REVIEWS

The immunopathogenesis of
seropositive rheumatoid arthritis:
from triggering to targeting
Vivianne Malmström, Anca I. Catrina and Lars Klareskog
Abstract | Patients with rheumatoid arthritis can be divided into two major subsets characterized
by the presence versus absence of antibodies to citrullinated protein antigens (ACPAs) and
of rheumatoid factor (RF). The antibody-positive subset of disease, also known as seropositive
rheumatoid arthritis, constitutes approximately two-thirds of all cases of rheumatoid arthritis and
generally has a more severe disease course. ACPAs and RF are often present in the blood long
before any signs of joint inflammation, which suggests that the triggering of autoimmunity may
occur at sites other than the joints (for example, in the lung). This Review summarizes recent
progress in our understanding of this gradual disease development in seropositive patients. We
also emphasize the implications of this new understanding for the development of preventive
and therapeutic strategies. Similar temporal and spatial separation of immune triggering and
clinical manifestations, with novel opportunities for early intervention, may also occur in other
immune-mediated diseases.

Rheumatoid factor
(RF). Antibody reactive with the
Fc part of self or non-self IgG.
Different isotypes of RF exist,
but most clinical assays that
are used to determine whether
an individual is RF positive
measure IgM RF.
CTLA4–Ig
Also known as abatacept. This
fusion protein of the cytotoxic
T lymphocyte associated
protein 4 (CTLA4) extracellular
domain and the Fc region of
IgG1 binds to the co‑stimulatory
receptors CD80 and CD86 on
effector T cells to prevent their
activation. Abatacept is an
approved treatment for
rheumatoid arthritis.
Rheumatology Unit,
Department of Medicine at
Solna, Karolinska University
Hospital, Karolinska Institute,
171 76 Stockholm, Sweden.
Correspondence to L.K.
Lars.klareskog@ki.se
doi:10.1038/nri.2016.124
Published online 5 Dec 2016
Rheumatoid arthritis (RA) is traditionally character‑
ized and defined by its clinical manifestations of joint
inflammation and bone destruction1,2. However, accu‑
mulating evidence now shows that patients with RA can
be divided into at least two major subsets on the basis of
the presence versus absence of autoanti­bodies specific
for autoantigens modified by citrullination (antibodies
to citrullinated protein antigens (ACPAs)) and autoanti‑
bodies specific for self IgG‑Fc (known as rheumatoid factor
(RF)). The presence of these anti­bodies defines the sero‑
positive form of RA, which constitutes approximately
two-thirds of all disease cases. The two RA subsets have
different disease courses (BOX 1), with the seropositive
form typically causing more bone and joint destruction.
Moreover, seropositive RA typically responds better to
B cell depletion using CD20‑specific antibodies3 and
toco‑stimulation blockade with CTLA4–Ig4 compared
with the seronegative form. Most notably, however, the
aetiology of these two forms of RA is different in regard
to both genetic and environmental determinants of dis‑
ease in that only the seropositive form of disease is asso‑
ciated with MHC class II‑restricted adaptive immune
responses5,6 (FIG. 1). This Review focuses on the seroposi­
tive subset of RA, for which substantial progress has been
made in our understanding of the gradual development
of disease. The aetiology and development of the sero­
negative subset of RA are less well understood, and this
subset of disease is probably more heterogeneous than
the seropositive variant.
Whereas much research focus and therapeutic pro‑
gress in RA has been on the synovial pathology of estab‑
lished disease and its cytokine-mediated regulation,
less attention has been given to the longitudinal devel‑
opment of disease and the interactions between innate
and adaptive immune responses in this process. In this
Review, we summarize and discuss some recent progress
in our emerging understanding of the triggering and
gradual development of adaptive immunity, in particu‑
lar to citrullinated autoantigens, in the long process that
often precedes the onset of joint inflammation in RA.
We also discuss data that suggest how immune responses
to citrullinated antigens and other RA‑associated factors
may ultimately target bones and joints to initiate the
destructive joint inflammation that we recognize as RA.
The resulting model represents an attractive hypothesis,
obviously not excluding other and complementary mech‑
anisms. We also acknowledge the obvious caveat that any
pathogenic model should be subjected to empirical test‑
ing by selective therapies before being accepted. Notably,
the scenario that we discuss may offer opportunities for
specific clinical trials that — if successful — will provide
several new options for prevention and early interven‑
tion that would significantly improve the prospects for
patients with RA or individuals at high risk for RA.

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Box 1 | Rheumatoid arthritis

Although rheumatoid arthritis (RA) is defined by clinical criteria2, there is increasing evidence to indicate that several
different aetiologies and pathogenic mechanisms are active in different subsets of the disease. Patients with RA can be
subclassified in several ways, and the pathogenic relevance of these subsets can also be tested for in several ways. The
major method for validating the pathogenic relevance of disease subsets in this Review is based on the combination of
genetic and environmental determinants of disease (FIG. 1). Other methods can be used to validate RA subsets, such as the
response to therapy or the appearance of clinical features. The table summarizes the different approaches to subclassifying
patients and the implications for our current understanding of the different molecular pathways underlying RA.

Classification Feature ACPA- ACPA- Pathogenic implications Refs

method positive negative
patients patients
Subclassifying HLA alleles Major risk Small or no • Involvement of MHC 5,110
by genetic and genes effect class II‑dependent adaptive
environmental immunity in the ACPA-positive
determinants PTPN22 risk allele Second major Small or no subset 72,111
risk gene effect • No evidence of homogeneity in the
Smoking exposure Large impact Small or no ACPA-negative subset 5,6
impact • Possibly a small impact of adaptive
immunity and MHC class II genes
Silica exposure Moderate Small or no in minor subsets of ACPA-negative 43,112,
impact impact disease 113

Subclassifying Rituximab Good but Weaker but • ACPA-positive patients are more 114
by response to (CD20‑specific variable variable responsive to therapies directed
therapies antibody) response response to B cells and T cells, but there are
common final pathways in the two
Abatacept Good but Weaker but disease subsets 115
(CTLA4–Ig) variable variable • Major heterogeneities between
response response patients in each subset indicate
Anti-TNF Variable Variable not yet understood and variable 116
response response pathogenic pathways
Anti‑IL‑6 Variable Variable 116
response response
Subclassifying Joint destruction More, but Less, but • Major, not yet understood, 117
by clinical (severe disease) variable variable heterogeneities in terms of the
features variable joint involvement
Extra-articular More, but Less, but • There are indications that different 118,119
manifestations variable variable pathogenic mechanisms may lead to
(nodules, similar clinical manifestations
vasculitis, pleuritis)
ACPA, antibodies to citrullinated protein antigens; CTLA4−Ig, cytotoxic T lymphocyte associated protein 4−immunoglobulin; IL,
interleukin; TNF, tumour necrosis factor.

