REVIEWS

Pathogenesis of antineutrophil cytoplasmic

autoantibody-mediated disease
J. Charles Jennette and Ronald J. Falk
Abstract | Antineutrophil cytoplasmic autoantibodies (ANCAs) are the probable cause of a distinct form of
vasculitis that can be accompanied by necrotizing granulomatosis. Clinical and experimental evidence supports
a pathogenesis that is driven by ANCA-induced activation of neutrophils and monocytes, producing destructive
necrotizing vascular and extravascular inflammation. Pathogenic ANCAs can originate from precursor natural
autoantibodies. Pathogenic transformation might be initiated by commensal or pathogenic microbes, legal or
illegal drugs, exogenous or endogenous autoantigen complementary peptides, or dysregulated autoantigen
expression. The ANCA autoimmune response is facilitated by insufficient Tcell and Bcell regulation.
Aputative pathogenic mechanism for vascular inflammation begins with ANCA-induced activation of primed
neutrophils and monocytes leading to activation of the alternative complement pathway, which sets in motion
an inflammatory amplification loop in the vessel wall that attracts and activates neutrophils with resultant
respiratory burst, degranulation, extrusion of neutrophil extracellular traps, apoptosis and necrosis. The
pathogenesis of extravascular granulomatosis is less clear, but a feasible scenario proposes that a prodromal
infectious or allergic condition positions primed neutrophils in extravascular tissue in which they can be
activated by ANCAs in interstitial fluid to produce extravascular necrotizing injury that would initiate an innate
granulomatous inflammatory response to wall off the necrotic debris.

Jennette, J.C. & Falk, R.J. Nat. Rev. Rheumatol. advance online publication 8 July 2014; doi:10.1038/nrrheum.2014.103

Introduction
Antineutrophil cytoplasmic autoantibodies (ANCAs)
are associated with, and are the probable cause of, a
distinct form of vasculitis that can affect any organ in
the body. Although small vessels are the predominant
target, ANCA-associated vasculitis (AAV) can affect
many different types of vessels including art eries,
arterioles, capillaries, venules and veins (Figure1).1
Immunopathologically, AAV has an absence or paucity
of immunoglobulin deposition in injured vessels (pauci-
immune vasculitis), compared with the more extensive
deposition of immunoglobulin in immune-complex-
mediated small-vessel vasculitis.1 AAV can be limited
to one organ at the time of presentation (for example,
lung-limited disease or renal-limited disease) or involve
multiple organs. Frequent target tissues are the upper
and lower respiratory tract, kidneys, skin and periph-
eral nerves. Onset and exacerbations induce signs and
symptoms of high levels of circulating inflammatory
Department of cytokines, such as fever, arthralgia and myalgia. Inaddi-
Pathology and tion to these nonspecific systemic inflammatory mani-
Laboratory Medicine,
School of Medicine, festations, inflammation of vessel walls causes more
University of North specific signs and symptoms of vasculitis depending
Carolina at Chapel Hill,
308 Brinkhous-Bullitt
on which vessels and which organs are affected, for
Building, CB#7525, example purpura caused by dermal venulitis; pulmonary
Chapel Hill,
NC27599-7525,
USA (J.C.J., R.J.F.).
Competing interests
J.C.J. has acted as a consultant for Amicus Therapeutics,
Correspondence to: Genentech, GlaxoSmithKline and Protalix BioTherapeutics, and
J.C.J. has undertaken research in collaboration with ChemoCentryx.
jcj@med.unc.edu R.J.F. declares no competing interests.
haemorrhage caused by alveolar capillaritis; glomerulo-
nephritis caused by glomerular capillaritis; peripheral
neuropathy caused by epineural arteritis; and ocular
inflammation caused by vasculitis in small vessels in the
eye and orbit. In addition to vasculitis, some variants of
ANCA-associated disease have extravascular necrotiz-
ing granulomatous inflammation (granulomatosis)
seemingly not arising from vascular inflammationthat
most often affects the upper and lower respiratory tracts
but can affect any organ.
On the basis of the distribution of vascular inflam-
mation and the presence or absence of granulomatosis
and asthma, systemic AAV is categorized as microscopic
polyangiitis (MPA) if there is vasculitis but no evidence
of granulomatosis or asthma, granulomatosis with poly-
angiitis (GPA) if there is granulomatosis but no asthma,
and eosinophilic granulomatosis with polya ngiitis
(EGPA; also known as ChurgStrauss syndrome) if
there is granulomatosis, asthma and blood eosinophilia.1
Organ-limited disease, including isolated pauci-immune
necrotizing and crescentic glomerulonephritis, can be
considered limited variants of MPA. Limited variants of
GPA and EGPA can be confined to the respiratory tract.
