CHAPTER 56 
Autoantibodies in
Rheumatoid Arthritis
Felipe Andrade • Erika Darrah • Antony Rosen
KEY POINTS
Autoantibodies in rheumatoid arthritis (RA) are important
tools for diagnosis and for defining pathogenic pathways in
this disease.
Rheumatoid factors (RFs) are autoantibodies that recognize
the Fc portion of immunoglobulin G molecules. They were
the first autoantibodies described in RA.
RFs have numerous causes in addition to RA and therefore
have limited specificity for RA.
Autoantibodies targeting citrullinated proteins are hallmarks
of the immune response in RA.
The anti–cyclic citrullinated peptide (anti-CCP) assay broadly
detects antibodies against anti-citrullinated protein
antibodies (ACPAs) and has high specificity and excellent
sensitivity in RA.
ACPAs precede the onset of clinical RA and are associated
with more severe disease, making them useful as diagnostic
and prognostic tools.
Peptidylarginine deiminase enzymes, which catalyze the
conversion of peptidyl-arginine to peptidyl-citrulline, play
important roles in generating citrullinated autoantigens
in RA.
Genetic factors such as “shared epitope” human leukocyte
antigen–DR subregion alleles appear to interact with
environmental factors, such as smoking and infection, to
initiate and drive autoimmunity in RA.
Autoantibodies have proven to be very useful tools for diag-
nosis and prediction in autoimmune rheumatic diseases.
Emerging data pertaining to several autoimmune rheumatic
diseases have demonstrated that the clinical evolution of
disease from the pre-clinical phase to overt clinical disease
is marked by a change in the specificity of the immune
response, with autoantibodies directed against distinct
antigenic targets at different disease phases. Whereas
numerous autoimmune rheumatic diseases traditionally
have been marked by highly phenotype-specific autoanti-
body responses (e.g., anti–double-stranded DNA antibodies
in systemic lupus erythematosus [SLE] and high-titer anti–
topoisomerase-1 antibodies in diffuse scleroderma), the
discovery of highly specific autoantibody markers of rheu-
matoid arthritis (RA) lagged significantly behind these
other diseases. During the past decade, enormous progress
has occurred in this area, largely fueled by the discovery that
citrullinated proteins are specific targets of autoantibodies
in RA. This finding has highlighted post-translational mod-
ifications (PTMs) as critical determinants targeted by auto-
antibodies in RA, which has led to the identification of
novel autoantibody systems and pathogenic mechanisms in
this disease (Table 56-1). In this chapter we will review the
autoantibodies in RA, with emphasis on rheumatoid factors
(RFs), anti-citrullinated protein autoantibodies (ACPAs),
and the anti–peptidylarginine deaminase (PAD) autoanti-
bodies. The implications of these specificities for diagnosis
and prediction of outcome, as well as for understanding
pathogenic events in the disease, will be highlighted.
RHEUMATOID FACTOR
The earliest assays (later called the Rose-Waaler agglutina-
tion test) that suggested the existence of autoantibodies in
RA were developed in the early 1940s, when sera from
patients with RA were shown to cause agglutination of
sheep blood cells that had been sensitized with sub-
agglutinating doses of rabbit anti–sheep erythrocyte anti-
bodies.1,2 These assays were later shown to be detecting
immunoglobulin (Ig)M antibodies from patients with RA
that recognized the Fc portion of IgG.3 Numerous modifica-
tions of the agglutination assay evolved, particularly the use
of IgG-coated latex beads instead of sheep erythrocytes.4,5
Subsequent developments established RF assays in radioim-
munoassay, enzyme-linked immunosorbent assay (ELISA),
and nephelometry formats, with increased convenience but
similar performance in terms of sensitivity and specificity.
RFs may be positive in healthy control subjects (1% in
younger persons and up to 5% in persons older than 70
years) and in patients with numerous other non-RA diseases
(including other rheumatic diseases such as Sjögren’s syn-
drome and cryoglobulinemia), as well as chronic infections.6-8
The pretest probability of diagnosing RA therefore greatly
influences the performance of the RF test, with sensitivities
and specificities both in the 50% to 90% range, depending
on the patient groups studied.9,10 The existence of IgG and
IgA RFs and evidence of somatic hypermutation have
suggested that some RFs in RA are T cell dependent.11,12
Although some studies have suggested that IgG and IgA
RFs increase the specificity of RFs for RA,13 a recent meta-
analysis showed that assays of the different RF isotypes per-
formed very similarly in terms of sensitivity and specificity
and therefore do not add much compared with standard
RF assays.14
Importantly, patients with RA also vary in terms of
timing of the appearance of RF, with RF preceding symp-
tomatic disease in a significant subpopulation15-18 and fol-
lowing disease onset with variable kinetics in other patients.