Seropositivity in RA including homo-­citrullination 16 and acetylation 17,

Citrullinated autoantigens
Proteins and/or peptides in
which the amino acid arginine
is changed to the amino acid
citrulline. The change is
mediated by one of the four
enzyme variants of
peptidyl-arginine deiminases
(PADs).
Cyclic citrullinated peptide
(CCP) assay
An assay that captures the
majority of antibodies reactive
to various citrullinated
autoantigens (these antibodies
are referred to generically as
ACPAs). The assay is built on
CCPs, and the most commonly
used commercial variant is
named CCP2.
The classical immune feature associated with RA is the
presence of RF7 in the blood; however, RF is not spe‑
cific for RA but occurs in the context of many other
inflammatory conditions, particularly in the context of
T cell-mediated immune responses to immune com‑
plexes containing IgG8,9. The identification of anti‑
bodies reactive to citrullinated autoantigens followed
from a systematic analysis of RA‑associated antibodies
to perinuclear factors10, keratin11 and filaggrin12; it was
subsequently discovered that these antibodies target
post-translationally modified (citrullinated) residues of
filaggrin13,14. This discovery was then translated into the
current major diagnostic test for RA, the cyclic ­citrullinated
peptide (CCP) assay15. Citrullinated autoantigens are more
specific than RF to patients with RA, occurring in less
than 2% of healthy individuals and only at low levels in
other inflammatory diseases. Today, the generic name
for these antibodies is ACPAs. Recently, antibodies
specific for other post-translational modifications,
have also been associated with RA. Substantial cross-­
reactivity exists between antibodies to these different
post-­translationally modified antigens18,19. The spec‑
trum of ACPA specificities found in patients with RA
represents a growing family of both ubiquitous and more
tissue-­specific and/or inflammation-restricted antigens,
including citrullinated epitopes on fibrinogen, vimentin,
histones, α­ -enolase, type II c­ ollagen and tenascin C20–24.
Use of genetic epidemiology in defining disease
One finding that has transformed our understanding of
the origins of the pathogenic immunity in RA is that
RA‑associated antibodies can be present in the blood
long before the first signs of joint inflammation. This
finding was first shown for RF and subsequently for
ACPAs25–27. Interestingly, the gradual development of
RA‑associated antibodies28–31 may occur over several
years, with epitope spreading and an increase in anti‑
body titre occurring in conjunction with disease onset

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Expression quantitative
trait loci
Genomic loci where variations
in nucleotide sequence (usually
in a single nucleotide)
determine the expression level
of a particular mRNA.
High-resolution computed
tomography
A sensitive imaging technique
that, in the context of this
Review, is used to visualize
changes in the lungs, including
inflammatory infiltrates.
Peptidyl-arginine
deiminases
(PADs). Citrullination (also
known as deimination) of
peptides and proteins is
mediated by the
calcium-dependent PADs.
Periodontitis
An inflammatory disease
affecting the gum
(peridontium). Severe
periodontitis leads to alveolar
bone loss and subsequent loss
of teeth.
(FIG. 2). Very few individuals seroconvert after the onset
of joint disease32, which indicates that the observed anti‑
body response is more likely a cause than a ­consequence
of disease.
An obvious question following on from the obser‑
vation that the emergence of RA‑associated immune
responses and joint inflammation are separated in time
is where and how this immunity is triggered. Major
clues to this question have emerged from cohort and
genetic epidemiology studies. The genetic studies con‑
firmed that MHC class II genes are by far the greatest
genetic risk factor for seropositive RA but also showed
that many additional genetic variants and their corre‑
sponding expression quantitative trait loci confer disease
risk. These findings therefore point to a crucial role of
T cells in disease development. Studies of environmental
risk factors have shown that noxious airway exposure, in
particular to cigarette smoke, is a major risk factor for
seropositive RA (but not for the seronegative form of
the disease). Furthermore, a major gene–environment
interaction was demonstrated between distinct HLA‑DR
variants and exposure to smoke and other agents such
as silica dust, and this interaction was specific for the
ACPA-positive subset of RA. These findings gave rise
to the initial suggestion that the anti-citrulline immu‑
nity that precedes the onset of joint inflammation in
RA may be triggered in the lungs or elsewhere in the
airways 5. These combined cohort and genetic epi­
demiology ­studies have contributed to the redefining of
RA as a gradually developing disease and have created
two foci for further investigation: how is the observed
RA‑specific MHC-restricted immune response trig‑
gered and how does this immune response contribute
to the subsequent targeting of the bones and joints. The
remainder of this Review discusses these two events.
Triggering of RA‑associated immunity
Encouraged by the data from genetic epidemiology
studies, a series of clinical and immunological studies
have recently been carried out focusing on the lung and
related mucosal tissues as possible sites for the triggering
of RA‑specific immune responses. Three major findings
have substantiated the idea that RA‑specific immunity
may indeed be initiated in the lungs. First, it was shown
that ACPAs are present in the sputum of (serum) ACPA-
positive individuals without arthritis33 and that ACPAs
are enriched in the bronchoalveolar lavage34 of patients
with early ACPA-positive RA. Second, both macroscopic
lung changes (as assessed by high-resolution computed
tomography) and microscopic lung changes (infiltrates
of inflammatory cells in bronchial biopsies) are pres‑
ent in patients with early untreated ACPA-positive RA,
whereas these changes do not occur to the same extent
in healthy individuals or in patients with seronegative
early RA34,35. Lung changes visible by high-resolution
computed tomography have also been reported in
ACPA-positive healthy individuals at increased risk for
developing RA36. Notably, studies of lung biopsy sam‑
ples taken from ACPA-positive patients with established
RA showed binding of citrullinated fibrino­gen to B cells
and plasma cells in the inflammatory infiltrates in lung
parenchyma, further suggesting that ACPAs are pro‑
duced locally 37. Third, several studies have shown that
IgA immune responses are prominent and appear early
in the development of the immunity that precedes joint
inflammation26,38,39, and they remain an important com‑
ponent of the immune response in established seroposi­
tive RA40,41. The early development and persistence of
IgA ACPA and RF reactivities thus suggest that there is
ongoing mucosal stimulation of the immune ­reactions
that are hallmarks for emerging as well as chronic
seropositive RA.
The precise molecular mechanisms that might be
responsible for the triggering of anti-citrulline immunity
in the lungs are, so far, relatively unexplored. We know,
however, that lung exposure to noxious agents, includ‑
ing smoke, can induce increased expression and activa‑
tion of peptidyl-arginine deiminases (PADs). This process
possibly follows the triggering of Toll-like receptors (in
particular TLR4) by particles in the smoke, silica parti‑
cles or microbial agents that are known to be present in
the lungs of smokers42–44. Such increased expression of
PADs has been shown to be associated with an increased
presence of citrullinated proteins in bronchial biopsy tis‑
sues and bronchoalveolar lavage cells from both patients
with RA and otherwise healthy individuals45. It is also
known that several components of smoke, as well as sil‑
ica dust and other noxious agents, can contribute to the
accumulation and activation of several types of antigen-­
presenting cell (APC) in the lungs, including classical
dendritic cells and B cells.
Thus, an attractive possibility is that triggering of
T cell-mediated immunity to citrullinated (and other­
ise modified) self-antigens may occur in the lungs after
presentation of the neo-antigens by locally activated
APCs (FIG. 3). It is not yet known whether T cells ­reactive
with environmentally induced post-translationally
modi­fied antigens escape negative selection in the thy‑
mus, but this interesting possibility remains to be further
investigated.
A completely different example of how immune
­reactions that develop in the lung can cause autoimmun‑
ity comes from a recent study exploring COPA (which
encodes coatomer subunit-α). A genetic mutation in
COPA that leads to the impairment of endoplasmic
reticulum–Golgi transport caused the development of
autoantibodies, lung disease and arthritis46. This study
showed that the lung is sensitive to a generic alteration of
endoplasmic reticulum stress and autophagy responses,
and that events that occur first in the lungs could lead to
subsequent joint disease.
A potential role for other mucosal compartments. Many
seropositive patients with RA do not have any evidence
of lung inflammation or any history of exposure to
noxious airway agents. Thus, other tissues should also
be considered as triggering sites for the initiation of
autoimmunity. One alternative triggering site that has
received much attention is the gum, where particular
interest has been given to a potential link between RA
and p­ eriodontitis caused by Porphyromonas gingivalis
infection47,48. This bacterium harbours its own PAD