AAV can also be classified on the basis of auto
antigen specificity. In patients with vasculitis, the two
best-d ocumented autoantigen targets of ANCA are
myelop eroxidase (MPO) 2 and proteinase3 (PR3). 35
Classification of AAV by antigen specificity clearly
shows differences in clinical presentations and outcomes,

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REVIEWS
Key points
Antineutrophil cytoplasmic autoantibodies (ANCAs) are associated with and
probably cause pauci-immune systemic necrotizing small-vessel vasculitis
and glomerulonephritis
ANCAs specific for proteinase3 or myeloperoxidase occur with organ-
limited disease, microscopic polyangiitis, granulomatosis with polyangiitis
or eosinophilic granulomatosis with polyangiitis
ANCAs seem to cause vasculitis by activating circulating primed neutrophils and
causing them to attach to, penetrate and damage vessel walls by undergoing
respiratory burst, degranulation, NETosis, apoptosis and necrosis
ANCA-induced neutrophil activation also releases factors that activate the
alternative complement pathway, which establishes a destructive inflammatory
amplification loop that attracts and activates more neutrophils that, in turn,
further activates the complement system
ANCA-associated granulomatosis might result from the same pathogenic
sequence of events involving extravascular primed neutrophils and interstitial-
fluid ANCAs, followed by a granulomatous reaction to wall off the resulting
extravascular necrosis
Immune complex small-vessel vasculitis
Cryoglobulinemic vasculitis
IgA vasculitis (HenochSchnlein)
Hypocomplementemic urticarial vasculitis
(Anti-C1q vasculitis)
Medium-vessel vasculitis Anti-GBM disease
Polyarteritis nodosa
Kawasaki disease
ANCA-associated small-vessel vasculitis
Microscopic polyangiitis
Granulomatosis with polyangiitis (Wegener)
Eosinophilic granulomatosis with polyangiitis (ChurgStrauss)
Large-vessel vasculitis
Takayasu arteritis
Giant cell arteritis
Figure 1 | Diagram depicting the predominant types of vessels affected by major
categories of systemic vasculitis. Note that vasculitis that is associated with
ANCAs can target a broad range of vessel types including medium and small
arteries, arterioles, capillaries, venules and veins. Large arteries, such as main
visceral arteries, are rarely affected by AAV. Thus, the types of vessels affected
by AAV overlap with other variants of systemic vasculitis, which results in many
overlapping clinical signs and symptoms of disease. Abbreviations: AAV, ANCA-
associated vasculitis; ANCA, antineutrophil cytoplasmic antibody; GBM, glomerular
basement membrane. Reproduced with permission from John Wiley & Sons, Inc.
Jennette, J.C. etal. Arthritis Rheum. 65, 111 (2013).
organ system involvement, patterns of extravascular
inflammation, and genetic associations including HLA
associations.6,7 The most informative AAV classifica-
tion scheme includes both the ANCA antigen specific-
ity and the clinicopathological phenotype, for example
MPOANCA MPA or PR3ANCA MPA.1
MPO and PR3 are not the only autoantigens recog-
nized by ANCAs. For instance, ANCAs specific for
elastase occur, and are most often identified, in patients
with drug-induced ANCA-associated disease, including
disease induced by cocaine adulterated with levamisole.8,9
A controversial autoantigen specificity is for lysosomal-
associated membrane protein 2 (LAMP2). Kain etal.10
reported finding LAMP2-ANCA in ~90% of patients
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with pauci-immune necrotizing glomerulonephritis,
including patients who were positive for MPO-ANCA
or PR3-ANCA and others who were negative for both.
However, Roth etal.11 were unable to confirm this high
frequency of LAMP2-ANCA in these patients. Similarly,
Kawakami etal. did not detect LAMP2-ANCA in
patients with MPA, although they did report evidence for
LAMP2-ANCA in some patients with cutaneous poly
arteritis nodosa12 and IgA vasculitis (formerly known
as HenochSchnlein purpura).13 ANCAs also occur
in patients with inflammatory bowel disease, and are
detected by indirect immunofluorescence assay in ~60%
of patients with ulcerative colitis and ~20% of those with
Crohn disease.1416 In this setting, ANCAs only rarely
have specificity for PR3, MPO or elastase: the major
autoantigens are catalase, enolase, lactotransferrin
and bactericidal permeability-increasing protein. 1416
ANCA, usually MPOANCA, occurs in a small minor-
ity of patients with systemic lupus erythematosus who
seem to to have vasculitic features of ANCA-associated
disease. 17 Approximately 30% of patients with anti
glomerular basement membrane disease have ANCA,
usually MPOANCA.18
An important role for ANCAs in the pathogenesis of
vasculitis and glomerulonephritis is supported by clinical
evidence and by invitro and invivo experimental data
(summarized in Box1). The leading theory proposes that
circulating neutrophils and monocytes that have been
primed by inflammatory stimuli display ANCA antigens
at or near the cell surface, and that interaction of these
antigens with ANCAs results in neutrophil activation
and initiation of vascular inflammation. An extension
of this theory proposes that primed extravascular neutro-
phils interact with interstitial ANCAs, causing necrotiz-
ing inflammation and resultant reactive granulomatous
inflammation. This Review summarizes observations
that support these pathogenic theories as well as puta-
tive mechanisms for the origin of the pathogenic ANCA
autoimmune response.
Pathology of ANCA-associated disease
Any valid theories about pathogenic mechanisms in
ANCA-associated disease must explain the development
of the observed pathologic lesions. The acute and chronic
lesions described in this section provide g uidance in
formulating pathogenic theories.
Vasculitis and glomerulonephritis
In patients1921 and experimental animal models,22 the
acute vascular and extravascular lesions of ANCA-
associated disease begin as neutrophil-rich necrotizing
inflammation. In vessels, the earliest lesions have mar-
gination and diapedesis of predominantly neutrophils
with admixed monocytes (Figure2a,b). Areas of segmen-
tal vascular necrosis also develop perivascular infiltrates
that initially have numerous neutrophils.