831
 832 PART 7    DIAGNOSTIC TESTS AND PROCEDURES IN RHEUMATIC DIEASES

TABLE 56-1  Native and Modified Targets of Autoantibodies in Rheumatoid Arthritis

Prevalence of
Type of Antigen Type of PTM Antigen Generation Protein Autoantibodies
Native None Unknown IgG (Fc) 50%-90%
hnRNP-A2 (RA33) 35%
G6PI 12%-29%
PAD4 30%-40%
Post-translationally Citrullination Enzymatic reaction mediated by Vimentin, fibronectin, actin, HSP90, Up to 80%
modified PADs; arginine residues are histones, α-enolase, eEF1a, CAP-1,
changed to citrulline CapZalpha-1, asporin, cathepsin D,
histamine receptor, PDI, ER60
precursor, ALDH2, collagen type I and
II, eIF4GI, aldolase, PGK1, calreticulin,
HSP60, FUSE-BP1 and 2, ApoE, MNDA
Carbamylation Nonenzymatic reaction of cyanate Unknown 45%
with lysine residues; lysine is
changed to homocitrulline
MAA adducts Nonenzymatic reaction; peroxidation Unknown 29%-93%
of membrane lipids generate
reactive aldehydes that modify
lysine residues, generating MAA
adducts

ALDH2, Mitochondrial aldehyde dehydrogenase; ApoE, apolipoprotein E; CAP1, adenylyl cyclase–associated protein-1; CapZalpha-1, F-actin capping
protein alpha-1 subunit; eEF1a, elongation factor 1-alpha; eIF4GI, eukaryotic translation initiation factor-4G1; FUSE-BP, far upstream element binding
protein; G6PI, glucose-6-phosphate isomerase; IgG, immunoglobulin G; hnRNP, heterogeneous nuclear ribonucleoprotein; HSP, heat shock protein; MAA,
malondialdehyde-acetaldehyde; MNDA, myeloid nuclear differentiation antigen; PAD, peptidylarginine deaminase; PDI, protein disulfide-isomerase; PGK1,
phosphoglycerate kinase-1; PTM, post-translational modification.

Indeed, the earlier onset of RF in patients with RA has been
associated with more severe disease, highlighting a possible
contribution of these antibodies to amplification.19 The
observation that RF only appears after disease onset in some
patients suggests that it may mark distinct, sequential events
in pathogenesis, which are very close together in the sub-
group in whom RF occurs early, or are separated in time in
patients in whom RF follows symptoms. The mechanisms
that drive the production of RF in persons with RA are not
fully understood. The presence of somatic hypermutation in
RFs and the evidence that these antibodies are produced by
plasma cells in the rheumatoid synovium have suggested
that the RF response is driven locally by antigen.11,12
However, it is still unclear how the IgG becomes self-
immunogenic in the rheumatoid joint. In contrast to other
autoantigens in RA, there is no evidence that the IgG
targeted by RFs requires modification (e.g., PTM) for anti-
body recognition. Nevertheless, it is still possible that the
initial event that breaks tolerance to IgG may be initiated
through an abnormally modified molecule, as occurs for
other autoantigens in RA. The possible mechanisms
whereby RF may play an amplifying role in RA are numer-
ous and include amplification of antigen capture, signaling,
and effector functions, among others.20
ANTIBODIES AGAINST CITRULLINATED
ANTIGENS
Antikeratin Antibodies and Antiperinuclear
Factor: Initial Insights into a Novel Group of
Autoantibodies in Rheumatoid Arthritis
Although RFs have been clinically useful in diagnosis of
RA—particularly in cases in which the index of clinical
suspicion is high—these autoantibodies have two shortcom-
ings: (1) their lack of specificity for RA and (2) an unclear
kinetic and mechanistic connection to pathogenesis. Thus
defining additional autoantibodies specific for RA has been
a major priority. A significant discovery in this regard was
made initially by Nienhuis and Mandema in 196421 (Figure
56-1). These investigators recognized an autoantibody that
stained keratohyalin granules surrounding the nucleus in
cells of human buccal mucosa, which they called antiperi-
nuclear factor (APF). These APF antibodies were found in
49% to 91% of patients with RA,21-25 and they have reported
specificities of 73% to 99%.21,24-27 The assay was difficult to
standardize because not every donor of buccal mucosal cells
demonstrated the staining pattern,28-31 and thus the assay
did not find its way into routine clinical practice, but the
specificity for RA suggested that the assay might be report-
ing on a potentially important pathogenic pathway. A
second research group subsequently demonstrated that RA
sera recognized antigens in the stratified squamous layer of
esophageal epithelium—a staining pattern they termed
antikeratin antibodies (AKAs).32 Subsequent iterations of
this assay utilized other forms of stratified squamous epithe-
lium, including human skin.33 Although this AKA assay
also had very good specificity properties, it was of limited
sensitivity34-37 and was not particularly convenient or easy
to standardize. Thus neither assay was broadly used in the
clinical diagnostic arena. Like the LE cell in SLE, however,
the assays provided a framework for antigen discovery that
would later lead to highly specific and convenient tools for
RA diagnosis and classification.
Discovery of Autoantibodies That
Recognize Peptidylcitrulline
The initial discovery of antibodies that recognized keratin-
ized squamous epithelium in patients with RA prompted
 CHAPTER 56    Autoantibodies in Rheumatoid Arthritis 833

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sorbent assay.

