Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis


Gunnels Wood Road, Stevenage, Herts, SG1 2NY, UK.


Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is an autoimmune disease in which the contributions of genetic, epigenetic and environmental factors to aetiology and pathogenesis are being unravelled. The ANCA immunoglobulin G targeting proteinase 3 and myeloperoxidase affects several neutrophil functions, usually to augment or dysregulate these, promoting a proinflammatory phenotype whereby neutrophils have enhanced capabilities of causing collateral damage to endothelial and other cells. In addition, B cells are intimately involved in pathogenesis as anti-B cell therapies are highly effective, but the manner of this involvement still needs to be delineated. Similarly, the T cell compartment is disturbed in ANCA vasculitis and numerous alterations in T cell subsets have been described, but recognition of a novel CD8+ T cell transcription signature which can predict likelihood of relapse in ANCA vasculitis indicates that more needs to be learnt about the influence of T cells in the disease process. Finally, the role of the alternative complement pathway and the potential therapeutic value of its neutralization is under active investigation after compelling studies in murine models have demonstrated that C5 and factor-B knock-out mice are protected.


Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is a complex disease with a strong underlying autoimmune diathesis. Its precise aetiology remains unknown, but contributions from both heritable and environmental factors seems certain. The pathogenic mechanisms that are then triggered involve diverse cell types, inflammatory mediators and signalling cascades. What have we learned from this bewildering array of altered biological processes about the pathogenesis of the disease over the past 2 years?


Turning first to the genome, familial segregation of Wegener's granulomatosis (WG) with a 1·56 relative risk for first-degree relatives of patients with WG, suggests a genetic basis [1]. Indeed, new associations between ANCA vasculitis and genetic polymorphisms are reported almost monthly from candidate gene association studies. The pattern that is emerging points to a polygenic contribution from relatively common variants that are found throughout the population, each of which may only provide a modest effect. Many of the genes described so far encode proteins that are involved in the immune response, such as human leucocyte antigen (HLA) proteins, PTPN22, CTLA4 and others (reviewed in [2]). Genomewide association studies that are in progress will doubtless provide further insights.


Environmental factors appear to contribute variously (reviewed in [3]). Multiple reports attest to the abilities of drugs such as the anti-thyroid agent propylthiouracil to induce myeloperoxidase (MPO)-ANCA and, in a minority of individuals, to trigger overt vasculitis. Environmental toxins have been implicated, with the strongest epidemiological evidence emerging around silica, a potential activator of the inflammasome complex that generates, among other activities, the active cytokine interleukin (IL)-1 [4]. Infections have been linked repeatedly to pathogenesis of vasculitis. Clinical association studies have shown an enhanced likelihood of relapse in nasal carriers of Staphylococcus aureus; α-toxin from S. aureus is also a potent activator of the NLRP3 inflammasome, suggesting potential links between different environmental agents and their proinflammatory effects in vasculitis [5]. Infection has also been implicated in the formation of the most recently described type of ANCA, namely lysosomal-associated membrane protein 2 (LAMP-2); Kain has suggested that anti-LAMP-2 antibodies are important in the pathogenesis of vasculitis and has provided evidence of molecular mimicry between LAMP-2 and the bacterial adhesion protein Fim-H [6]. Links with infection via homology between the middle portion of the complementary proteinase 3 (cPR3) sequence and S. aureus proteins were suggested originally by Pendergraft, such that exposure to S. aureus may induce anti-complementary PR3 antibodies that, in turn, induce anti-PR3 antibodies via an anti-idiotypic response and ANCA vasculitis. These observations were extended recently when it was shown that vasculitic sera also contain antibodies to the C-terminus of PR3, but not the N-terminus; further, epitope determination showed that a common motif, ‘PHQ’, characterized the reactivity to the middle and C-terminus of cPR3, a motif that was reported to form the basis of the cross-reactivity of anti-cPR3 middle portion antibodies with plasminogen [7].