Within hours, neutrophils undergo apoptosis and
necrosis (Figure2a,b). The early destruction of neutro-
phils by apoptosis and necrosis during ANCA-associated
glomerulonephritis and vasculitis is supported by the
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Box 1 | Evidence that ANCAs are pathogenic
Clinical evidence
Strong association with ANCA (MPA >90%, GPA >90%, EGPA >40%*)
Partial correlation of ANCA titre with disease activity
Correlation of ANCA epitope specificity with disease activity (MPOANCA only)
Disease induction by transplacental transfer of ANCA (one MPOANCA case report)
Similar disease associated with drug-induced ANCA
HLA genetic associations with MPOANCA and PR3ANCA-associated disease
Response to immunosuppressive therapy that targets Bcells
Invitro evidence
Activation of cytokine-primed neutrophils by ANCA IgG
Endothelial injury by ANCA-activated neutrophils
Alternative complement pathway activation by ANCA-activated neutrophils
Evidence from animal models
Induction of pauci-immune vasculitis, glomerulonephritis and granulomatosis
in mice and rats by anti-MPO IgG
Prevention of murine anti-MPO IgG-induced disease by deficiency of neutrophils
Prevention of murine anti-MPO IgG-induced disease by blockade of alternative
complement pathway activation or blockade of C5a receptors
*More than 75% of patients with EGPA and glomerulonephritis have ANCA. Convincing animal
models of MPOANCA-associated disease have been described, but only less-convincing
animal models of PR3ANCA-associated disease have been described. Abbreviations: ANCA,
antineutrophil cytoplasmic autoantibody; EGPA, eosinophilic granulomatosis with polyangiitis;
GPA, granulomatosis with polyangiitis; MPA, microscopic polyangiitis; MPO, myeloperoxidase;
PR3, proteinase3.
frequent observation of leukocytoclasia (fragmentation
of nuclei) at sites of acute fibrinoid necrosis (Figure2d).
Conspicuous leukocytoclasia with numerous nuclear
fragments could result from the intensity of neutrophil
infiltration or reduced clearance of neutrophil fragments,
or both. Neutrophil cytoplasmic and nuclear constitu-
ents, including neutrophil extracellular traps (NETs),
are present at sites of inflammation and acute necro-
sis.2628 NETs comprise extracellular fibrillary material
containing chromatin and granule proteins extruded by
activated neutrophils.
Necrotizing injury to vessel walls results in haemor-
rhage and release of plasma proteins into the vessel walls
and adjacent extravascular tissue. These proteins include
coagulation factors, which are activated by thrombogenic
cellular and tissue debris, and tissue factor, which results
in the formation of fibrin within areas of fibrinoid necro-
sis (Figure2c,d). Fibrin formation also might be facili-
tated by tissue factor present in NETs.29 In glomeruli,
foci of segmental fibroid necrosis develop adjacent cel-
lular reactions (crescents) composed predominantly
of monocytes, macrophages and activated epithelial
cells(Figure2d).
Within days, the initial acute neutrophil-rich inflam-
mation and necrosis is replaced by inflammation with
a predominance of monocytes and macrophages and,
eventually, Tcells (Figure2c). Vascular and extravascu-
lar inflammatory infiltrates also often contain eosino-
phils, which are most numerous in EGPA. In glomerular
lesions of ANCA-associated glomerulonephritis, at the
time of biopsy, leukocytes are predominantly mono-
cytes and macrophages with admixed Tcells and usually
only scant neutrophils.2325 Within glomeruli in animal
models of ANCA-associated disease, the neutrophils
that are predominant during the first several days of
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REVIEWS
necrotizing inflammation are subsequently replaced by
monocytes and macrophages.22
Within 12weeks, vascular segments transition
through the monocyte and/or macrophage-rich phase of
inflammation to a phase of scarring that involves inter-
stitial deposition of collagen. The extent of scarring, and
the likelihood of return to normal structure and func-
tion, depends on the severity and duration of the inflam-
matory injury. Areas of mild segmental vascular injury
can undergo complete remodelling to restore normal
architecture, whereas severe vascular injury could result
in irreversible total vascular occlusion.
Granulomatosis
Patients with GPA and EGPA have nodular extra
vascular inflammatory lesions that contain numerous
macrophages, often including multinucleated giant cells
(Figure3).1921 In an early granulomatous pulmonary
lesion, the centre is in essence a microabscess contain-
ing neutrophils in varying stages of necrosis and apop-
tosis, and the margin has a thin band of macrophages
that are responding to the necrosis (Figure3a). In a later
stage of granulomatosis, the centre of the lesion contains
amorphous necrotic debris that is walled off from the
adjacent lung parenchyma by a well-defined band of
epithelioid macrophages (macrophages with elongated
nuclei and abundant acidophilic cytoplasm, resembling
epithelial cells) (Figure3b). At this stage, neutrophils
are no longer conspicuous, and the granulomatous
inflammation contains progressively more lymphoid
cells including effector Tcells, memory Tcells, Bcells,
dendritic cells, macrophages and plasma cells.30 At a still-
later stage, the necrotic debris is replaced by scar tissue,
and lymphocytes, including organized lymphoid folli-
cles, become components of the chronic granulomatous
inflammation. End-stage lesions are predominantly scar
tissue, often with no definitive histological features of the
earlier granulomatous inflammation.
Genesis of the ANCA autoimmune response
The evidence suggests that multiple genetic and environ-
mental factors converge to induce the onset of ANCA-
associated disease, and these factors have modulatory
effects on the clinical and pathological phenotype of
disease. These factors differ among patients; for example,
in a given patient the pivotal aetiological event could be
an infection, a drug, impaired immune regulation or
dysregulation of genomic expression of autoantigens,
or combinations of these and other factors.
Natural ANCAs
Although Ehrlich described the horror of autotoxicus
and Burnet theorized the elimination of forbidden
clones, all healthy individuals seem to have numerous
circulating autoantibodies with beneficial homeostatic
functions.31 Thus, pathogenic autoimmunity might arise
from dysregulation of homeostatic autoimmunity.
