independent mechanism is, however, also operative. In experimental animal models, it has been demonstrated that immunisation with myeloperoxidase (MPO) induces MPO-ANCA. The induction of ANCA, however, is not sufficient to induce vasculitis in rats since immune complexes first have to be deposited along the vessel wall before lesions develop. When MPO-deficient mice are, however, immunised with murine MPO, anti-MPO immunoglobulins are purified and subsequently injected into mice that are not deficient for MPO, systemic vasculitis and glomerulonephritis is induced. These experiments suggest that ANCA indeed induces vasculitis. Risk factors for breaking self-tolerance to ANCA antigens are genetic factors, drugs, chemical substances and/or infectious agents.