studies to define the target of these antibodies. It was ini-
tially shown that APF only stained a fully differentiated
squamous epithelial layer and that APF staining was identi-
cal to the pattern produced by a monoclonal antibody spe-
cific for human (pro)filaggrin in indirect immunofluorescence
of permeabilized squamous epithelial cells.23 Although the
antigen could not be definitively identified using this
co-localization alone, it suggested that the differentiation
state might affect antigen recognition. Subsequently, RA
sera were shown to recognize a 40 kDa protein extracted
from human epidermis, which was identified as a neutral/
acidic isoform of filaggrin.38 IgG from RA sera affinity-
purified against the 40 kDa filaggrin protein was reactive in
both the APF and AKA tests, demonstrating that these
autoantibodies were very similar or identical.39
Filaggrin (filament-aggregating protein) is produced
during the late stages of terminal differentiation of epithe-
lial cells. It is synthesized as a heavily phosphorylated pre-
cursor protein (profilaggrin) that consists of 10 to 12 filaggrin
repeats.40 Profilaggrin is deposited in granules, and filaggrin
is released by proteolytic cleavage during differentiation of
the cells by proteases that remain to be fully defined. Coin-
cident with cleavage, the protein is dephosphorylated, and
a significant proportion (~20%) of the arginine residues are
deaminated (i.e., converted to citrulline),41,42 a PTM medi-
ated by the PAD enzymes (see later section Citrullinated
Antigen Generation in Rheumatoid Arthritis).43 Because
RA sera appeared to specifically target the neutral isoform
of filaggrin (which is present and citrullinated in fully dif-
ferentiated squamous epithelia), van Venrooij and col-
leagues addressed whether RA sera that were positive in the
AKA/APF assays specifically recognized citrullinated pep-
tides.44 Thus regions within the deduced amino acid
sequence of human profilaggrin with a high antigenicity
index and the largest number of arginine residues were
selected to generate synthetic peptides where arginine resi-
dues were substituted with citrulline. Using these citrulli-
nated peptides, van Venrooij and colleagues demonstrated
that AKA/APF antibodies are directed against citrullinated
filaggrin. It is important to highlight that antibodies against
citrullinated proteins in RA do not recognize free citrulline
but only citrulline residues (i.e., peptidylcitrullines) within
the context of peptides or protein sequences.
These initial studies became the basis for the major dis-
covery that protein sequences containing citrulline residues
are one of the most prominent targets for autoantibodies
in RA. However, because the epidermis is not a target of
rheumatoid inflammation and there is no evidence that
(pro)filaggrin is expressed in articular tissues, this molecule
cannot be the autoantigen that drives the AKA/APF
response in the joint. The fact that anti-citrullinated filag-
grin antibodies were enriched in synovial tissue compared
with the serum or synovial fluid45 and that these antibodies
are synthesized locally by plasmocytes within the rheuma-
toid pannus46 strongly suggests that anti-citrullinated filag-
grin cross-reacts with an antigen enriched in the RA joint/
synovial tissue. Because citrullination is a very frequent
event in different tissues,47-51 great caution must be exer-
cised when attempting to ascribe a primary role for a specific
antigen in driving the antipeptidylcitrulline antibody
response. The relevance of filaggrin in RA is therefore
largely historic and unlikely to be of pathogenic relevance.
Instead, its conformation and high content of citrulline
residues make it an excellent surrogate with which to detect
antibodies against citrullinated molecules. Many other
citrullinated autoantigens found in the rheumatoid joint
that are currently being characterized are much more likely
to be of pathogenic relevance (see later section Anti-
citrullinated Protein Antibodies).
Anti–Cyclic Citrullinated Peptide Antibodies
In the initial characterization of AKA/APF antibodies, van
Venrooij and colleagues used a single C-terminal peptide
derived from filaggrin (amino acids 306 to 324) to generate
nine variants in which five arginine residues were changed
to citrullines, either individually or in pairs, and these pep-
tides were all assayed by ELISA.44 Interestingly, although
 834 PART 7    DIAGNOSTIC TESTS AND PROCEDURES IN RHEUMATIC DIEASES
the peptides were almost identical (the only difference being
that the citrulline residues had different positions within
the peptide), remarkable differences were found in the serum
reactivity patterns toward each peptide (from 20% to 48%
positivity), suggesting that although citrullination plays a
critical role in antigen recognition by AKA/APF antibodies,
the modification per se is not the only determinant that
confers antibody binding. Instead, the data suggested that
AKA/APF represent a pool of antibodies that recognize
citrulline residues depending on the context of their sur-
rounding amino acids. Indeed, when data from all peptides
were pooled, the sensitivity increased to as much as 76%.44
Because peptides often adopt a β-turn conformation
within the antibody–peptide complex52 and cysteine-
bridged cyclic peptides have been shown to mimic the β-
turn structure of the original antigenic determinant and can
bind with enhanced affinity to antibodies,53 van Venrooij
and colleagues engineered a cyclic peptide (substituting the
terminal serine residues with cysteines and cyclizing the
peptide through the formation of a disulfide bond) to which
anti–citrullinated peptide antibodies reacted with higher
affinity.54 Using this cyclic citrullinated peptide (later
named CCP1) and its linear counterpart, it was demon-
strated that the cyclic structure increased the sensitivity of
the assay (68% vs. 49%, without affecting specificity),44,54
although it was still less sensitive than the assays using the
combination of linear peptides (i.e., 76% vs. 68%).44 Nev-
ertheless, CCP1 became the antigen for the first generation
of ELISAs designed to detect antibodies against citrulli-
nated autoantigens.