Healthy individuals have circulating autoantibodies
against MPO and PR3.3236 In asymptomatic individuals,
such autoantibodies are often designated natural or
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a b
E L
N
A method was used; this epitope was blocked from reacting
A
c d
Figure 2 | Vascular and glomerular pathology of AAV. a | Leukocytoclastic venulitis
in the nasal septal mucosa of a patient with PR3ANCA-associated disease
(magnification 400, H&E staining) demonstrates features of early AAV lesions:
numerous neutrophils (arrows) are marginating along and penetrating the wall of
avenule, and accumulating in the perivascular interstitium, with some undergoing
apoptosis and karyorrhexis (leukocytoclasia). b | Another venule from the same
specimen demonstrates neutrophils in the lumen, marginating neutrophils,
endothelial cells and apoptotic bodies derived from neutrophils (magnification
1,000, H&E staining). c | Necrotizing arteritis of a small artery in the kidney of
apatient with microscopic polyangiitis involves a circumferential zone of fibrinoid
necrosis (arrow) and perivascular leukocytes that, at this phase, contain
predominantly mononuclear leukocytes (magnification 400, H&E staining).
d | A glomerulus from a patient with ANCA-associated glomerulonephritis shows
segmental fibrinoid necrosis (lower arrow) and an adjacent cellular crescent
composed of mononuclear leukocytes and epithelial cells (upper arrow)
(magnification 400, H&E staining). Abbreviations: A, apoptotic bodies;
AAV,ANCA-associated vasculitis; ANCA, antineutrophil cytoplasmic antibody;
E,endothelial cells; H&E, haematoxylin and eosin; L, lumen; N, marginating
neutrophils; PR3, proteinase 3.
nonpathogenic autoantibodies. Presumably, quanti
tative or qualitative differences determine autoantibody
pathogenicity. Compared with pathogenic MPOANCA,
natural MPOANCA has lower titres, lower avidity, less
subclass diversity and less capability to activate neutro-
phils invitro.34 In autoimmune diseases that have auto
antigens with multiple epitope specificities, the epitope
specificity of natural autoantibodies can differ from that
of pathogenic autoantibodies. Roth etal.35 identified
more than 20 MPO epitopes that can be recognized by
MPOANCA in patients with ANCA-associated dis
ease. MPOANCA IgG from patients with active disease
recognized the largest number of epitopes, including
epitopes that were never recognized by MPOANCA
IgG from patients with ANCA-associated disease in
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remission or from healthy individuals. Healthy indi
viduals had very low titres of MPOANCA that recog
nized only a few MPO epitopes. Some patients with
clinical and pathological features of ANCA-associated
disease who were determined to be ANCA-negative
using conventional clinical assays reacted with a specific
MPO epitope when a highly sensitive epitope-excision
with ANCA IgG in serum because of competitive binding
by a fragment of ceruloplasmin, which is a natural inhibi-
tor of MPO.35 Modulation of epitope specificity (epitope
spreading) can be influenced by endogenous or exo
genous stimuli, causing nonpathogenic autoantibodies
to becomepathogenic.
The detection and characteristics of autoantibodies in
asymptomatic individuals might be a predictor of sub-
sequent development of disease. This predictive value
has been evaluated using the US Department of Defense
serum repository, which contains thousands of serum
samples taken from healthy military service members at
the time of enlistment as well as during their subsequent
time in the military.36 When compared with individuals
who did not develop GPA, a greater percentage of those
with GPA had at least one elevated PR3ANCA titre
before diagnosis (63% versus 0%) and a greater rate of
increase in titre (62% versus 0%). The rises in PR3ANCA
titre typically occurred before the onset of elevations in
serum Creactive protein level or signs and symptoms of
GPA. These findings are consistent with the concept that
endogenous or exogenous influences might modify quan-
titative or qualitative characteristics of natural ANCAs to
render them pathogenic.
Pathogenic ANCAs
Factors that could induce the development of patho-
genic ANCAs include exposure to exogenous anti-
gens that influence ANCA epitope specificity (such
as drugs or microbes), newly expressed or modified
ANCA autoantigens (such as alternatively spliced tran-
scripts or antisense transcripts), immunogenic display
of ANCA autoantigens (for example, on apoptotic cells
or NETs), or loss of effective suppression of the ANCA
autoimmune response (for example, because of defec-
tive Tcells, Bcells or myeloid-derived suppressor cells).
This pathogenic transformation could be multifactorial,
with genetic, environmental and immunological events
conspiring to break autoimmune homeostasis.
Mapping of epitopes that are the targets of natural and
pathogenic MPOANCAs has demonstrated that these
two classes of epitopes occur in close proximity on the
3D structure of MPO.35 This finding suggests that epitope
spreading of ANCA specificity, from nonpathogenic to
pathogenic epitopes, occurs as disease develops.
