1. NATURE REVIEWS | RHEUMATOLOGY VOLUME 10 | AUGUST 2014 | 463
Department of
Pathology and
Laboratory Medicine,
School of Medicine,
University of North
Carolina at Chapel Hill,
308 Brinkhous-Bullitt
Building, CB#7525,
Chapel Hill,
NC 27599-7525,
USA (J.C.J., R.J.F.).
Correspondence to:
J.C.J.
jcj@med.unc.edu
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 T‑cell and B‑cell regulation.
A putative 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. 10, 463–473 (2014); published online 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 ar­teries,
arterioles, capillaries, venules and veins (Figure 1).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
cytokines, such as fever, arthralgia and myalgia. In addi-
tion to these nonspecific systemic inflammatory mani-
festations, inflammation of vessel walls causes more
specific signs and symptoms of vasculitis depending
on which vessels and which organs are affected, for
example purpura caused by dermal venulitis; pulmonary
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 inflammation—that
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 poly­angiitis
(EGPA; also known as Churg–Strauss 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-­documented autoantigen targets of ANCA are
myelo­peroxidase (MPO)2
and proteinase 3 (PR3).3–5
Classification of AAV by antigen specificity clearly
shows differences in clinical presentations and outcomes,
Competing interests
J.C.J. has acted as a consultant for Amicus Therapeutics,
Genentech, GlaxoSmithKline and Protalix BioTherapeutics, and
has undertaken research in collaboration with ChemoCentryx.
R.J.F. declares no competing interests.
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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 specifi­
city and the clinicopathological phenotype, for example
MPO‑ANCA MPA or PR3‑ANCA 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 (LAMP‑2). Kain et al.10
reported finding LAMP‑2-ANCA in ~90% of patients
Key points
■■ Antineutrophil cytoplasmic autoantibodies (ANCAs) are associated with and
probably cause pauci-immune systemic necrotizing small-vessel vasculitis
and glomerulonephritis
■■ ANCAs specific for proteinase 3 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
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 et al.11
were unable to confirm this high
frequency of LAMP‑2-ANCA in these patients. Similarly,
Kawakami et al. did not detect LAMP‑2-ANCA in
patients with MPA, although they did report evidence for
LAMP‑2-ANCA in some patients with cutaneous poly­
arteritis nodosa12
and IgA vasculitis (formerly known
as Henoch–Schönlein 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.14–16
In this setting, ANCAs only rarely
have specificity for PR3, MPO or elastase: the major
autoantigens are catalase, α‑enolase, lacto­transferrin
and bactericidal permeability-increasing protein.14–16
ANCA, usually MPO‑ANCA, 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 MPO‑ANCA.18
An important role for ANCAs in the pathogenesis of
vasculitis and glomerulonephritis is supported by clinical
evidence and by in vitro and in vivo experimental data
(summarized in Box 1). 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 ­guidance in
­formulating pathogenic theories.
Vasculitis and glomerulonephritis
In patients19–21
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 (Figure 2a,b). Areas of segmen-
tal vascular necrosis also develop perivascular infiltrates
that initially have numerous neutrophils.
Within hours, neutrophils undergo apoptosis and
necrosis (Figure 2a,b). The early destruction of neutro-
phils by apoptosis and necrosis during ANCA-associated
glomerulonephritis and vasculitis is supported by the
Medium-vessel vasculitis
Polyarteritis nodosa
Kawasaki disease
Immune complex small-vessel vasculitis
Cryoglobulinemic vasculitis
IgA vasculitis (Henoch–Schönlein)
Hypocomplementemic urticarial vasculitis
(Anti-C1q vasculitis)
ANCA-associated small-vessel vasculitis
Microscopic polyangiitis
Granulomatosis with polyangiitis (Wegener)
Eosinophilic granulomatosis with polyangiitis (Churg–Strauss)
Large-vessel vasculitis
Takayasu arteritis
Giant cell arteritis
Anti-GBM disease
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. et al. Arthritis Rheum. 65, 1–11 (2013).
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3. NATURE REVIEWS | RHEUMATOLOGY VOLUME 10 | AUGUST 2014 | 465
frequent observation of leukocytoclasia (fragmentation
of nuclei) at sites of acute fibrinoid necrosis (Figure 2d).
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.26–28
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 (Figure 2c,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 (Figure 2d).