To improve the CCP1 test, libraries of citrullinated pep-
tides were used to construct the second-generation anti-
CCP assay (CCP2), which was broadly adopted for clinical
use.55,56 In 2005, a third generation of anti–cyclic citrulli-
nated peptide (CCP3) was made available for the laboratory
diagnosis of RA. These assays have been reported to recog-
nize additional citrullinated epitopes that are not identifi-
able with the second-generation CCP assays. In studies
directly comparing second- and third-generation anti-CCP
assays, the assays performed very similarly,57,58 with a slightly
increased sensitivity of CCP3 in some studies (e.g., sensi-
tivities of 82.9% vs. 78.6% for CCP2, with specificities in
the 93% to 94% range).58
ANTI-CITRULLINATED PROTEIN
ANTIBODIES
The finding that autoantibodies in RA recognize peptide
sequences containing citrulline residues and that filaggrin
was unlikely to be the physiologic target of these antibodies
prompted the search for the primary protein targets of the
antibodies detected by the CCP assay. It was believed that
the identification of these antigens may provide additional
information with regard to patient subtyping (compared
with the general anti-CCP assay), as well as novel insights
into disease etiology and pathogenesis. With the initial dis-
covery of some of these citrullinated antigens, the name
anti-citrullinated protein antibodies (ACPAs) was introduced
to refer to RA autoantibodies for which detection was per-
formed using citrullinated proteins/peptides from putative
RA-associated autoantigens instead of filaggrin-derived or
commercial CCPs.
Using protein extracts from rheumatoid synovial mem-
branes and affinity purified anti-citrullinated filaggrin anti-
bodies, Masson-Bessière and colleagues showed that RA
synovial tissue contains several citrullinated proteins and
defined the α and β chains of fibrin(ogen) as the first local
targets for ACPAs in RA synovial tissue.59 Because circulat-
ing fibrinogen is not citrullinated, the presence of citrulli-
nated fibrin in the rheumatoid synovium strongly suggested
that after fibrin deposition, citrullination occurs in situ
through the activity of locally expressed PAD. This proposal
(i.e., that the joint is the site for autoantigen citrullination
in RA) has been further supported and extended to many
other citrullinated autoantigens in RA.60
To date, several different approaches have been used to
identify potential ACPA targets in RA. Although these
approaches all utilize protein sequencing to identify auto-
antigens detected by ACPAs using immunoblotting assays,
they vary in terms of the source of the antigens. Thus, in
addition to RA pannus, identification of citrullinated auto-
antigens have been extended to rheumatoid synovial fluid,
as well as to cell lysates in which purified PADs have been
used to generate in vitro citrullinated proteins.
Using these approaches, several citrullinated autoanti-
gen candidates have also been identified. These candidates
include vimentin61,62; fibronectin63; actin64; heat shock
protein (HSP)9065; histones66; α-enolase, elongation factor-
1α, and adenylyl cyclase–associated protein-167; F-actin
capping protein α-1 subunit, asporin, cathepsin D, hista-
mine receptor, protein disulfide-isomerase, ER60 precursor,
and mitochondrial aldehyde dehydrogenase68; collagen type
I69 and II70; eukaryotic translation initiation factor 4G171;
aldolase, phosphoglycerate kinase-1 (PGK1), calreticulin,
HSP60, and the far upstream element binding proteins
(FUSE-BP) 1 and 272; and apolipoprotein E and myeloid
nuclear differentiation antigen.73 Interestingly, some of
these molecules (e.g., vimentin, α-enolase, collagen type II,
HSP60, aldolase, and calreticulin) had been previously
identified as autoantigens in RA, before it was recognized
that protein citrullination played a role in their recognition
by autoantibodies.67,74 Although RA sera containing ACPAs
may recognize both the native and citrullinated forms of the
antigen, the modified form is usually preferentially recog-
nized. Although this group of citrullinated antigens repre-
sents proteins that are recognized by RA antibodies, the
number of citrullinated proteins found in the rheumatoid
joint is more extensive and includes several dozen molecules
that have not yet been characterized.73,75,76 Whether unique
ACPAs exist for each one of these molecules or whether
only a few citrullinated autoantigens are responsible for
driving the complete ACPA response in RA is still unknown.
Among the citrullinated candidate antigens present in
the joint, the best characterized clinically and pathogeni-
cally are fibrin(ogen), vimentin, collagen type II, and
α-enolase. ELISA-based assays have been developed using
citrullinated proteins (e.g., fibrinogen) or citrullinated pep-
tides (e.g., vimentin, collagen type II, and α-enolase)
derived from these putative RA autoantigens to detect
ACPAs. In the case of vimentin, the citrullinated sequence
from a mutated isoform isolated from RA synovial fluid
(named mutated citrullinated vimentin [MCV]) has been
more commonly used instead of the native sequence.77
Although it was greatly anticipated that the detection of
 CHAPTER 56    Autoantibodies in Rheumatoid Arthritis 835
ACPAs targeting single antigens would provide additional
diagnostic information compared with the anti-CCP ELISA,
studies in this regard are inconclusive. Thus, in the absence
of convincing evidence that measuring individual ACPAs
provides any additional information compared with the
anti-CCP assay, it appears that the use of CCPs is still a
more reliable test to confirm the presence of antibodies
against citrullinated proteins. In the case of patients who
are anti-CCP negative, the use of antigen-specific assays
may provide an additional tool to detect ACPAs.