Modulation of existing epitope specificities is known
to occur during immune responses to infectious patho-
gens, and several microorganisms have been implicated
in the induction and modulation of ANCA-associated
disease, such as Staphylococcus aureus 37,38 and Ross
River virus.39 A novel model for induction of the ANCA
autoimmune response theorizes that the initial immune
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 REVIEWS

a b peptides have structures and physicochemical character-

istics that give them affinity to bind together. A classic
complementary peptide pair comprises the sense and
antisense transcripts of a gene; however, peptide mimics
of an antisense peptide are similarly complementary
to the sense peptide. Codon-directed amino acids in
sense peptides have specific pairwise interactions with
the corresponding complementary amino acids in
complementary peptides.42
An immune response to one complementary protein
can result in the production of an immune response to
the paired complementary protein; thus, immunization
with an antisense complementary peptide can induce
Figure 3 | Pathology of ANCA-associated necrotizing granulomatosis. a | The edge an antibody response to the sense peptide.42,43 Patients
of an early acute pulmonary lesion in a patient with PR3ANCA-positive GPA is positive for PR3ANCA have circulating antibodies
characterized by a central zone of necrosis containing numerous neutrophils in reactive not only to PR3 sense peptides but also to PR3
varying stages of leukocytoclasia, and by an adjacent band of monocytes and complementary antisense peptides.40,41 In addition to
macrophages (arrows) (magnification 400, H&E staining). b | A later stage of antibodies that recognize complementary PR3 antigens,
granulomatous inflammation in the same pulmonary lesion specimen demonstrates
Yang etal.44 detected anticomplementary PR3-specific
a central zone of amorphous necrotic debris containing a few pyknotic leukocytes,
and an adjacent marginal zone of epithelioid macrophages (arrows) (magnification
memory Tcellsdirected against a PR3 complementary
400, H&E staining). Abbreviations: ANCA, antineutrophil cytoplasmic antibody; peptidein patients with PR3ANCA. The stimulus for
GPA, granulomatosis with polyangiitis; H&E, haematoxylin and eosin; PR3, the antibodies to complementary PR3 could have been
proteinase 3. endogenous PR3 antisense peptides45 or exogenous pep-
tides that mimic the antisense peptides, such as peptides
brought in by commensal or pathogenic microbes.40
Endogenous source
Sense
Interestingly, the bioinformatics software tool BLAST
ANCA antigen
transcription peptide (basic local alignment search tool) identifies multiple
microbial peptide homologues of complementary PR3,
Sense DNA Idiotypic Idiotope
strand antibody mimicking the including peptides from microbes that are known to be
autoantigen associated with PR3ANCAs such as Ross River virus
Neutrophil and S.aureus.40,41
A genome-wide association study has indicated that
Antisense
different HLA specificities correlate with MPOANCA
Antisense
DNA strand
peptide
Anti-idiotypic and PR3ANCA, and a gene encoding PR3 was associ-
antibody
(autoantibody) ated with PR3ANCA but not MPOANCA. 7 These
Antisense
transcription findings suggest that antigen-recognition capabilities
are important in ANCA-associated disease, and that the
Exogenous source initial antigen recognized by antigen-presenting cells is
different in PR3ANCA-associated disease as opposed
T cell to MPOANCA-associated disease. HLA specificities
seemed to be involved in the ability of ANCA to recog
nize both sense and antisense antigen peptides. 46 The
HLA DRB1*15 allele is over-represented in African
American patients with PR3ANCA-associated disease
Mimic of (odds ratio 73.3).46 The DRB1*1501 protein binds with
Microbes antisense peptide
high affinity to both sense-PR3 peptide as well as the
Figure 4 | Diagram of the induction of an ANCA-mediated autoimmune response by complementary antisense-PR3 peptide.46 This finding
an initial immune response to a peptide that is complementary to an autoantigen suggests that one factor in the induction of ANCA-
peptide. This complementary peptide immunogen could arise from antisense associated disease could be the presence of HLA antigen-
transcription of the antisense strand of the autoantigen gene, or could be a mimic binding sites that recognize the autoantigen and a
of an antisense peptide that is produced by a symbiotic or pathogenic microbe.
complementary protein.
The anticomplementary peptide antibody idiotopes would engender an anti-
idiotypic antibody response that crossreacts with the autoantigen epitopes that Thus, the theory of autoantigen complementarity sug-
are complementary to the initial immunogenic peptide. Abbreviation: ANCA, gests that a patient with a genetically determined pre-
antineutrophil cytoplasmic antibody. disposing antigen-recognition capability could produce
pathogenic ANCA as a result of an initial appropriate
response is not against the autoantigen but rather a immune response against a microbial mimic of a com-
peptide that has a complementary structure relative to plementary peptide, or against an endogenously pro-
the autoantigen, and that the anti-idiotypic response duced antisense transcript, that would be transformed
to this initial immune response results in antibodies that into an autoimmune response by an anti-idiotypic res
recognize the autoantigen (Figure4).40,41 Complementary ponse (Figure4). In support of this theory, anti-idiotypic

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REVIEWS
antibodies raised against autoantibodies have been
shown in multiple animal models to induce anti-anti-
idiotypic antibodies that possess characteristics of the
initial a utoantibodies and cause autoimmune disease.47
Induction of pathogenic ANCAs by microbial molecu-
lar mimicry of autoantigens has been proposed as a cause
for ANCAs with specificity for LAMP2.10 The human
LAMP2 epitope has complete homology with the bacte-
rial adhesin FimH, and human LAMP2-ANCA cross-
reacts with this microbial protein. Kain etal.10 reported
that rats immunized with FimH develop antibodies to
rat and human LAMP2, and develop pauci-immune
necrotizing glomerulonephritis that resembles human
ANCA-associated glomerulonephritis. This study pro-
poses that infection with fimbriated bacteria can cause
an immune response against FimH that crossreacts with
human LAMP2 and mediates ANCA-associated disease.
However, Roth etal.11 were not able to reproduce the
findings from this e xperimental animalmodel.
Another exogenous stimulus that can induce a
pathogenic ANCA immune response is drugs, includ-
ing hydralazine, minocycline, propylthiouracil and
levamisole-adulterated cocaine.8,9 The mechanisms by
which drugs induce ANCAs could shed light on patho
genic mechanisms that do not involve drugs. Hydralazine-
induced ANCA-associated disease is accompanied by
dysregulation of ANCA-antigen expression by neutro-
phils.8 Hydralazine is a DNA methylation inhibitor that
might reverse epigenetic silencing of PR3 and MPO,
resulting in increased expression of both autoantigens in
neutrophils. This increased autoantigen expression
in drug-induced disease is similar to a phenomenon
observed in patients with ANCA-associated disease who
have abnormal epigenetically deregulated overexpres-
sion of MPO and PR3 in neutrophils.48,49 Increased auto
antigen expression in neutrophils could either facilitate a
loss of tolerance to these proteins, or enhance the activa-
tion of neutrophils by ANCAs, or both. Organized lym-
phoid configuration in areas of granulomatosis that bring
together antigen-presenting cells with Tcells and Bcells,30
as well as effective presentation of neutrophil antigens on
apoptotic neutrophils50,51 and NETs,27 could also augment
ANCA autoimmune responses.
