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Page 4 of 14 Almeida et al. Vessel Plus 2021;5:44 https://dx.doi.org/10.20517/2574-1209.2021.66

ANCA PATHOGENICITY
ANCA patterns
Two major types of ANCA can be detected by indirect immunofluorescence of ethanol-fixated neutrophils.
Perinuclear ANCAs (p-ANCAs) stain around the nucleus and are mainly composed of myeloperoxidase.
Conversely, some ANCAs stain diffusely in the cytoplasm, namely cytoplasmatic ANCAs (c-ANCAs), of
which the most important is PR3. Other “minor” ANCAs have been described: against α-enolase,
azurocidin, bactericidal permeability-increasing protein (BPI), cathepsin G, elastase, defensin, lactoferrin,
lysosome-associated membrane glycoprotein 2 (LAMP2), and moesin. These antibodies are mainly p-
ANCAs and are rarely associated with vasculitis. Nowadays, enzyme-linked immunosorbent assay (ELISA)
[15]
tests detect MPO and PR3 instead of p-ANCAs and c-ANCAs, which correspond to the staining pattern .

ANCA production
Neutrophils participate in the innate immune defense. These cells form networks of extracellular fibers,
primarily composed of DNA from neutrophils, which bind pathogens , the so-called neutrophil
[23]
extracellular traps (NETs). An imbalance in NETs’ production is known to participate in ANCA
production. These traps are degraded by serum DNAse I. Nevertheless, in some circumstances, as in the
case of propylthiouracil administration, NETs become resistant to DNAse I. The overexposure to NETs
promotes the production of autoantibodies against neutrophils. Meanwhile, the traps are formed, and the
content of neutrophilic granules, including PR3 and MPO, becomes mixed with chromatin fibers, which
bind to DNA. By this physiological step, the antigenicity of PR3 and MPO might suffer modifications .
[24]
However, only a second step that prevents a complete degradation of NETs seems to be specific to some
autoimmune diseases, such as AAVs and systemic lupus erythematosus [24,25] . This impaired NET
degradation coincides with the lower serum DNAse I activity observed in patients with AAV when
[26]
compared to healthy individuals . Moreover, a later step triggers the autoimmune reaction by presenting
misfolded proteins/antigens, in which cryptic epitopes are then recognized in class II MHC molecules by
[27]
the immune system .
PATHOGENESIS OF AAV
Facing a bacterial or viral infection, dendritic cells produce tumor necrosis factor (TNF)-β and interleukin-6
(IL-6). These inflammatory markers stimulate the differentiation of T cells into T helper 17 (Th17). In turn,
Th17 cells produce IL-17, an important cytokine that can induce the production of TNF-α or IL-1β by
macrophages. These proinflammatory cytokines then prime the innate immune system, namely neutrophils
and the formation of ANCAs. Additionally, the autoreactive response is sustained by imbalances of T
regulatory cells (Treg) that regulate the differentiation of Th17 and are associated with increased risk of
AAV [28,29] .

The priming of the neutrophil also occurs by the complement system, namely C5a by the altern
complement pathway, as described in animal models , which is related to hypercoagulability states in
[30]
patients with AAV due to tissue factor release .
[31]
Upon priming, neutrophils express surface ANCA antigens. These ANCAs, besides binding to the
corresponding antigens, also bind to the Fcγ receptor in neutrophils. This positive feedback induces an
excessive activation of these neutrophils, promoting cytokine production and reactive oxygen species and
lytic enzymes release , further incrementing the inflammatory status. ANCA-stimulated neutrophils
[32]
release NETs that contribute to the local inflammatory response and epithelial damage [Figure 1]. At the
same time, more neutrophils are activated either directly by NETs or indirectly by circulating ANCA.