Because single ACPAs appear to have limited utility in
RA, further assays have been introduced to measure more
than one ACPA simultaneously. For example, autoantibod-
ies targeting putative RA-associated citrullinated autoanti-
gens are measured using custom Bio-Plex bead-based
autoantibody assays (Bio-Rad Laboratories, Hercules, Calif.)
in which antigens (usually peptide sequences) are conju-
gated to spectrally distinct beads.78 Autoantibody binding
to antigen-coated beads is detected with anti–human
phycoerythrin–labeled antibody and analysis with a Lu-
minex 200 instrument (Luminex, Austin, Tex.). This assay
is slightly more sensitive than the CCP ELISA, detecting
ACPAs in approximately 10% of patients negative for anti-
CCP antibodies (representing 3.7% of the total patient
cohort),79 a difference that might be explained by the array
of peptides used on the assay. In addition, the bead-based
multiplex assay has been useful to define the epitope spread-
ing of ACPAs in pre-clinical RA.78 However, although the
multiplex assay provides additional information with regard
to unique ACPA specificities present in the patient, know-
ing these specificities has not yet demonstrated any advan-
tages for diagnosis or prognosis in RA.
Anti–Cyclic Citrullinated Peptide Antibodies/
Anti-citrullinated Protein Antibodies:
Clinical Relevance
The clinical importance of autoantibodies against citrulli-
nated antigens in RA stems from the following favorable
features (Figure 56-2):
PADs convert peptidylarginine
to citrulline
Anti-PAD4 and ACPAs
predict disease severity
Anti-PAD4 and ACPAs
precede disease onset
PAD4 polymorphisms are associated
with RA and anti-PAD4 antibodies
in some cohorts
A subtype of anti-PAD4 antibodies
enhance PAD4 activity
Figure 56-2  Protein citrullination as a hub in rheumatoid arthritis (RA) pathogenesis. ACPAs, Anti-citrullinated protein antibodies; PAD, peptidylar-
ginine deiminase.
1. Anti-CCP antibodies have high specificity for the diagnosis of
RA. In systematic reviews and meta-analyses, anti-CCP
antibody positivity has been shown to be as sensitive as
but more specific than RF in distinguishing RA from
other forms of inflammatory arthritis.14
2. The presence of anti-CCP autoantibodies is an important
predictor of development of RA. RA developed within 3
years in more than 90% of the patients with undifferenti-
ated arthritis who tested positive for anti-CCP, in con-
trast to only 25% of the patients who tested negative for
anti-CCP.80
3. Anti-CCP positivity has been associated with a more severe
and destructive disease course, although the independent role
of the antibody in comparison with RF has not yet been
confirmed. Some investigators have shown that both RF
and anti-CCP antibodies are independent predictors of
severity, whereas others have shown that anti-CCP anti-
bodies rather than RF are the better predictor of radio-
graphic progression,81-88 particularly in patients who were
seronegative for RF.88
4. Anti-CCP/ACPAs are associated with RA-related interstitial
lung disease (ILD) and cardiovascular disease (CVD).89-95
ILD and CVD are extra-articular manifestations with
high mortality in persons with RA. Although the mech-
anistic significance of these associations is still unclear,
one possibility is that these antibodies are markers of
systemic inflammation that contributes to lung and car-
diovascular damage. Alternatively, it is noteworthy that
citrullination has been found in lung, myocardium, and
atherosclerotic plaque,51,96-98 suggesting the possibility
that these antibodies are directly pathogenic by targeting
extra-articular tissues containing citrullinated antigens.
The presence of anti-CCP antibodies in patients who
have ILD but do not have RA is intriguing and has
focused attention on the lung as a possible extra-articular
site where RA may be initiated99,100 (see section Poten-
tial Environmental Factors in Rheumatoid Arthritis
Pathogenesis).