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and hence contains citrullinated proteins49. Thus, an
attractive molecular mimicry mechanism has been sug‑
gested whereby the RA‑associated ACPA response
originates from a protective immune response against
P. gingivalis 50. The fact that smoking is strongly linked
to the presence of perio­dontitis and to P. gingivalis
infection provides circumstantial evidence in favour of
this hypothesis. However, recent epidemiological data
have failed to show a clear relationship between peri‑
odontitis and RA51, although an increased presence of

a ACPA-positive RA ACPA-negative RA
60
50 HLA-DR-SE
40
30
20
Genome-wide
–Log10 (P)

10
significance 8
7 7
6 PTPN22 6

–Log10 (P)
5 5
4 4
3 3
2 2
1 1
0 0
Chr1 Chr2 Chr3 Chr4 Chr5 Chr6 Chr7 Chr8 Chr9 Chr10 Chr11
Chr12 Chr13 Chr14 Chr15 Chr16 Chr17 Chr18 Chr19 Chr20 Chr21 Chr22

b ACPA-positive
ACPA+ RA RA ACPA-negative RA
13.3% 13.3%

10% 10%
Lifetime risk of RA

Lifetime risk of RA

6.7% 6.7%

Environment Environment
3.3% 3.3%
High-level smoker High-level smoker
(no/low alcohol intake) (no/low alcohol intake)
Low-level smoker Low-level smoker
(no/low alcohol intake) (no/low alcohol intake)
Genes High-level smoker Genes High-level smoker
(high alcohol intake) (high alcohol intake)
Any HLA-DR-SE risk allele, Any HLA-DR-SE risk allele,
Non-smoker Non-smoker
any PTPN22 risk allele any PTPN22 risk allele
(no/low alcohol intake) (no/low alcohol intake)
Any HLA-DR-SE risk allele, Low-level smoker Any HLA-DR-SE risk allele,
no PTPN22 risk allele (high alcohol intake) Low-level smoker
no PTPN22 risk allele
(high alcohol intake)
No HLA-DR-SE risk allele, Non-smoker No HLA-DR-SE risk allele, Non-smoker
any PTPN22 risk allele (high alcohol intake) any PTPN22 risk allele (high alcohol intake)
No HLA-DR-SE risk allele, No HLA-DR-SE risk allele,
no PTPN22 risk allele no PTPN22 risk allele

ACPA-positive RA
c 10–5

10–4

10–3
P values

0.01

Dendritic Memory CD8+ T cells NKT cells

0.1 cells CD4+ T cells

B cells CD4+ T cells NK cells

1
Cell type

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Molecular mimicry certain P. gingivalis-specific antibodies (for example,
Occurs when sequence to gingipains) was observed in patients with ACPA-
similarity can lead to immune positive RA52. Thus, a relationship between RA and
system cross-reactions, such as bacterial infection in the mucosa — that is not neces‑
a foreign antigen triggering a
response that is subsequently
sarily related to periodontitis — may exist and warrants
propagated by a self-antigen. further investigation.
The sequences do not need to The intestinal tract is another mucosal organ that has
be identical, but share certain long been implicated in the pathogenesis of RA53,54. This
crucial residues.
potential connection has been subject to renewed inter‑
est recently on the basis of observations that changes
in the gut microbiota may be related to the presence of
RA55. There is also evidence from animal experiments
that experimental perturbations of the gut microbiota
and the gut barrier may contribute to the development of
arthritis56–58. However, compared with the epidemiologi‑
cal evidence and data on gene–environment interactions
that describe the relationship between RA and immu‑
nity triggered in the lungs, evidence is currently lack‑
ing to indicate that RA‑specific immunity is triggered
in the gut. There is, however, evidence that inflamma‑
tion and inflammatory diseases, including RA, can be
modified by events in the gut, such as changes in the gut
microbiota59.
Progression to systemic immunity
As described above, longitudinal studies of historical
serum biobanks have shown that various RA‑associated
antibodies can be present in the serum long before the
onset of joint inflammation. However, these studies were
carried out retrospectively in individuals known to have
RA. Only a few studies have so far investigated the pres‑
ence and evolution of ACPAs and related autoantibodies
in populations not selected for the future development
of RA. Typically, sera from individuals in whom no
◀ Figure 1 | Genetic epidemiology of RA. a | Genome-wide association studies have
demonstrated major differences between the two subsets of rheumatoid arthritis (RA)
as defined by the presence versus absence of antibodies to citrullinated protein antigens
(ACPAs). Genetic association of RA with the HLA region (in particular, certain HLA‑DR
alleles; also known as the shared epitope (HLA‑DR‑SE) alleles) and with PTPN22 is mainly
confined to the ACPA-positive subset of patients5,120,121. In the ACPA-negative subset, the
overall heritability of disease is much lower122, and the influence of HLA genes is limited
and different from ACPA-positive disease113. The P value for genome-wide significance is
indicated by the horizontal blue lines. b | Pronounced gene–gene and gene–environment
interactions are present in ACPA-positive RA but absent in ACPA-negative disease. The
figure presents the combined effects of two genetic factors (HLA‑DR‑SE and PTPN22)
and two environmental factors (smoking and alcohol consumption) on the lifetime risk
of RA in the two disease subsets, and illustrates the increasingly complex pattern of
determinants of disease. The data represent a combination of data on the lifetime risk
of RA123 and on gene–gene and gene–environment interactions72,124. c | The information
regarding RA‑associated genes, environmental factors and their interactions can be
combined with information on active molecular pathways in different cell types and in
different organs as studied, for example, by expression quantitative trait loci analyses.
The graph illustrates such cell type-specific expression of genes that have been
associated with disease through an analysis of single nucleotide polymorphisms (SNPs)
in different cell types125. The graph shows a preferential expression of genes related to
T cells in the context of SNPs related to seropositive RA. Hereby, CD4+ T cells, particularly
effector T cells, have been implicated in the pathogenesis of ACPA-positive RA125−127. This
combined view lends support to further studies of T cell specificity and effector function
in patients with seropositive RA, including (but not necessarily limited to) T cells of T
helper 1, T follicular helper and regulatory T cell phenotypes. Chr, chromosome;
NK, natural killer. Part a is reproduced from REF. 120. Part c is reproduced from REF. 125.
evidence was found for future development of RA had
lower titres of ACPAs and had reactivity against fewer
citrullinated peptides or epitopes compared with sera
from individuals who later developed RA60,61. Notably,
one study carried out in a large group of twins indicated
that environmental triggers (including smoking) are
major determinants of the initial triggering of auto­
immunity, whereas genetic factors are more impor‑
tant as determinants of high ACPA titres and the large
number of citrullinated epitopes that are recognized in
individuals who later developed RA or already had RA61.
These data suggest that environmental and stochastic,
partly T cell-independent, events are important for the
initial triggering of ACPAs and RF. By contrast, genetic
determinants, in particular HLA alleles, have a greater
relative role in the transformation from autoimmunity
to ­autoimmune disease.
In addition to epitope spreading of the antibody
response within the group of citrullinated autoanti‑
gens during the progression to systemic immunity, the
induction of several other antibody types, modification
of antibodies and occurrence of other biomarkers have
been observed during this phase62. Thus, different iso‑
types of RF, including IgA RF, may occur early during
the systemic response, and the simultaneous presence
of RF and ACPAs in the serum is highly predictive of
future RA development 27. Notably, a change in glycosy­
lation of the antigen-binding part of ACPAs, with cur‑
rently unknown functional consequences, has also been
observed during this early evolution of immunity before
the onset of joint disease63.
We currently have a limited understanding of the
mechanisms that drive the gradual development of
autoimmunity from the first triggering of anti-­citrulline
immunity until the occurrence of a more vigorous
immune response that is seen immediately before the
onset of joint inflammation. A better understanding of
this process might facilitate the development of agents
that can inhibit the potentially pathogenic immunity at
a very early stage.
Specificity of B cell and T cell responses
Following the discovery of the potentially pathogenic
autoimmunity to citrullinated and other antigens in sero‑
positive RA, research has increasingly been devoted to
the specificity and regulation of this immune response.
HLA class II alleles. The connection between HLA
class II variants and susceptibility to RA goes back to
the early days of tissue and HLA typing 64. However, a
more precise understanding of MHC-restricted immune
responses in RA pathogenesis, in particular in rela‑
tion to antigen specificity, was lacking for a long time.
The finding of shared consensus amino acids lining
the peptide-binding grooves of several RA‑associated
HLA alleles provided circumstantial evidence that the
peptide-­binding capacity of these HLA molecules is of
pathogenic significance. More precisely, a positively
charged P4 pocket in HLA‑DR (as influenced by amino
acids 13, 71 and 74 in the β‑chain65) is a strong suscepti‑
bility factor for ACPA-positive RA. Notably, a positively