Regulation of the ANCA immune response
The pathogenic ANCA autoimmune response seems to
be enabled by impaired Tcell and Bcell suppression,
and possibly by enhanced Bcell stimulation by ANCA-
activated neutrophils. Patients with ANCA-associated
disease have dysfunction of regulatory T (TREG) cells that
could contribute to loss of tolerance and emergence of
a pathogenic ANCA response.5254 TREG cells are charac-
terized by high expression of CD25 and FOXP3. Even
though patients with active GPA have increased numbers
of CD4+ CD25+ Tcells, the numbers of FOXP3+ cells are
decreased. 52 The suppressive function of TREG cells
from patients with ANCA-associated disease has been
reported to be markedly decreased in active disease and
more normal during remission.53 Free etal.54 described
an increase in a proinflammatory suppression-resistant
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effector CD4 + Tcell subset in patients with active
ANCA-associated disease, in comparison with patients
in remission and with healthy individuals. Although
circulating TREG cell numbers were increased in active
disease, these cells had decreased suppressive function
because of expression of a FOXP3 isoform that lacks
exon2.54 A study by Wilde etal.55 showed that increased
expression of the negative co-stimulator programmed
cell death protein1 (PD1) on circulating Tcells in
patients with GPA might oppose Tcell activation; how
ever, Tcells in renal lesions in these patients mostly
lacked PD1 expression.
B-cell regulation might also influence production of
pathogenic ANCAs.56,57 Bcells that have high expression
of CD5 and produce IL10 have regulatory capabilities.
Patients with active ANCA-associated disease have a
reduced percentage of circulating CD5+ Bcells whereas
most patients in remission have a normal percentage.52
In addition, the degree of normalization of circulating
CD5+ Bcells after therapy with rituximab correlates with
more-persistent remission.56
ANCA-activated neutrophils might augment the
production of ANCAs by stimulating Bcells. Acti
vated neutrophils release ligands for Bcell-activating
factor (BAFF; also known as TNF ligand superfamily
member13B or B lymphocyte stimulator), which stimu-
lates Bcell proliferation and inhibits apoptosis.58 Patients
with ANCA-associated disease have serum levels of
BAFF that are elevated during active disease and decline
during remission.5962
Thus, Bcell-stimulating factors released by ANCA-
activated neutrophils along with permissive Tcell and
Bcell regulation seem to facilitate the production of
pathogenic ANCAs.
Pathogenesis of vascular inflammation
Neutrophil and monocyte activation by ANCAs
Invitro studies have shown that both MPOANCA and
PR3ANCA IgG can activate neutrophils that have been
primed by proinflammatory stimuli, such as TNF,63 bac-
terial lipopolysaccharide64 or the complement anaphyla-
toxin C5a.65 Priming causes neutrophils to release ANCA
target antigens (for example, MPO and PR3) at the cell
surface and into the microenvironment that can then
interact with ANCAs. Full neutrophil activation results
from engagement of constitutively expressed Fc recep-
tors (FcRs) by immune complexes containing ANCA
and antigen66,67 and from binding of F(ab')2 fragments of
ANCA to antigen expressed on neutrophil surfaces.68,69
Phosphorylation of p38 mitogen-activated protein kinase
(p38 MAPK) and the extracellular signal-regulated
kinase (ERK) are involved in neutrophil priming and
activation, and blockade of MAPK prevents this priming
and activation.7072
ANCA-activated neutrophils generate a respiratory
burst with production of toxic oxygen radicals, release
destructive enzymes through degranulation, and extrude
NETs that have proinflammatory properties. 27,28,6374
These ANCA-activated cells have been shown to cause
injury to and death of cultured monolayers of endothelial
www.nature.com/nrrheum

Unprimed Priming
neutrophil
Alternative
pathway
activation
Activation
Acute Chronic inflammation
inflammation and scarring
Epithelial cell
NETosis
C5a ANCA antigen Neutrophil
C5a receptor
ANCA Apoptotic body Fibroblast
Cytokine
Fc receptor Macrophage Fibrin
Cytokine
receptor T cell Monocyte Collagen
Figure 5 | Putative sequence of pathogenic events in ANCA-mediated vasculitis.
Circulating neutrophils are primed for activation by ANCA by inflammatory cytokines
or C5a derived from complement activation (note that monocytes can be similarly
primed and activated but are not illustrated). Primed neutrophils release ANCA
antigens at the cell surface and into the microenvironment, where they interact
with ANCA. Fc receptor engagement by ANCA bound to ANCA antigens as well as
F(ab')2 binding to ANCA antigens on neutrophil surfaces cause neutrophil
activation. ANCA-activated neutrophils release factors that activate the alternative
complement pathway, generating C5a. C5a and ANCA create an inflammatory
amplification loop, with C5a attracting and priming more neutrophils for activation
by ANCA, which causes, in turn, further activation of the alternative complement
pathway and production of more C5a. ANCA-activated neutrophils marginate and
penetrate vessel walls and undergo respiratory burst, degranulation, NETosis,
apoptosis and necrosis. Disruption of endothelium allows plasma to spill into
vascular and perivascular tissue where activation of the coagulation cascade
produces the fibrin strands of fibroid necrosis. The innate inflammatory response
orchestrates the conversion of the initial acute neutrophil-rich inflammation into
mononuclear leukocyte-rich inflammation and subsequent collagen deposition
and fibrosis. Abbreviation: ANCA, antineutrophil cytoplasmic antibody.