The early and accurate diagnosis of RA, together with
effective treatment with disease-modifying anti-rheumatic
Citrullinated proteins accumulate
in the target tissue together
with IgG and complement
Citrullination is induced in
PAD-expressing cells during
activaton/damage
PADs can autocitrullinate
 836 PART 7    DIAGNOSTIC TESTS AND PROCEDURES IN RHEUMATIC DIEASES
drugs, decreases accrual of joint damage in RA. Because
anti-CCP/ACPAs are markers of disease severity and are
detectable early in the disease course, they are powerful
tools to classify patients with early stage inflammatory
arthritis who will benefit from treatment.101-103
Kinetics of Appearance of Anti–Cyclic
Citrullinated Peptide Antibodies/
Anti-citrullinated Protein Antibodies in
Rheumatoid Arthritis
Recent studies have demonstrated that autoantibodies may
precede the onset of clinical disease in both tissue-specific
and systemic autoimmune diseases.17,104-110 Defining events
prior to clinical onset is challenging, and several different
study designs have been used to define the appearance of
autoantibodies and their relationship to disease. The two
general categories include (1) stored blood samples col-
lected prior to disease onset (blood banks or military
cohorts) and (2) prospective studies examining emergence
of autoantibodies and disease in high-risk persons (often
relatives of affected individuals).111 In RA, the former
approach has clearly shown that autoantibodies precede RA
diagnosis in many persons in whom seropositive RA subse-
quently develops, often by 2 to 6 years.109,110,112,113 In several
studies, 20% to 60% of patients with RA were RF positive
prior to diagnosis, and 30% to 60% of patients were positive
for anti-CCP.109,110,112,113 With one exception,113 the preva-
lence of anti-CCP prior to diagnosis was almost twice as
high as RF. Interestingly, whereas RF remained present con-
sistently, anti-CCP could vary over time, even disappearing
in some persons. In one study, pre-clinical anti-CCP posi-
tivity was strongly associated with the development of
erosive RA (odds ratio [OR], 4.64; 95% confidence interval,
1.71 to 12.63; P < 0.01), whereas RF was not (P = 0.60).113
The evolution of autoantibody epitope spreading in pre-
clinical RA has been addressed using a multiplex assay. The
study confirmed that ACPA positivity predicts progression
to RA in asymptomatic persons and showed that the number
of ACPA specificities accumulate over time during the pre-
clinical phase of the disease.78 Moreover, the expansion of
the ACPA response precedes the elevation of many inflam-
matory cytokines, including tumor necrosis factor (TNF),
interleukin (IL)-6, IL-12p70, and interferon (IFN)-γ, sug-
gesting that ACPAs may be part of the driving inflammatory
process in RA.78
Together, these data highlight that the development of
autoimmunity against citrullinated antigens is often asymp-
tomatic and generally precedes the onset of clinical disease.
This presymptomatic phase is potentially of great impor-
tance, because it may identify persons in whom important
precursor conditions for the subsequent development of RA
have been satisfied. The subsequent events that convert this
RA precursor into a chronic, self-sustaining process are not
yet known, but defining them is of great importance.
Genetic Associations with Anti–Cyclic
Citrullinated Peptide Antibodies/
Anti-citrullinated Protein Antibodies
Although the association of specific human leukocyte
antigen–DR region (HLA-DR) alleles and RA has been
known for decades, the relationship between genetics and
the development of RA autoantibodies is just beginning to
be elucidated.114 A subset of HLA-DR alleles termed the
“shared epitope” (SE) alleles includes HLA-DR*0101,
*0102, *0401, *0404, *0405, *0408, *0410, *1001, and
*1402 and is united by a conserved sequence of amino acids
(QRRAA, QKRAA, or RRRAA) on the alpha helix of
the DR-β chain peptide-binding groove.115 SE alleles are
strongly associated with the development of anti-CCP/
ACPA but are not independently associated with RF.80 Fur-
thermore, there appears to be a gene dosage effect on the
relative risk of anti-CCP development with an OR of 3.3
to 4.7 for patients with one SE allele and an OR of 11.8 to
13.3 for patients with two SE alleles.80,116,117 Analysis of
individual SE alleles has revealed that anti-CCP/ACPAs
are predominantly associated with HLA-DR4 rather than
HLA-DR1 SE alleles.118 Interestingly, smoking has been
shown to skew the genetic association of SE alleles with
anti-CCP development, with HLA-DR1 and HLA-DR10
SE alleles being more important in this patient group (see
Anti-citrullinated Protein Antibodies in Rheumatoid Ar-
thritis: Insights into Disease Mechanism section).119
In addition to SE alleles, a single nucleotide polymor-
phism in the PTPN22 gene (1858C/T) is also associated
with anti-CCP antibodies with an OR of 3.80.120 PTPN22
encodes for lymphoid protein tyrosine phosphatase and has
been shown to be associated with several autoimmune dis-
orders, including RA.121 The combination of PTPN22
1858C/T genotype and anti-CCP antibodies was 100% spe-
cific for RA and confers a relative risk of 130.03. The
1858C/T polymorphism was not associated with RF isotypes
and appeared to act independently of SE alleles.120 Genetic
factors such as the SE and PTPN22 may therefore play a
role in the pre-clinical phase of RA, predisposing individu-
als to the generation of anti-CCP antibodies and suscepti-
bility to the overt disease phenotype.
Citrullinated Antigen Generation in
Rheumatoid Arthritis
The Peptidylarginine Deiminase Enzymes
Citrulline is not part of the natural pool of amino acids used
for protein synthesis, and therefore citrulline residues need
to be generated post-translationally once the protein has
been synthesized. This PTM, termed arginine deamination or
citrullination, is mediated by the calcium-dependent PAD
enzymes. PADs target arginine residues in proteins and
mediate hydrolysis of the arginine guanidinium side chain,
resulting in the generation of citrulline residues and
ammonia (Figure 56-3A). The PADs belong to a larger
group of guanidino-modifying enzymes called the ami­
dinotransferase (AT) superfamily.122,123 This superfamily of
enzymes is expressed both in prokaryotes and eukaryotes
and includes the arginine deaminases, the dimethylarginine
dimethylaminohydrolases, and the dihydrolases.123 PADs
are found in numerous species from bacteria to humans.