Within days, the initial acute neutrophil-rich inflam-
mation and necrosis is replaced by inflammation with
a predominance of monocytes and macrophages and,
eventually, T cells (Figure 2c). 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 T cells and usually
only scant neutrophils.23–25
Within glomeruli in animal
models of ANCA-associated disease, the neutrophils
that are predominant during the first several days of
necrotizing inflammation are subsequently replaced by
­monocytes and macrophages.22
Within 1–2 weeks, 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
(Figure 3).19–21
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 (Figure 3a). 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) (Figure 3b). At this stage, neutrophils
are no longer conspicuous, and the granulomatous
inflammation contains progressively more lymphoid
cells including effector T cells, memory T cells, B cells,
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 PR3.32–36
In asymptomatic indi­viduals,
such autoantibodies are often designated ‘natural’ or
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 (MPO‑ANCA only)
■■ Disease induction by transplacental transfer of ANCA (one MPO‑ANCA case report)
■■ Similar disease associated with drug-induced ANCA
■■ HLA genetic associations with MPO‑ANCA and PR3‑ANCA-associated disease
■■ Response to immunosuppressive therapy that targets B cells
In vitro 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 MPO‑ANCA-associated disease have been described, but only less-convincing
animal models of PR3‑ANCA-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, proteinase 3.
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nonpathogenic autoantibodies. Presumably, quanti­
tative or qualitative differences determine autoantibody
pathogenicity. Compared with pathogenic MPO‑ANCA,
natural MPO‑ANCA has lower titres, lower avidity, less
subclass diversity and less capability to activate neutro-
phils in vitro.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 et al.35
identified
more than 20 MPO epitopes that can be recognized by
MPO‑ANCA in patients with ANCA-associated dis­
ease. MPO‑ANCA IgG from patients with active disease
recognized the largest number of epitopes, including
epitopes that were never recognized by MPO‑ANCA
IgG from patients with ANCA-associated disease in
remission or from healthy individuals. Healthy indi­
viduals had very low titres of MPO‑ANCA 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
method was used; this epitope was blocked from reacting
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 become pathogenic.
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 PR3‑ANCA titre
before diagnosis (63% versus 0%) and a greater rate of
increase in titre (62% versus 0%). The rises in PR3‑ANCA
titre typically occurred before the onset of elevations in
serum C‑reactive 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 T cells, B cells 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 MPO‑ANCAs 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 aureus37,38
and Ross
River virus.39
A novel model for induction of the ANCA
autoimmune response theorizes that the initial immune
a
c d
b
E L
A
A
N
Figure 2 | Vascular and glomerular pathology of AAV. a | Leukocytoclastic venulitis
in the nasal septal mucosa of a patient with PR3‑ANCA-associated disease
(magnification ×400, HE staining) demonstrates features of early AAV lesions:
numerous neutrophils (arrows) are marginating along and penetrating the wall of
a venule, 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, HE staining). c | Necrotizing arteritis of a small artery in the kidney of
a patient with microscopic polyangiitis involves a circumferential zone of fibrinoid
necrosis (arrow) and perivascular leukocytes that, at this phase, contain
predominantly mononuclear leukocytes (magnification ×400, HE 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, HE staining). Abbreviations: A, apoptotic bodies;
AAV, ANCA-associated vasculitis; ANCA, antineutrophil cytoplasmic antibody;
E, endothelial cells; HE, haematoxylin and eosin; L, lumen; N, marginating
neutrophils; PR3, proteinase 3.