cells. 7578 Release of NETs (a process referred to as
NETosis) by ANCA-activated neutrophils27,28 contributes
to endothelial injury and death.79
In considering pathogenic pathways in ANCA-
associated disease, it is important to note that monocytes
as well as neutrophils contain ANCA antigens (including
PR3 and MPO) and can be activated invitro by engage-
ment of FcRs by ANCA IgG. 80,81 ANCA-activated
monocytes release proinflammatory cytokines, includ-
ing monocyte chemoattractant protein1 (MCP1, also
known as CCL2)80 and IL8.81 IL8 attracts and activates
neutrophils and thus could amplify neutrophil-mediated
injury. MCP1 attracts monocytes and macrophages and
could participate in the transition of ANCA-induced
vascular injury from predominantly neutrophil-rich
inflammation to predominantly monocyte and/or
macrophage-rich inflammation, including granulo
matous inflammation. Monocytes are conspicuous
at sites of ANCA vascular inflammation, 24 especially
NATURE REVIEWS | RHEUMATOLOGY ADVANCE ONLINE PUBLICATION | 7
2014 Macmillan Publishers Limited. All rights reserved
REVIEWS
after the first day or two, and there is evidence for sys-
temic activation of monocytes in patients with active
ANCA-associateddisease.82
Induction of necrotizing vascular inflammation
Pathogenic mechanisms of ANCA-induced vascular
inflammation must be in accord with observed lesions
in tissue specimens from patients with ANCA-associated
disease or in animal models. Earlier in this Review, we
described the pathological features of AAV. The histo-
pathology of the earliest acute vascular lesions reveals
a very destructive localized neutrophil-rich necrotizing
inflammation that quickly transforms into monocyte/
macrophage-rich inflammation. Immunohistology
reveals a paucity, but usually not an absence, of immuno
globulin at sites of inflammation. Small amounts of
ANCA and ANCA-antigen are found at sites of vascular
inflammation, including glomerulonephritis, in patients
biopsies15,26,83 and in experimental animal models of
ANCA-associated disease.84 Invitro studies have dem-
onstrated that ANCA-induced neutrophil activation
releases into the microenvironment ANCA-antigens that
can bind to nearby structures to act as targets for insitu
immune-complex formation. 76 However, the amount
of immunoglobulin deposited at sites of inflammation in
ANCA-associated disease typically is far less than is seen
with conventional forms of immune-complex-mediated
vascular inflammation. 1 This observation supports
the concept that the vascular inflammation of ANCA-
associated disease is caused primarily by direct activation
of neutrophils and monocytes by ANCA, with somewhat
incidental deposition of immune complexes containing
ANCA and ANCA antigen at the sites of injury.
The most compelling evidence that ANCAs are
pathogenic and the most informative experiments that
elucidate the underlying pathogenic mechanisms come
from animal models of ANCA-associated disease.8587
Multiple convincing models of MPOANCA-associated
disease have been described and confirmed.8487 How
ever, no convincing models of PR3-ANCA-associated
disease have been widely accepted, although several
have beenproposed.8891
Mouse models of AAV and ANCA-associated glo-
merulonephritis induced by anti-MPO antibodies have
been used most extensively to elucidate pathogenic
mechanisms. 64,65,71,84,9298 Disease can be induced by
injecting mouse anti-MPO IgG into immunocompetent
or immunodeficient mice,84 injecting splenocytes con-
taining anti-MPO Bcells into immunodeficient mice,84
or transplanting bone marrow that contains MPO-
positive myeloid cells into MPO-knockout mice.93 In
these models, a pathogenic level of anti-MPO antibodies
induces necrotizing and crescentic glomerulonephritis
in all mice of susceptible strains, and systemic necrotiz-
ing small-vessel vasculitis and granulomatous inflam-
mation in some, but not all, animals. The inflammatory
lesions in these mouse models closely mimic human
ANCA-associated disease.
Several conclusions can be drawn from experiments
using mouse models of MPOANCA-associated disease:
REVIEWS
Priming
Unprimed
neutrophils
Activation
Granulomatosis
Microabscess
formation
C5a ANCA antigen Neutrophil
C5a receptor ANCA
Multinucleated
Cytokine Macrophage
Cytokine
Fc receptor giant cell
receptor T cell Monocyte Fibrin
Figure 6 | Putative sequence of pathogenic events in ANCA-mediated necrotizing
granulomatosis. An inflammatory prodrome, such as an infectious or allergic
inflammatory respiratory tract disease, positions increased numbers of primed
neutrophils in extravascular interstitial tissue. ANCA immunoglobulin in the
interstitial fluid would activate primed neutrophils and initiate an inflammatory
amplification loop that would attract and activate more neutrophils, resulting in the
formation of a necrotizing microabscess. This acute inflammation and necrosis
would initiate an innate inflammatory response that would wall off the necrotic zone
with granulomatous inflammation containing a predominance of monocytes and
macrophages with admixed lymphocytes. Abbreviation: ANCA, antineutrophil
cytoplasmic antibody.
antibody to MPO alone (in the absence of functional
Tcells) is sufficient to cause acute disease; 84 bone-
marrow-derived cells are sufficient and necessary to
induce disease;92 neutrophils are required;92 genetic back-
ground determines the susceptibility to and severity of
disease;94 circulating proinflammatory factors exacerbate
disease;64 and activation of the alternative complement
pathway is required to induce vascular inflammation.96
Studies of these mouse models also showed that disease
can be prevented or ameliorated by hydrolysis of anti-
MPO IgG glycans, which affects FcRantibody inter-
actions;95 by inhibition of p38 MAPK, which prevents
priming of neutrophils by inflammatory stimuli;71 and
by blockade of C597 or C5a receptor,65,98 which prevents
activation of the alternative complement pathway.