Interestingly, the bacterial PADs are not dependent on
calcium for activity and only contain an approximately
40 kDa catalytic domain, whereas vertebrate mammals
have larger multidomain enzymes (~75 kDa) whose activity
is regulated by calcium. Five PAD enzymes have been
 CHAPTER 56    Autoantibodies in Rheumatoid Arthritis 837

Citrullination

PAD + Ca2+

H2N Protein Protein

O
+ NH H2O NH3 NH
NH2 NH2
A Peptidylarginine Peptidylcitrulline

Carbamylation

Cyanate
Protein Protein
+ NH3 NH
NH2
O
B Peptidyllysine Peptidylhomocitrulline

MAA adducts
Acetaldehyde (AA) FAAB MDHDC
+ H
Malondialdehyde (MDA) O
H
+ NH3 O NH CH3 N
Protein H Protein Protein
O CH3 O
H H
C Primary amine Malondialdehyde acetaldehyde adducts (MAA)
Figure 56-3  Post-translational modifications targeted by antibodies in rheumatoid arthritis (RA). A, During citrullination, the calcium-dependent
peptidylarginine deiminase (PAD) enzymes target arginine residues in proteins, resulting in the generation of citrulline residues and ammonia.
B, Carbamylation results from the reaction of cyanate with the primary amine group of peptidyllysine that is converted to peptidylhomocitrulline.
C, Malondialdehyde-acetaldehyde (MAA) adducts (FAAB and MDHDC) are generated from the reaction of malondialdehyde (MDA) and acetaldehyde
(AA) with primary amine groups of amino acid residues, preferentially peptidyllysine. FAAB, 2-formyl-3-(alkylamino)butanal; MDHDC, 4-methyl-1,
4-dihydropyridine-3,5-dicarbaldehyde.

identified in mammals, and the PADI genes are located at
a single cluster on chromosome 1p36.1.43,124 For historical
reasons, these isozymes have been designated PAD1 to PAD4
and PAD6. Human PAD4 was initially named PAD5 but
was later renamed PAD4 to reflect the fact that it is a true
ortholog of this isoform.125 Although initial evidence
suggested that PAD4 was the only nuclear PAD,126 recent
evidence suggests that some PAD2 may also reside in the
nucleus.127
The PADs are highly conserved and share 50% to 55%
sequence identity with each other,128 with different PADs
having distinct substrate preferences and expression in spe-
cific tissues. PAD1 is primarily expressed in uterus and skin.
PAD2 is more widely expressed in muscle, skin, brain,
spleen, secretory glands, monocytes, and neutrophils. PAD3
is expressed in skin and neutrophils, PAD4 is expressed in
hematopoietic cells (e.g., neutrophils and monocytes), and
PAD6 is broadly expressed in germ cells, peripheral blood
leukocytes, lung, small intestine, liver, spleen, and skeletal
muscle.43,61,124,125,129-131 Because of their prominent expres-
sion in rheumatoid synovial tissue and fluid,60,132,133 PAD2
and PAD4 have gained prominence as potential candidates
that drive citrullination of self-antigens in RA.
Peptidylarginine Deiminase Structure, Activity,
and Regulation
The three-dimensional structure of PAD4128,134 and PAD2135
has been solved and is likely representative of the broader
family. PAD2 and PAD4 are dimers formed by head-to-tail
contact between the N-terminal domain of one molecule
and the C-terminal domain of the second. The N-terminal
domain includes two immunoglobulin-like subdomains
(named subdomain 1 and 2) that are proposed to mediate
protein-protein interactions and/or substrate targeting.128
Five and six Ca2+-binding sites were identified in the struc-
ture of PAD4 and PAD2, respectively, with Ca2+ binding
inducing conformational changes required to generate the
active site cleft. Interestingly, the calcium binding residues
are highly conserved among PADs, except for PAD6, which
lacks some of these sites; the catalytic cysteine is also dif-
ferent in PAD6, and together these findings have suggested
that PAD6 is not a deaminating enzyme.
PAD1 to PAD4 rely strongly on the presence of Ca2+ for
activity and require millimolar amounts of calcium (~2
to 5 millimolar) to be activated in vitro.136 The resting
concentration of calcium in the cytoplasm is normally
maintained in the range of 50 to 100 nM and only rises to
200 to 800 nM upon cell activation with physiologic
stimuli.137-140 The large discrepancy between the in vitro and
cellular calcium requirements for PAD activation suggests
that additional mechanisms, such as allosteric effects of
PTMs or partner binding, may regulate PAD activity in vivo
by modifying the calcium requirement of the enzyme.136 It
is also important to note that whereas PAD activation pro-
vides one level of regulation, the activity of the PADs may
also be negatively regulated through autocitrullination.