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5. NATURE REVIEWS | RHEUMATOLOGY VOLUME 10 | AUGUST 2014 | 467
response is not against the autoantigen but rather a
peptide that has a complementary structure relative to
the auto­antigen, and that the anti-idiotypic response
to this initial immune response results in antibodies that
recognize the autoantigen (Figure 4).40,41
Complementary
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
an antibody response to the sense peptide.42,43
Patients
positive for PR3‑ANCA have circulating antibodies
reactive not only to PR3 sense peptides but also to PR3
complementary antisense peptides.40,41
In addition to
antibodies that recognize complementary PR3 antigens,
Yang et al.44
detected anticomplementary PR3-specific
memory T cells—directed against a PR3 complementary
peptide—in patients with PR3‑ANCA. The stimulus for
the antibodies to complementary PR3 could have been
endogenous PR3 antisense peptides45
or exogenous pep-
tides that mimic the antisense peptides, such as peptides
brought in by commensal or pathogenic microbes.40
Interestingly, the bioinformatics software tool BLAST
(basic local alignment search tool) identifies multiple
microbial peptide homologues of complementary PR3,
including peptides from microbes that are known to be
associated with PR3‑ANCAs such as Ross River virus
and S. aureus.40,41
A genome-wide association study has indicated that
different HLA specificities correlate with MPO‑ANCA
and PR3‑ANCA, and a gene encoding PR3 was associ-
ated with PR3‑ANCA but not MPO‑ANCA.7
These
findings suggest that antigen-recognition capabilities
are important in ANCA-associated disease, and that the
initial antigen recognized by antigen-presenting cells is
different in PR3‑ANCA-associated disease as opposed
to MPO‑ANCA-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 PR3‑ANCA-associated disease
(odds ratio 73.3).46
The DRB1*1501 protein binds with
high affinity to both sense-PR3 peptide as well as the
complementary antisense-PR3 peptide.46
This finding
suggests that one factor in the induction of ANCA-
associated disease could be the presence of HLA antigen-­
binding sites that recognize the autoantigen and a
complementary protein.
Thus, the theory of autoantigen complementarity sug-
gests that a patient with a genetically determined pre-
disposing antigen-recognition capability could produce
pathogenic ANCA as a result of an initial appropriate
immune response against a microbial mimic of a com-
plementary peptide, or against an endogenously pro-
duced antisense transcript, that would be transformed
into an autoimmune response by an anti-idiotypic res­
ponse (Figure 4). In support of this theory, anti-idiotypic
a b
Figure 3 | Pathology of ANCA-associated necrotizing granulomatosis. a | The edge
of an early acute pulmonary lesion in a patient with PR3‑ANCA-positive GPA is
characterized by a central zone of necrosis containing numerous neutrophils in
varying stages of leukocytoclasia, and by an adjacent band of monocytes and
macrophages (arrows) (magnification ×400, HE staining). b | A later stage of
granulomatous inflammation in the same pulmonary lesion specimen demonstrates
a central zone of amorphous necrotic debris containing a few pyknotic leukocytes,
and an adjacent marginal zone of epithelioid macrophages (arrows) (magnification
×400, HE staining). Abbreviations: ANCA, antineutrophil cytoplasmic antibody;
GPA, granulomatosis with polyangiitis; HE, haematoxylin and eosin; PR3,
proteinase 3.
Endogenous source
Exogenous source
Microbes
Neutrophil
Anti-idiotypic
antibody
(autoantibody)
Mimic of
antisense peptide
Antisense
transcription
Sense
transcription
Sense DNA
strand
Antisense
DNA strand
ANCA antigen
peptide
Antisense
peptide
T cell
Idiotypic
antibody
Idiotope
mimicking the
autoantigen
Figure 4 | Diagram of the induction of an ANCA-mediated autoimmune response by
an initial immune response to a peptide that is complementary to an autoantigen
peptide. This complementary peptide immunogen could arise from antisense
transcription of the antisense strand of the autoantigen gene, or could be a mimic
of an antisense peptide that is produced by a symbiotic or pathogenic microbe.
The anticomplementary peptide antibody idiotopes would engender an anti-
idiotypic antibody response that crossreacts with the autoantigen epitopes that
are complementary to the initial immunogenic peptide. Abbreviation: ANCA,
antineutrophil cytoplasmic antibody.
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antibodies raised against autoantibodies have been
shown in multiple animal models to induce anti-­anti-
idiotypic antibodies that possess characteristics of the
initial ­autoantibodies 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 LAMP‑2.10
The human
LAMP‑2 epitope has complete homology with the bacte-
rial adhesin FimH, and human LAMP‑2-ANCA cross-
reacts with this microbial protein. Kain et al.10
reported
that rats immunized with FimH develop antibodies to
rat and human LAMP‑2, 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 LAMP‑2 and mediates ANCA-associated disease.
However, Roth et al.11
were not able to ­reproduce the
findings from this ­experimental animal model.
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 T cells and B cells,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 T‑cell and B‑cell suppression,
and possibly by enhanced B‑cell 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.52–54
TREG
cells are charac-
terized by high expression of CD25 and FOXP3. Even
though patients with active GPA have increased numbers
of CD4+
CD25+
T cells, 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 et al.54
described
an increase in a proinflammatory suppression-­resistant
effector CD4+
T‑cell 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
exon 2.54
A study by Wilde et al.55
showed that increased
expression of the negative co-stimulator programmed
cell death protein 1 (PD‑1) on circulating T cells in
patients with GPA might oppose T‑cell activation; how­
ever, T cells in renal lesions in these patients mostly
lacked PD‑1 expression.