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Environmental stimuli Mucosal surfaces Gums

Textile dust, silica
Smoking Lungs

Bacteria

Triggering
Genetic risk factors
Gut

HL

P TP
A-D

N2
R

2
Epitope spreading and increased titres of autoantibodies

RF
T cell
Lymph node

CD40 TCR

Maturation CD40L HLA

BCR
B cell

ACPA

Bone erosion and arthralgia

CXCR1 CXCL8
or CXCR2
RF

Osteoclast
Targeting

Bone
Autoimmune response

Nociceptive
ACPA nerve ending
Additional
hit

Citrullinated
Macrophage histone

NETosis

Neutrophil
Fulminant ACPA
disease TLR TNF

IL-6
MMPs
Fibroblast-like
synoviocyte

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Nature Reviews | Immunology
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ACR/EULAR criteria charged P4 pocket is an unfavourable binding site for
Classification criteria for arginine-containing peptides, whereas it easily accom‑
rheumatoid arthritis from modates citrullinated variants of the same peptides66.
the American College of Thus, an arginine to citrulline conversion by PADs will
Rheumatology (ACR) and
European League Against
enhance HLA binding and therefore the presentation
Rheumatism (EULAR). The of peptides that would not be bound in their native
latest criteria include positive form (FIG. 4).
status for antibodies to
citrullinated protein antigens.
CD4+ T cells. Analyses of T cell reactivity to candidate
PTPN22 autoantigens in RA, including their citrullinated vari‑
This gene encodes the protein ants, have so far been focused on autoantigens that were
tyrosine phosphatase LYP. previously identified by autoantibodies from the same
A functional polymorphism patients. Subsequently, candidate peptides for T cell
in PTPN22 shows genetic
association to several
activation in response to these autoantigens were iden‑
inflammatory diseases, tified by their ability to specifically bind to the grooves
including rheumatoid arthritis. of the HLA‑DR alleles associated with ACPA-positive
RA. This approach has obvious limitations in terms of
its ability to identify all targets for T cell activation in
RA, which may not even be part of the same proteins as
those recognized by antibodies. For example, in c­ oeliac
disease, T cells that are reactive to gluten-derived pep‑
tides provide help to B cells that are reactive to both
the autoantigen tissue transglutaminase 2 and gluten
(reviewed in REF. 67). Nevertheless, in the absence of
any obvious additional T cell targets in RA, studies
of T cell reactivities to citrullinated peptides from the
major autoantigens recognized by B cells and anti­bodies
from patients with ACPA-positive RA have yielded
­interesting results.
So far, citrulline-reactive CD4+ T cells from patients
with RA have been validated for several ACPA targets
(for more details, see FIG. 4). Both in vitro cultures and
◀ Figure 2 | From triggering to targeting: a longitudinal perspective on the
development of seropositive RA. The figure illustrates a model for the longitudinal
disease course of rheumatoid arthritis (RA). Interestingly, the pathology associated with
anti-citrulline immunity often does not begin with arthritis; instead, the pathology can
begin with bone loss and arthralgia before the onset of synovitis128,129. The figure illustrates
four schematic phases of disease development. Phase 1 (triggering): autoimmunity is
triggered as a result of environmental stimuli acting together with the genotype at
mucosal surfaces such as the lungs or possibly the gums or gut. Phase 2 (maturation): the
autoimmune response gradually matures and involves epitope spreading and increasing
titres of autoantibodies (antibodies to citrullinated protein antigens (ACPAs) and
rheumatoid factor (RF)). This autoimmune response is restricted to sites outside the joints,
possibly in the regional lymph nodes and other peripheral lymphoid organs. This phase is
contingent on MHC class II‑dependent T cell activation (as evidenced from genetic
linkage studies) and the resulting interaction between T cells and B cells. Phase 3
(targeting): symptoms such as bone loss and joint pain (arthralgia) develop owing to
certain ACPAs acting in synergy with RF. The binding of ACPAs to osteoclasts induces the
production of the chemokine CXCL8, which functions as an autocrine growth factor for
osteoclasts. CXCL8 also has the capacity to bind to its receptors CXCR1 and CXCR2 on
nociceptive nerves and thereby cause pain. Phase 4 (fulminant disease): synovitis and the
classical symptomatology of RA as defined by ACR/EULAR criteria develop. ACPAs may also
promote the formation of neutrophil extracellular traps (NETosis) after binding to
citrullinated histones in NETs. Moreover, citrullinated fibrinogen fragments, and
potentially other citrullinated proteins, can bind to Toll-like receptors (TLRs) on synovial
cells, leading to the production of interleukin-6 (IL‑6), tumour necrosis factor (TNF) and
matrix metalloproteinases (MMPs). The four phases depicted in the figure may occur
sequentially; importantly, they may also co‑occur or revert. The major message is that
disease-inducing immunity may be triggered at one site by one mechanism, and
thereafter target another site or organ much later, potentially using a different
mechanism. We also postulate that an ‘additional hit’ may contribute to the targeting of
the joint (see FIG. 5 for details). BCR, B cell receptor; TCR, T cell receptor.
ex vivo analyses of in vivo-activated T cells with HLA
class II tetramers have shown the presence of effector
and/or memory CD4+ T cells specific for citrullinated
epitopes in patients with RA66,68,69. A parallel analysis
in HLA-matched healthy controls showed much lower
levels of T cell activation against these antigens, whereas
reactivity to an unrelated antigen (from influenza virus)
was similar between healthy controls and patients with
RA68,69. These data provide compelling evidence in
favour of a pathogenic role of specific T cell reactivi‑
ties to citrullinated peptides; however, we clearly need
more studies of the frequencies and phenotypes of rare
antigen-specific T cells in patients with RA, for exam‑
ple in clinical trial settings, to understand the relative
­contribution of autoreactive T cells to disease.
Although a major focus of this Review is on adaptive
immune responses with specificities that are dependent
on the genetic and environmental risk factors for ACPA-
positive RA, several other more general features of T cells,
irrespective of their specificity, have been described in
patients with RA. One such feature is determined by a
polymorphism of PTPN22, in which the presence of the
minor risk allele influences the threshold for activation of
T cells70 and also reduces the negative selection of B cells71,
leading to a more autoimmune-prone B cell and T cell rep‑
ertoire. Importantly, the PTPN22 risk allele displays strong
gene–gene interaction with the RA‑associated HLA‑DR
alleles. This finding implies that the combined presence
of an HLA‑DR variant that presents certain peptides to
induce specific immunity and a PTPN22 gene variant that
promotes a generally wider T cell repertoire provides a
permissive environment for the specific autoimmunity
in RA, in particular in the context of smoking 72 (FIG. 1).
Another more recently described feature of T cells in RA
concerns their cellular metabolism, whereby CD4+ T cells
from patients with RA have biased metabolic activity that
favours proliferation and memory conversion73. However,
the studies of immunometabolism were all carried out on
cells from patients with established RA; thus, it is likely
that the observed changes are influenced by the o ­ ngoing
disease. It will be interesting to investigate whether such
metabolic dysregulation develops before the clinical onset
of disease in patients with RA.
In summary, accumulating data indicate that specific
T cell-mediated immune responses to candidate auto­
antigens are formed outside the joints and before joint
inflammation. It is also likely that additional factors, not
directly linked to T cell specificity, may further enhance
the activation of antigen-specific T cells in patients with
emerging RA.
Autoreactive B cells. The roles of antibodies and B cells
in RA have been investigated and discussed over many
years, initially mainly in terms of RF and immune
complexes 74. More recently, the focus has been on
antigen-specific B cell responses and on the diverse
functions of B cells in addition to being precursors to
antibody-producing plasma cells.
Studies showing the occurrence of antibodies to cit‑
rullinated and other modified autoantigens before and
after the diagnosis of RA have now been extensively