Potentially linking the genome with the environment is epigenetic modification of histone marks. Ciavatta et al. have demonstrated that levels of the chromatin modification H3K27me3, which is associated with gene silencing, were depleted at PR3 and MPO loci in ANCA patients compared with healthy controls [8]. In parallel with these changes, JMJD3, the demethylase specific for H3K27me3, was expressed preferentially in ANCA patients versus healthy controls. Describing a new mechanism for recruiting the H3K27 methyltransferase enhancer of zeste homologue 2 (EZH2) to PR3 and MPO loci, namely a RUNX3 dependent mechanism, Ciavatta went on to show that RUNX3 message was decreased in patients compared with healthy controls, possibly because it was also under epigenetic control. Indeed, DNA methylation was increased at the RUNX3 promoter in ANCA patients. Collectively, these data indicate that epigenetic modifications associated with gene silencing are perturbed at ANCA autoantigen-encoding genes, potentially contributing to inappropriate expression of PR3 and MPO in ANCA patients, and suggest that epigenetic influences may be extremely important during development of autoimmunity.


A defining feature in patients with WG and microscopic polyangiitis is the presence of ANCA with specificity to PR3 or MPO. While the ability of these antibodies to induce functional affects from neutrophils has been recognized for many years, a more refined understanding of structure to function has begun to emerge. Antibody immunoglobulin G (IgG) subclass, defined by the Fc portion, glycosylation status and precise epitope recognition by the Fab antibody portions, may all affect the abilities of ANCA to activate neutrophils and the type of functional response induced. Thus, ANCA IgG4 antibodies have been shown to activate a neutrophil respiratory burst, despite the fact that this IgG subclass is often regarded as being immunologically inert [9]. While earlier studies showed that glycosylation status affected the activating potential of ANCA in vitro, in vivo studies involving a murine model have confirmed that induction of vasculitis is attenuated if anti-myeloperoxidase IgG is pretreated with the bacterial enzyme endoglycosidase S, which deglycosylates the IgG and abolishes its ability to bind to neutrophil Fc receptors, without affecting the antigen-binding capacity of the antibodies [10]. Although epitope specificity has long been suspected of impacting upon the functional effects of ANCA, this was confirmed recently for PR3-specific antibodies using an epitope-based capture enzyme-linked immunosorbent assay (ELISA) system which showed that inhibition of enzymatic function of PR3 by anti-PR3 antibodies depended upon the precise PR3 epitope that was recognized [11]. At a higher level, ANCA IgG can also cross-react with other proteins, as demonstrated clearly by the ability of anti-PR3 antibodies to recognize both plasminogen and tissue plasminogen activators, leading to retardation of fibrinolysis and increased likelihood of the development of fibrinoid necrosis within glomeruli [12].


Of the many soluble mediators implicated in ANCA vasculitis, components of the alternative complement pathway are emerging as forerunners since the elegant demonstration of protection from disease in C5 and factor-B knock-out mice [13]. Increasingly it is recognized that ANCA vasculitis in the kidney is not quite so pauci-immune as was once thought [14], while the anaphylatoxin, C5a, not only primes neutrophils for an ANCA-induced respiratory burst, but C5a receptor-deficient animals are protected for development of glomerulonephritis [15].


A central cell in the development of vasculitis remains the neutrophil, as it both contains the target antigens for ANCA (PR3, MPO and LAMP-2) as well as contributing to vascular damage. PR3 and MPO are contained predominantly, but not exclusively, within azurophilic granules. Antigens become expressed at the neutrophil cell membrane following neutrophil activation and, in addition, are captured within the neutrophil extracellular traps (NETS) that contain serine proteases, MPO and chromatin [16]. PR3 and elastase containing NETs have been detected in affected human glomeruli [17], where inefficient dismantling of these NETs may result in renal damage [18]. Engagement of surface target antigens by ANCA IgG leads to functional responses by the neutrophil after engagement of intracellular signal transduction pathways. The pathways involved are being unravelled and have been shown recently to include diacylglycerol kinase, important in adhesion and degranulation [19] and phosphoinositol-3-kinase-γ, important in the superoxide response and degranulation where inhibition of signalling mitigated glomerulonephritis [20]. Ultimately, interplay between ANCA IgG, chemokines and neutrophils leads to preferential recruitment of neutrophils to microvascular sites [21–23].