B-cell regulation might also influence production of
pathogenic ANCAs.56,57
B cells that have high expression
of CD5 and produce IL‑10 have regulatory capabilities.
Patients with active ANCA-associated disease have a
reduced percentage of circulating CD5+
B cells whereas
most patients in remission have a normal percentage.52
In addition, the degree of normalization of circulating
CD5+
B cells after therapy with rituximab correlates with
more-persistent remission.56
ANCA-activated neutrophils might augment the
production of ANCAs by stimulating B cells. Acti­
vated neutrophils release ligands for B‑cell-activating
fac­tor (BAFF; also known as TNF ligand superfamily
mem­ber 13B or B lymphocyte stimulator), which stimu-
lates B‑cell 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.59–62
Thus, B‑cell-stimulating factors released by ANCA-
activated neutrophils along with permissive T‑cell and
B‑cell regulation seem to facilitate the production of
pathogenic ANCAs.
Pathogenesis of vascular inflammation
Neutrophil and monocyte activation by ANCAs
In vitro studies have shown that both MPO‑ANCA and
PR3‑ANCA 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 Fcγ recep-
tors (FcγRs) 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.70–72
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.27,28,63–74
These ANCA-activated cells have been shown to cause
injury to and death of cultured monolayers of endo­thelial
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7. NATURE REVIEWS | RHEUMATOLOGY VOLUME 10 | AUGUST 2014 | 469
cells.75–78
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 in vitro by engage-
ment of FcγRs by ANCA IgG.80,81
ANCA-activated
monocytes release proinflammatory cytokines, includ-
ing monocyte chemoattractant protein‑1 (MCP‑1, also
known as CCL2)80
and IL‑8.81
IL‑8 attracts and activates
neutrophils and thus could amplify neutrophil-­mediated
injury. MCP‑1 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
after the first day or two, and there is evidence for sys-
temic activation of monocytes in patients with active
ANCA-associated disease.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
In vitro 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 in situ
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.85–87
Multiple convincing models of MPO‑ANCA-associated
dis­ease have been described and confirmed.84–87
How­
ever, no convincing models of PR3-ANCA-associated
disease have been widely accepted, although several
have been proposed.88–91
Mouse models of AAV and ANCA-associated glo-
merulonephritis induced by anti-MPO antibodies have
been used most extensively to elucidate pathogenic
mechanisms.64,65,71,84,92–98
Disease can be induced by
injecting mouse anti-MPO IgG into immunocompetent
or immunodeficient mice,84
injecting splenocytes con-
taining anti-MPO B cells 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 MPO‑ANCA-associated disease:
C5a
C5a receptor
Cytokine
Cytokine
receptor
Neutrophil
Apoptotic body
Macrophage
Monocyte
Unprimed
neutrophil
Priming
Activation
Alternative
pathway
activation
NETosis
Acute
inflammation
Chronic inflammation
and scarring
ANCA antigen
ANCA
Fc receptor
T cell
Fibroblast
Fibrin
Collagen
Epithelial cell
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.
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antibody to MPO alone (in the absence of functional
T cells) 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 FcγR–antibody 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
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 factor B 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 C5b‑9, 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 (Figure 5).
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. FcγR 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 ­mononuclear-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 Klinger102
and Wegener103,104
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
pathways—one initiated by a T‑cell mediated (type IV)
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
Unprimed
neutrophils
Activation
Microabscess
formation
Granulomatosis
Priming
C5a
C5a receptor
Cytokine
Cytokine
receptor
Neutrophil
Macrophage
Monocyte
ANCA antigen
ANCA
Fc receptor
T cell
Multinucleated
giant cell
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.
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9. NATURE REVIEWS | RHEUMATOLOGY VOLUME 10 | AUGUST 2014 | 471
walled off.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 type IV
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 (Figure 3a) or subacute lesions
with central necrosis (Figure 3b) to chronic lesions with
a central zone of scarring and chronic inflammation.20
Chronic lesions contain substantial accumulations of
lymphocytes and dendritic cells, including T cells and
B cells 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 in vitro, 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 (Figure 6).
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 30 years 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-DK058335‑11).
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.
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