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Replacement mutations replicated (see above). Less is known, however, about the are present among plasmablasts in the circulation of
In contrast to silent mutations, B cells and plasma cells that produce these antibodies. patients with RA75,76, among memory B cells and plasma‑
replacement mutations lead to In this regard, new insights have recently been provided blasts in synovial fluid of patients with RA77, and among
amino acid changes. When by several groups who produced human recombinant B cells retrieved from the synovial tissue of patients with
these changes occur in the
complementarity-determining
monoclonal antibodies based on the sequencing and RA undergoing arthroplastic surgery 78. Importantly, no
regions of antibodies, they are re‑expression of paired heavy and light chains from citrulline-reactive immunoglobulins could be generated
likely to affect how antigen is ­single B cells or plasma cells from patients with RA. from B cells or plasma cells from seronegative patients
recognized by the antibody. These studies have shown that citrulline-reactive B cells with RA or from healthy donors70,77.
The emerging picture gained from these studies is
that citrulline-reactive B cells are relatively frequent in
Bronchus number in patients with ACPA-positive RA and that they
accumulate in the inflamed joints. Moreover, the variable
Microorganism genes encoding the citrulline-reactive immunoglobulins
have an accumulation of replacement mutations in their
complementarity-determining regions, which is indica‑
Increased tive of T cell help. Also, the monoclonal ACPAs have a
expression of PADs high degree of cross-reactivity with many citrullinated
H2N NH2 H2N NH2 autoantigens but no reactivity to the non-modified forms
PAD
H2O Ca2+
of these antigens77,79.
NH NH Taken together, these studies indicate that B cells
reactive to post-translationally modified autoantigens,
NH4
and helped by HLA class II‑dependent T cells, either
Citrullination
migrate to or proliferate in the joints to constitute a size‑
Peptidyl- Peptidyl- able proportion of the total B cells in the inflamed tis‑
arginine citrulline sue. If some, or all, of these B cells and plasma cells and
their immunoglobulin products are able to promote joint
TLR stimulation inflammation and bone destruction, they would indeed
HLA B cell TLR constitute major effector functions in RA, possibly in
synergy with effector T cells reactive to peptides from
HLA
the same m ­ odified proteins.
DC
TLR
HLA
Targeting the bones and joints
APC Textile dust,
activation silica particles or Although the epidemiological data from longitudinal
B cell smoke particles studies of the development of RA (reviewed above) indi‑
maturation
T cell activation cate that anti-citrulline immunity may be pathogenic,
Macrophage to self-antigen particularly when combined with the presence of RF,
CD80/
CD86 CD28
Figure 3 | Local early immune activation in the lungs.
APC T cell Various noxious agents (such as smoke particles, silica
iBALT
particles, textile dust and microorganisms) can cause
HLA TCR insults in the lungs and, in the context of the susceptibility
genes, trigger rheumatoid arthritis (RA)‑associated
Antigen presentation
immune reactions. Events inside the lungs involve the
activation of dendritic cells (DCs), macrophages and B cells
T cell help by Toll-like receptor (TLR) stimuli from particles in cigarette
smoke, silica dust or textile dust or from components of an
aberrant microbial flora42–44. This process may lead to
CD40L CD40 peptidyl-arginine deiminase (PAD) activation and local
extracellular citrullination and thereby the appearance of
RA‑associated neo-antigens. Another consequence may
be the differentiation of B cells, whereby several different
HLA TCR stimuli and specific recognition events can cause initial
Lung tissue
somatic mutations of immunoglobulin genes, enabling
B cells to bind and present post-translationally modified
Local Antigen presentation autoantigens to T cells. Germinal centre-like structures
antibody (induced bronchus-associated lymphoid tissue (iBALT))
production
Class switching are formed in the lungs early in the development of
RA–associated immune responses and disease35,36, and
T cell-dependent B cell activation promotes the local
production of antibodies to citrullinated protein
ACPA IgA antigens (ACPAs) in lungs34. APC, antigen-presenting cell;
ACPA IgG
TCR, T cell receptor.

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mediated both by direct recognition (because antigen-­