Evidence from animal models of the involvement of
alternative complement pathway activation in the patho-
genesis of ANCA-associated disease prompted studies to
8 | ADVANCE ONLINE PUBLICATION
2014 Macmillan Publishers Limited. All rights reserved
identify supportive evidence in human disease. Exami
nation of glomerular and vascular inflammatory lesions
in biopsy specimens from patients with ANCA-associated
disease revealed markers of alternative complement path
way activation including factorB and properdin, as well
as factors shared by the classical and alternative path
ways including C3d and membrane attack complex, but
the absence of components of the classical and lectin
pathways such as C4d and mannose-binding lectin.99
Although not yet substantiated by other investigators,
one research group has reported evidence of alternative
complement pathway activation in patients with active
ANCA-associated disease, including elevated plasma
levels of C3a, C5a, soluble C5b9, and Bb; reduced levels
of properdin; and normal levels of C4d.100,101
On the basis of observations in experimental animal
models and supportive findings in patients, a pathogenic
scenario can be proposed for the interaction of patho-
genic ANCAs with neutrophils that have been primed
by a synergistic proinflammatory condition (Figure5).
Circulating neutrophils are primed by, for example,
inflammatory cytokines or C5a derived from com-
plement activation. Primed neutrophils release small
amounts of ANCA antigens at the cell surface and in
the microenvironment, where these antigens can inter-
act with ANCAs. FcR engagement by ANCAs bound
to ANCA antigens as well as F(ab')2 binding to ANCA
antigens expressed on neutrophil surfaces cause neu-
trophil activation. ANCA-activated neutrophils release
factors that activate the alternative complement pathway
with generation of C5a. Neutrophils, C5a and ANCAs
create an inflammatory amplification loop, with C5a
attracting and priming more neutrophils for activation
by ANCAs, which in turn causes further activation of
the alternative complement pathway and consequent
production of more C5a. ANCA-activated neutrophils
marginate and penetrate vessel walls, and undergo res-
piratory burst, degranulation, NETosis, apoptosis and
necrosis. Disruption of endothelium enables plasma to
spill into vascular and perivascular tissue where acti-
vation of the coagulation cascade produces the fibrin
strands of fibroid necrosis. Modulated by the severity and
reversibility of the acute injury, the innate inflammatory
response orchestrates the conversion of the acute inflam-
mation into m ononuclear-leukocyte-rich inflammation
and subsequent fibrosis.
Pathogenesis of granulomatosis
There has been substantial controversy over the nature
of the granulomatosis seen in GPA and EGPA. The term
granulomatosis has been used to describe the multiple
foci of extravascular inflammation that were described
initially by Klinger 102 and Wegener 103,104 in GPA, and
by Churg and Strauss in EGPA.105 Controversy over the
pathogenesis of this granulomatous inflammation derives
from the fact that it can arise from at least two distinct
pathwaysone initiated by a Tcell mediated (typeIV)
immune response specific for PR3 or MPO, and another
by an antigen-independent innate response of macro
phages to something in the tissue that needs to be
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 REVIEWS

walledoff.106 The latter pathway is an exaggerated version
of the innate monocyte and macrophage response that
closely follows any acute inflammatory event. Although
many investigators have proposed that ANCA-associated
granulomatosis is caused by an antigen-specific typeIV
adaptive immune response, we favour the theory that
the granulomatosis is an innate inflammatory response
to acute extravascular inflammation that is initiated by
ANCA-induced neutrophil activation.
The concept that the granulomatosis of GPA is a
response to acute neutrophil-mediated necrosis is not
new. For example, based on careful histopathological
examination of lesions in patients with GPA and EGPA,
Fienberg and his associates concluded that micro
necrosis, usually with neutrophils (microabscesses),
constitutes the early phase in the development of the
pathognomonic organized palisading granuloma. 21
Lungs of patients with active GPA and EGPA often have
a range of granulomatous lesions, from acute lesions with
central microabscesses (Figure3a) or subacute lesions
with central necrosis (Figure3b) to chronic lesions with
a central zone of scarring and chronic inflammation.20
Chronic lesions contain substantial accumulations of
lymphocytes and dendritic cells, including Tcells and
Bcells with specificity for ANCA antigens that could be
a source for autoantibody production.108
On the basis of this apparent sequence of pathologi-
cal events, and the knowledge that ANCA IgG activates
primed neutrophils, even invitro, a reasonable hypothesis
is that if an inflammatory condition (perhaps infection in
GPA and allergy in EGPA) positions primed neutrophils
in the extravascular tissue (for example, the submucosa
of the respiratory tract), these primed neutrophils would
be activated by ANCA IgG in the interstitial fluid of a
patient with circulating ANCAs. ANCA-induced neutro-
phil activation, possibly fuelled by activation of the alter-
native complement pathway, would induce microabscess
formation that, in turn, would engender a granulomatous
reaction to wall off the zone of necrosis (Figure6).
Conclusions
Pauci-immune necrotizing small-vessel vasculitis and
extravascular granulomatosis are associated with, and
might be caused by, ANCAs. The distinctive, extremely
destructive necrotizing vascular inflammation of AAV
seems to be fuelled by an amplification loop in which
ANCA-activated neutrophils activate the alterna-
tive complement pathway, which, in turn, leads to the
attraction and activation of more neutrophils. Promising
therapies are those that can target one or multiple com-
ponents of this vicious cycle (for example ANCAs,
ANCA antigens, neutrophil activation and complement
activation). The pathogenesis of ANCA-associated
necrotizing extravascular granulomatous inflammation
is not known, but might be fuelled by the same inflam-
matory amplification loop occurring in the extravas-
cular compartment, involving primed neutrophils and
interstitial ANCAs.
Review criteria
This Review is based primarily on full-text, peer-reviewed,
English-language medical literature from the 1980s into
2014 reporting on vasculitis and glomerulonephritis
caused by antineutrophil cytoplasmic autoantibodies,
as well as the authors knowledge of the field acquired
over more than 30years of involvement in research on
glomerulonephritis and vasculitis.

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Acknowledgements
The authors are supported by a grant from the NIH
National Institute of Diabetes and Digestive and
Kidney Diseases (P01-DK05833511).
Author contributions
J.C.J. researched data for the article, and both
authors contributed equally to discussions of
content,writing and review/editing of the manuscript
before submission.