While monocyte/macrophages are also believed to play important roles in the development of ANCA vasculitis their precise importance has been difficult to establish, but studies continue to suggest that down-regulating their activities can be beneficial. A recent example of this approach has been the use of a p38 mitogen-activated protein kinase inhibitor that, aside from reducing ANCA-activation of neutrophils, also reduced glomerular macrophage accumulation and crescent formation [24].

T lymphocytes

The wider adaptive immune system is believed to be fundamental to the development of autoimmune responses in vasculitis, as well as contributing to the effector pathways of tissue damage. Multiple changes in circulating T cell populations have been described, with markedly low numbers of CD4+ T helper cells, skewing towards effector memory T cells, altered expression of co-stimulatory molecules and increased numbers of activated T cells (reviewed in [25]). Translation of circulating T cell alterations to understand their impact within tissues remains problematic. Interest in T regulatory cells (Tregs) suggests that while expanded CD4+CD25+ T cell populations are predominantly activated effector cells rather than Tregs, there is evidence for a numerical reduction of Treg numbers [26] and/or functional deficiency [27]. The T helper type 17 (Th17) subset, dysfunctional in several autoimmune disease settings, may also contribute, as there is evidence for its enhanced activity with increased serum IL-17 and IL-23 levels during acute disease, and increased autoantigen-specific IL-17-producing cells during disease remission compared to healthy controls [28]. In animal models of autoimmune anti-MPO glomerulonephritis, mice deficient in IL-17A are protected [29]. That events in the T cell compartment may influence the course of the disease has been demonstrated clearly by observations that a novel CD8+ T cell transcription signature can predict the likelihood of relapse in ANCA vasculitis [30].

B lymphocytes

Interest in B cells increased markedly after efficacy in ANCA vasculitis of the B cell-depleting agent, rituximab, was demonstrated. The precise role of B cells in vasculitis still needs to be clarified, whether as precursors to antibody-producing plasma cells, antigen-presenting cells, providers of cytokines and growth factors or other roles. That B lymphocyte stimulator (BLyS) levels are increased in patients with active ANCA vasculitis may also be important, given that autoimmune B cells may be more dependent than non-autoimmune cells on this growth factor [31,32]. The promise of new techniques to determine specificity of immunoglobulins from distinct B cells out of WG has yet to be incorporated fully into our thinking; to date, specificity for a tetraspanin and for a lysosomal transmembrane protein 9B, a regulator for TNF-α activation, has been demonstrated [33].

Endothelial cells

Vascular endothelial cells become activated during ongoing vasculitic activity, up-regulating adhesion molecules and developing prothrombotic phenotypes. Increased numbers of activated cells and their microparticles are released into the circulation. Enumerating circulating cells or their microparticles is complex, so it is of interest that elevated serum levels of angiopoietin-2, which leads to disassembly of cell–cell junctions after binding to the Tie2 receptor, correlate closely with circulating endothelial cell numbers in ANCA vasculitis [34]. Increasingly, thought is being given to repair mechanisms to counteract ongoing endothelial damage. Low numbers of circulating endothelial progenitor cells appear to be associated with an enhanced likelihood of disease relapse, but are not predictive of progression of renal disease, number of organs involved or death from any cause [35].

In summary, advances in understanding the pathogenesis of ANCA vasculitis on all fronts has progressed apace in the past 2 years. Translating this knowledge into better therapies for patients will be the next challenge.


The author is currently employed by GlaxoSmithKline.