Naive binding Fab fragments also promoted osteoclast
T cell matur­ation)82 and by the activation of Fc receptors on
TCR
osteoclasts83. No effects on osteoclasts were seen with
IgG antibodies from ACPA-positive patients depleted of
CD28 TCR
-2 -1 2 3 5 7 8 ACPAs or with IgG from patients with seronegative RA82.
Citrullinated 1 4 6 9 These results indicate that antibodies reactive to citrul‑
CD80/
CD86 peptide Citrulline
P1 P4 P6 P9 linated autoantigens can exert a specific function on
HLA-DR osteo­clasts at the earliest stages of disease ­development
Peptide-binding groove of HLA-DR
APC and considerably before joint inflammation.
HLA-DR Subsequent investigations have suggested that osteo‑
clasts and osteoclast precursors might be the major cell
Figure 4 | Involvement of adaptive immunity. The figure depicts the interaction types that express citrullinated antigens on their surface
between the peptide–MHC complex on an antigen-presenting cell (APC) and the T cell in the normal, non-inflamed bone and joint compart‑
Nature
receptor (TCR) on a T cell. Citrullinated peptides (as indicated byReviews
red rings)| Immunology
have been ment. Thus, these precursor cells are prime targets for
shown to bind the P4 pocket of the peptide-binding groove of the HLA‑DR molecules circulating ACPAs82,84. This feature of osteoclasts is
associated with rheumatoid arthritis (RA); these genetically defined HLA-DR alleles associated with a non-redundant role of PADs in their
are known as the shared epitope (HLA‑DR‑SE) alleles (FIG. 1). Neutral citrulline can be differentiation from macrophage precursors, which may
accommodated in the positively charged P4 pocket of the HLA‑DR variants that be a major role of PADs in cell differentiation once the
predispose for RA, whereas the positively charged amino acid arginine is unlikely to fit immune system has matured. Osteoclast differentiation
into this structure66. Specific binding to the RA‑associated P4 pocket has been is accompanied by the induced expression of PADs, and
demonstrated for peptides from vimentin, fibrinogen, α‑enolase, aggrecan and
differentiation can be inhibited in vitro by PAD inhibi‑
cartilage intermediate-layer protein66,68,69. In addition, arginine or citrulline can be
found at other positions in the antigenic peptide, including those facing towards the tors such as chloramidine82. The exact role of PADs and
TCR. Examples of citrulline being recognized directly by the TCR at positions –2, –1 citrullination in osteoclast differentiation remains to be
and 2 come from peptides of α‑enolase and type II collagen69,130,141. Together, these determined. However, these findings provide a poten‑
findings indicate the importance of specific genetic associations of HLA‑DR‑SE alleles tial explanation for how antibodies triggered against
with RA in presenting disease-associated T cell epitopes. However, it is becoming certain protein modifications initially encountered
increasingly clear from studies of both B cell and T cell responses in RA that this outside the joint may then target a joint component
disease is not the result of an aberrant immune response to a single autoantigen but (here, the bone) where the same modifications occur as
to many autoantigens, of which we still do not understand the relative importance in the triggering organ (such as the lungs), but arising
and/or hierarchy. through a different mechanism. Notably, of the cell types
that have been tested so far (fibroblast-like synoviocytes,
experimental evidence for such a pathogenic role has T cells and B cells), only osteoclasts are dependent on
been lacking. Moreover, there has been a conceptual PADs, which supports the idea that this dependency may
problem in terms of linking immunity to citrullinated explain the unique targeting of osteoclasts by ACPAs in
proteins, which are ubiquitously present during inflam‑ a ­non-inflamed context.
mation, to disease in a specific organ system such as the The concept of a pathogenic role of ACPA-dependent
bones and joints. Furthermore, there is no indication osteoclast activation has been further strengthened by
that antibodies transferred from mothers with high titres several different findings. One key observation was
of ACPAs and/or RF to their fetuses can cause disease that the chemokine CXCL8 (also known as IL‑8) is a
(which is the case for other antibody-mediated diseases major effector molecule produced by ACPA-exposed
such as neonatal lupus80). osteoclasts. CXCL8 (and its murine analogue CXCL1)
In the face of these questions, significant progress has, can function as an autocrine factor for osteoclasts85,
Osteoclasts however, been made recently to suggest that immunity to and the CXCL8‑dependent differentiation of osteo‑
Bone lining cells that absorb
citrullinated and otherwise modified autoantigens may clasts is enhanced by ACPAs82. This role of CXCL8 was
bone during growth and
healing. They are identified by indeed contribute to the development of joint disease non-redundant for ACPA-dependent osteoclast dif‑
their multiple nuclei and when the longitudinal perspective and the concept of ferentiation in both humans and mice, as neutralizing
positive staining for tartrate ‘additional hits’ are taken into account (FIG. 5). CXCL8‑specific antibodies (in vitro in humans)82 and
resistant acid phosphatase antagonists of a receptor for CXCL1 (CXCR1; in vivo
(TRAP). Osteoclasts
differentiate in the presence of
Osteoclast-dependent bone loss. The first clue that in mice) blocked osteoclast differentiation and osteo­
the cytokine RANKL from ­ steoclasts might have an important role in ACPA-
o clast functions both with and without ACPAs84. The
myeloid cells. dependent pathology came from studies of polyclonal interaction of physiologically developing osteoclasts
ACPAs purified from the serum of patients with ACPA- with ACPAs is thus an attractive possibility to explain
Osteopaenia
positive RA. These polyclonal ACPAs were able to pro‑ how these antibodies specifically may target bones and
Loss of bone density.
mote osteoclast differentiation and activation in vitro, ­possibly also joints (see below).
Fibroblast-like synoviocytes and cause bone erosion and osteopaenia in vivo 81. In
The specialized fibroblasts that subsequent experiments that confirmed and extended Osteoclast-dependent arthralgia. Arthralgia (joint
are part of the normal lining of these findings, some monoclonal ACPAs generated pain) is a common symptom that often precedes the
the joint synovial membrane.
These cells can undergo
from B cells found in the synovial fluid of patients development of joint inflammation in RA. Moreover,
large-scale proliferation in with RA were able to promote osteoclastogenesis and arthralgia together with seropositivity for ACPAs and/or
rheumatoid arthritis. bone resorption79,82. The effects of the antibodies were RF has been shown to be highly predictive for the future

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Joint • Mechanical
space hypersensitivity
• Thermal hypersensitivity
Bone • Decreased locomotor
activity

Synovial
membrane

CXCR1
or CXCR2 Nociceptor
Maturation Cartilage Maturation
CXCL8

Fc Cartilage and ACPA

receptor bone erosion
Citrullinated
target antigen
PAD
Immune Osteoclast
complex RANKL,
M-CSF

Monocyte
precursor

Figure 5 | Development of ACPA-mediated disease as a precursor to RA. Autoantibodies (and in particular

antibodies to citrullinated protein antigens (ACPAs)) generated at extra-articular sites and present in the circulation
Nature Reviews can
| Immunology
mediate bone destruction and pain before the occurrence of chronic joint inflammation. Osteoclasts develop from
monocyte precursors in the presence of receptor activator of nuclear factor-κB ligand (RANKL; also known as TNFSF11)
and macrophage colony-stimulating factor (M‑CSF). The physiological maturation of these precursor cells depends on
the enzymatic activity of peptidyl arginine deiminases (PADs), leading to an increased expression of citrullinated targets
in mature osteoclasts. Some ACPAs can bind these targets and activate osteoclasts to secrete CXCL8, which in turn
induces further osteoclast development and maturation82. Antibodies incorporated in immune complexes, especially
those that are a‑sialylated83,131, can also bind to the Fc receptors expressed on the surface of maturing osteoclasts to
increase osteoclast differentiation through the activation of immunoreceptor tyrosine-based activation motif (ITAM)
signalling. The activation of osteoclasts through antibodies, either alone or incorporated in immune complexes, finally
leads to bone erosion, cortical fenestration and loss of trabecular bone in the absence of any sign of inflammation.
ACPAs bound to osteoclasts trigger CXCL8 release, which in turn sensitizes or activates sensory neurons by binding to
CXCR1 and CXCR2 (receptors for CXCL8) expressed on nociceptors87,132–135. ACPAs, but not other antibodies, induce
mechanical and thermal hypersensitivity as well as a decrease in spontaneous locomotor activity, and these changes can
largely be reversed by CXCR1 and CXCR2 blockade84.

Collagen-induced arthritis
(CIA) model
A murine model of polyarthritis
that is induced after
immunization with collagen
type II, which is the major
structural component of
cartilage.
onset of RA86. Interestingly, a direct connection between
ACPAs and arthralgia was recently observed when mice
injected with ACPAs, but not with other anti­b odies
from patients with ACPA-positive RA, developed
pain-like behaviour involving mechanical and thermal
hyper­sensitivity and leading to a significant reduction
in spontaneous locomotor activity 84. However, despite
their pain-inducing capacity in vivo, ACPAs neither
bound to peripheral sensory neurons nor stimulated
them in vitro. Instead, after intravenous injection of
purified ACPAs to mice, these antibodies bound spe‑
cifically to CXCL1 (analogous to human CXCL8)-
expressing osteoclasts or osteoclast precursors found in
close proximity to sensory nerves expressing CXCR1
and/or CXCR2 (which are receptors for CXCL1). As
CXCL8 and its analogues have previously been shown
to activate nociceptive nerve signalling and pain by
binding to CXCR1 and/or CXCR2 (REF. 87), these find‑
ings suggested that the ACPA-induced pain behaviour
in mice might be mediated by CXCL1 produced by
osteoclasts. This concept was further supported by the
observation that blockade of CXCL1 using a receptor
antagonist completely abolished not only bone loss but
also ACPA-induced pain84. Based on these observations,
it is possible that the effect of ACPAs on osteoclasts,
mediated partly by CXCL8 or CXCL1, is a major con‑
tributor to arthralgia and bone loss, both of which can
precede joint inflammation ­during the ­development of
ACPA-positive RA.
Joint inflammation (synovitis). Injection of ACPAs
from patients with RA or of monoclonal ACPAs gener‑
ated from mice or from human B cells or plasma cells has
not by itself been shown to induce synovitis. However,
the transfer of such ACPAs can enhance synovitis that
is induced by other means, for example in a collagen-­
induced arthritis (CIA) model88; in that context, these anti‑
bodies contributed to a destructive joint inflammation

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 REVIEWS

that was reminiscent of human RA. Consequently,
an attractive scenario is that an individual with high
­levels of ACPAs and RF who develops an acute form
of joint inflammation that would otherwise normally
resolve within a short period of time instead develops a
chronic and destructive form of synovitis. Transient joint
inflammation due to trauma, virus infections or other
poorly characterized stimuli is a common occurrence
in humans.
One potential mechanism by which such ‘addi‑
tional hits’ may precipitate the development of chronic
arthritis in an ACPA-positive individual relates to the
potential effect of CXCL8 and ACPAs on neutrophils.
The local presence of CXCL8 may both attract and
promote the differentiation of neutrophils to a state in
which n­ eutrophil extracellular traps (NETs) are formed89.
Interestingly, such NETosis has recently been shown to
be enhanced under the influence of certain ACPAs, in
this case antibodies that cross-react with citrullinated
histones23. A potential scenario is that CXCL8 produced
by ACPA-activated osteoclasts enhances the activation of
and NETosis by neutrophils that have migrated into the
adjacent joint compartment, where ACPAs reactive to
histones could further enhance NETosis. This scenario
is particularly attractive in the context of the well-known
early infiltration of neutrophils into the joints of patients
with RA, and the fact that this feature of RA has so far
rarely been linked to other pathogenic features of the
disease, such as humoral immunity.
Notably, several additional effects of citrullinated
autoantigens, ACPAs and associated immune com‑
plexes may further promote inflammation. This pro‑
cess may occur through the TLR4‑mediated activation
of myeloid cells by citrullinated autoantigens, in par‑
ticular citrullinated fibrinogen fragments that bind
to TLR4 (REF. 90). Myeloid cell activation may be fur‑
ther enhanced by the binding of ACPAs and ACPA-
associated immune complexes, including RF, to the
target cells. Such complexes can trigger the production
of all classical pro-­inflammatory cytokines, including
tumour necrosis factor (TNF) and IL‑6, by macro­
phages91 and fibroblast-like synovio­cytes. The role of
fibroblast-like synoviocytes during the phase of disease
when the inflammation becomes chronic and addi‑
tional joints are engaged in a selective way is of increas‑
ing interest. For example, fibroblast-like synoviocytes
have the capacity to migrate between different joints
and contribute to the spread of synovitis to additional
joints92. Furthermore, recent studies have shown that
fibroblast-like synoviocytes derived from different
Neutrophil extracellular joints have different patterns of epigenetic regulation93
traps and engage different molecular pathways94, possibly
(NETs). Neutrophils can explaining in part why only certain joints are affected
extrude their nuclear content
by the polyarthritis in RA. A role for fibroblast-like
through the process of NETosis.
The released DNA–histone synoviocytes has even been suggested in the context of
complexes can have smoking, as epigenetic modifications in fibroblast-like
antibacterial effects, but also synoviocytes are observed in mice exposed to cigarette
lead to exposure of nuclear smoke95. Hence, we hypothesize that targeting of the
contents to the immune
system. NETs are abundant in
joints in individuals with pre-­existing ACPAs and RF
the synovial fluid of patients may depend on initial ACPA-driven events that spe‑
with rheumatoid arthritis. cifically target bones and joints. This process is further
enhanced by more general pro-­inflammatory events,
some of these also being dependent on anti-­citrulline
immunity, but ­o thers being dependent on addi‑
tional mechanisms such as the s­ elective activation of
­fibroblast-like synoviocytes (FIG. 6).
Other potential pathways towards RA
Several other mechanisms, in addition to anti-­citrulline-
driven events, may contribute to the development of
joint inflammation and symptoms of RA, either in sep‑
arate subsets of disease or as contributors to disease in
patients with seropositive RA. However, most of these
alternative or complementary mechanistic explanations
have been developed on the basis of animal models and
they lack the robust epidemiological basis that is pro‑
vided for the anti-citrulline model that is the main focus
of this Review.
The best-studied alternative or complementary
pathogenic model concerns immunity to type II colla‑
gen and related cartilage-derived autoantigens, which
may also involve citrullination. Although antibodies to
native (unmodified) type II collagen are relatively rare
in patients with RA and before the onset of RA, high
titres of such antibodies have been recorded in a small
subpopulation of patients96. It may be that these patients
constitute the human equivalent of the conventional CIA
model in mice and rats. Other recent data have shown
that many, but not all, ACPAs are cross-reactive with cit‑
rullinated epitopes of type II collagen97. If such epitopes
from the cartilage can be shown to be expressed and be
available to the immune system during the early stages of
RA development, this mechanism of targeting the joint
compartment would offer an attractive complement
to the osteoclast-focused mechanisms that have been
described in previous sections.
Additional mechanisms that have been extensively
described and studied in animal models include the for‑
mation of immune complexes with ubiquitously avail­
able autoantigens, such as glucose phosphate isomerase
(GPI)98. However, anti-GPI reactivity (including anti‑
bodies) is neither specific for RA nor common in this
disease99. Nevertheless, the principle of arthritis devel‑
opment in animal models in the context of immune
complex formation involving ubiquitously available
autoantigens has obvious implications for other anti‑
gen specificities, including citrullinated and otherwise
­modified antigens discussed above.
Finally, several MHC class II‑independent mech­
anisms are known to contribute to arthritis in animal
models, involving, for example, cytokine dysregulation
and the activation of synovial fibroblasts100. However,
the enigma concerning all of these mechanisms has
been to explain why the dysregulation of mechanisms
that are present in all types of inflammation would
preferentially target the joints. Therefore, we propose
that the adaptive immune responses described in this
Review may contribute to the initial targeting of joints
and bone (FIG. 6), and that such an initial spark may then
lead to the involvement of the alternative inflammatory
pathways that have been described in many previous
reviews101,102.

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Antigen presentation
T cell activation a ACPA
CD80/
CD86 CD28
Osteoclast
APC T cell
Maturation
HLA TCR
e
Citrullinated T cell help
antigen
CD40L
CD40 Destruction CXCL8
B cell
HLA TCR Recruitment

Antigen
presentation NETosis

Plasma cell Citrullinated

Articular cartilage Neutrophil histone
Citrullinated IL-6,
protein CXCL8
c ACPA
RF
b Inflammation
Citrullinated
protein
TNF
Synovial
membrane IL-6
Bone

Immune
complex
TLR4

Macrophage
Fc receptor

Fibroblast-like d
synoviocyte

MMPs

Figure 6 | The inflammatory cascade in established RA. This figure
illustrates how antibodies to citrullinated protein antigens (ACPAs) and
rheumatoid factor (RF) may contribute to joint inflammation. a | Binding of
ACPAs to osteoclasts can induce CXCL8 secretion, which promotes
neutrophil chemoattraction. Neutrophils can also migrate to joints in
response to non-specific inflammatory stimuli (caused by ‘additional hits’; not
shown). CXCL8 promotes neutrophils to release neutrophil extracellular traps
(NETs), which can be further enhanced by ACPAs binding to citrullinated
histones exposed in the NETs. b | Moreover, expression of citrullinated proteins
in articular cartilage might allow ACPAs97,136 to bind the cartilage surface and
initiate local inflammation through immune complex-mediated mechanisms.
c | ACPAs might also bind citrullinated proteins directly in the synovial
membrane following minor insults (such as trauma or infection). d | Immune
complexes between citrullinated proteins (such as fibrinogen)
Nature Reviews and ACPA-IgG
| Immunology
can drive inflammation (including the production of IL‑6 and tumour necrosis
factor (TNF), which are a hallmark of the synovial inflammation in rheumatoid
arthritis (RA)102) by engaging both Toll-like receptor 4 (TLR4) and Fc
receptors91. Irrespective of the initiating event, changes associated with
normally transient joint inflammation may be sufficient to allow ACPAs and
RF to exert their pro-inflammatory effects selectively in the joints, where
inflammation may promote the expression as well as citrullination of proteins.
e | As a consequence, both ACPA- and RF‑dependent events could contribute
to the initiation and propagation of chronic synovial inflammation, and
further synergize with T cell activation137 and enhance the local production
of autoantibodies77,138. APC, antigen-presenting cell; MMPs, matrix
metalloproteinases; TCR, T cell receptor.

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Table 1 | Preventive and therapeutic opportunities during the longitudinal development of RA

Stage of Patient subset General interference Examples Background
disease mechanism refs
Triggering Genetically susceptible Remove triggers via lifestyle Stop smoking, weight loss 139,140
individuals exposed to changes
distinct environmental
triggers Block inflammatory pathways at Local steroids, interference 104
triggering sites such as the lungs with adaptive immunity
Block the generation of modified PAD inhibitors 82,104
self-antigens at triggering sites
such as the lungs
Maturation ACPA-positive Interfere with the specific Antigen-specific tolerance 105–108
individuals without adaptive immune response procedures using citrullinated
synovitis peptides or autologous
synovial fluid
Interfere with B cell and T cell B cell depletion using 104
functions CD20‑specific antibodies
such as rituximab
T cell co‑stimulation blockade Ongoing
with CTLA4–Ig (abatacept) clinical trials
Targeting ACPA-positive Block the generation of modified PAD inhibitors 82,104
individuals with self-antigens at targeting sites
arthralgia such as the joints
Minimize pain and bone erosion, Inhibit the effects of CXCL8 82,84
delay development of disease by blocking CXCR1 and/or
CXCR2 or by CXCL8‑specific
neutralizing antibodies
Fulminant Diagnosis of RA with Several options for early and Cytokine blockade, T cell and 103,116
disease synovitis active treatment B cell-targeted therapy
ACPA, antibody to citrullinated protein antigen; CTLA4−Ig, cytotoxic T lymphocyte associated protein 4−immunoglobulin;
PAD, peptidyl-arginine deiminase; RA, rheumatoid arthritis.

Rituximab
A monoclonal antibody
targeting CD20 that is
a B cell-depleting agent.
B cell-depletion therapy is
an approved treatment for
rheumatoid arthritis.
Dissecting the molecular pathogenesis of RA
Future studies of RA must take into account two concepts
that have been discussed in this Review. The first concept
is the heterogeneity of the disease. In this regard, there is
the need to stratify patients with RA as well as individuals
at risk of RA into different subsets that take into account
the determinants of disease (genes and environment)
and the available immunological data. The second con‑
cept is the need to address the gradual development of dis‑
ease, which requires longitudinal studies beginning with
individuals at risk of disease who have not yet developed
joint inflammation. To allow immunologists to properly
study RA and to take these two fundamental aspects
into account, close collaboration will be necessary with
clin­icians in charge of cohorts and with biobanks, and a
means to stratify the patients will be required
Such a broad and clinically oriented approach has
facilitated the emerging understanding of the gradual
development of disease in conjunction with anti-­citrulline
immunity. Similar strategies are needed for the seronega‑
tive subset of RA, for which much less is known about the
triggering or targeting of potentially pathogenic immune
and inflammatory reactions. Obviously, we are also only
beginning to understand the different phases in the devel‑
opment of seropositive RA. There is still much more to
learn regarding the specificity and regulation of immune
reactions not only to the candidate antigens identified so
far but also to other immune targets, which may be both
autoantigens and foreign (mainly microbial) structures.
Finally, within the anti-citrulline model described here,
we need a better understanding of when and in which
contexts a­ utoimmunity is converted to autoimmune
disease.
Therapeutic implications
The interventions that are currently in clinical use for
patients with RA, which have been summarized in several
recent reviews (see, for example, REF. 103), are all directed
towards inflammatory events in established disease, and
most of these successful drugs have been developed based
on studies of the inflamed synovium.
This Review summarizes recent progress with a dif‑
ferent approach, in which the longitudinal and gradual
development of seropositive RA is the focus. Using this
approach, several previously unrecognized mechanisms of
disease and therapeutic opportunities become apparent,
and the indications for use of other therapies may also be
extended, in particular in the phase of disease develop‑
ment when symptoms such as arthralgia are present but
joint inflammation has not yet developed (TABLE 1). For
example, clinical trials are currently ongoing in ACPA-
positive individuals without synovitis to test whether
B cell depletion using rituximab or T cell co‑stimulation
blockade using CTLA4–Ig can prevent or delay the onset
of full-blown arthritis. Preliminary results from the rituxi­
mab trial indicate that onset of disease may be retarded
by this treatment104. Also, initial attempts to develop and
test tolerizing procedures using citrullinated peptides

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and autologous synovial fluid as tolerogenic antigens
have recently been reported in patients with established
RA105,106. We anticipate many more such tolerizing thera­
pies to follow as ACPA-positive RA is now emerging as
an attractive prototype disease for inducing tolerance107.
An example of such an approach would be the loading of
appropriate carriers, such as nanoparticles, with candidate
peptide antigens alone or bound to appropriate MHC
class II molecules107. Notably, the administration of pep‑
tides or MHC class II–peptide complexes alone without
carriers can induce antigen-specific tolerance in animals108
and in humans with multiple sclerosis109. Other therapeutic
approaches could involve early interference with trigger‑
ing events in the lungs, particularly those dependent on
PADs, using both PAD inhibitors and less specific, gen‑
eral anti-inflammatory agents that might be given locally
to the lungs. Other approaches could interfere with the
proposed mechanisms that target bones and joints, includ‑
ing the activation of CXCL8- and PAD-dependent mech‑
anisms in osteoclasts and neutrophils, for example, using
CXCR1- and CXCR2‑blocking agents or neutralizing
CXCL8‑specific antibodies. Notably, only therapeutic ­trials
in humans will be able to define the rela­tive ­importance of
these ­mechanisms in different subsets of RA.
Most importantly, however, the increasing insights
into the gradual development of RA make it obvious that
this disease may be targeted during the initial phases and
not only when joint inflammation and emerging joint
destruction have been established. Continuing insights
into the mechanisms of emerging RA should enable us to
follow the example of the cardiologists, who treat hyper‑
tension (comparable to the presence of autoimmunity)
to prevent stroke or ischaemic heart disease (equivalent to
joint inflammation).

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Acknowledgements
The authors thank numerous colleagues in their own and
many collaborating laboratories for providing insights into
the story of RA development told here. The authors give spe-
cial thanks to the following individuals: L. Alfredsson and
L. Padyukov, who made the initial gene–environment interac-
tion studies possible; C. Svensson, who provided data and
insights into pain mechanisms; and V. Joshua, who helped
with illustrations. Studies from the authors’ own laboratory
described in this Review were funded by the Swedish Medical
Research Council, the European Union (European Research
Council senior investigator grant to L.K., and several
Innovative Medicines Initiative and 7th Framework
Programme (FP7) grants, including BTCure, TEAM and
Osteoimmune), grants from the Swedish Strategic Foundation
and Margaretha af Ugglas Foundation, and the Swedish
Organization for Patients with Rheumatic Diseases.
Competing interests statement
The authors declare competing interests: see Web version for
details.

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