This is not dissimilar to other enzymes, where automodifica-
tion (e.g., autophosphorylation) modifies enzymatic func-
tion. Autocitrullination has been described for PADs 1, 2,
 838 PART 7    DIAGNOSTIC TESTS AND PROCEDURES IN RHEUMATIC DIEASES
3, and 4.141,142 In the case of PAD4, direct citrullination of
arginines surrounding the active site cleft appears to have
major impact on activity and substrate binding.141 Besides
cell activation, it has been proposed that PADs may also be
activated under conditions where intra-cellular concentra-
tions of calcium approach extra-cellular concentrations (~1
to 1.4 millimolar), which may occur in dying cells experi-
encing secondary necrosis or as result of membranolytic
damage.75
Structural and Functional Implications
of Protein Citrullination
Arginine residues play a central role in the maintenance of
secondary and tertiary protein structure. The positively
charged guanidino group allows the formation of intramo-
lecular hydrogen bonds to backbone carbonyl oxygens and
also intermolecularly between different proteins.143 During
the process of citrullination, the net charge of the protein
is reduced by the loss of a positive charge per citrulline
residue, producing changes in protein structure and affect-
ing intramolecular and intermolecular interactions.144
It is likely that these structural changes also have impor-
tant functional implications. Indeed, deamination has been
implicated in several physiologic processes. In the skin
(which expresses PADs 1, 2, and 348,130,145), deamination of
filaggrin (a component of the cornified cell envelope) is
critical for its degradation into free amino acids, which act
as a natural moisturizing factor in the stratum corneum.48,145
Interestingly, PAD2-deficient mice have normal develop-
ment and show no abnormalities in the skin,146 suggesting
functional redundancy in mouse development. Whether
PAD2 functions in the skin during injury or environmental
perturbations is currently unknown. In the immune system,
PAD2 (and/or potentially other PADs) citrullinates chemo-
kines, a process that modulates chemokine activity.147-150
Thus PAD2 may play important roles in controlling effector
functions related to environmental triggers.
Human PAD4 (first called PAD5) was first found in
human myeloid leukemia HL-60 cells induced to differenti-
ate into granulocytes by retinoic acid125 and later described
in peripheral blood granulocytes.129 Histones H2A, H3, and
H4 and nucleophosmin/B23 were the first PAD4 substrates
to be identified.151 PAD4 appears to function as a transcrip-
tional co-regulator, mediated by its ability to catalyze the
deamination of specific residues present in the N-terminal
tails of histones.152-154 This function, together with the
finding that PAD4 is expressed during granulocyte differen-
tiation, initially suggested that this enzyme may have a role
during granulopoiesis. However, mice deficient in PAD4
have normal development,155 suggesting that this enzyme
has no essential roles in steady-state cellular functions,
granulopoiesis, or other developmental processes. Recent
studies have demonstrated that PAD4-mediated citrullina-
tion of histones is required for neutrophil extra-cellular trap
(NET) formation and bacterial clearance,154,155 suggesting a
role for this enzyme in immune and inflammatory effector
functions.
PAD6, the most recently defined member of the PAD
family, was initially cloned from mouse oocytes and named
egg PAD (ePAD),156 although the enzyme is widely expressed
in different tissues. PAD6 is essential for the formation of
cytoplasmic lattices, and female mice deficient in PAD6 are
infertile but otherwise normal.131 The function of PAD6 in
other organs/tissues besides ovaries is unknown.
Peptidylarginine Deaminases in Rheumatoid Arthritis
Multiple PADs are likely responsible for autoantigen citrul-
lination in RA, particularly PADs 2 and 4.60,132,133 Although
PAD4 has some unique characteristics that have focused
significant attention on this protein, it is important to be
aware that no evidence exists to suggest that pathologic
citrullination in RA is exclusively or preferentially medi-
ated by this isoform.
It is of interest that several polymorphisms in PAD4
have been genetically associated with RA development,
particularly in Asian populations.157-160 In this regard, two
common haplotypes of the PADI4 gene were initially iden-
tified157 and designated “susceptible” or “nonsusceptible”
based on their relative frequency in patients with RA versus
control subjects. In the initial population, the OR for the
susceptible haplotype was almost 2; this effect was not
observed in most white populations. Changes in messenger
RNA stability of the susceptible PADI4 reported in the
initial paper were not associated with changes in enzyme
level. Furthermore, although the susceptible haplotype
generates a PAD4 molecule containing three amino acid
substitutions in the N-terminal region (i.e., Gly55-Ser,
Val82-Ala, and Gly112-Ala), current evidence suggests
that these changes have no effect on the function of the
protein.141,161 Indeed, they appear to affect conformation
only within the N-terminal domain but not the active site
located in the C-terminal domain.161 The fact that PAD4
acts as a head-to-tail dimer may nevertheless allow confor-
mational differences in the N-terminus of one molecule to
influence the C-terminal domain of another and influence
catalysis or even inhibition by autocitrullination. It has
been proposed that the PAD4 genotype may exert its effects
on RA disease susceptibility as a consequence of its immu-
nogenicity, rather than its enzyme activity.141 Consistent
with this hypothesis is the observation that, in addition to
its ability to citrullinate RA autoantigens, PAD4 itself is an
RA autoantigen (see next section Anti–peptidylarginine
deiminase autoantibodies).162,163 Moreover, it is noteworthy
that the PADI4 susceptible gene appears to contribute to
the development and progression of RA regardless of ACPA
status,164,165 supporting the notion that the snp variant may
drive susceptibility through other mechanisms besides the
production of citrullinated autoantigens.
ANTI–PEPTIDYLARGININE DEIMINASE
AUTOANTIBODIES
In addition to its prominent enzymatic role in the genera-
tion of citrullinated autoantigens, PAD4 itself is targeted by
autoantibodies in a subset of patients with RA.162,163 These
antibodies recognize the unmodified form of PAD4, but it
has been suggested that autocitrullinated PAD4 may be
more immunogenic in some persons.141 As has been observed
for anti-CCP and RF, anti-PAD4 antibodies are frequently
present prior to the onset of any disease symptoms.107 Inter-
estingly, in a cohort of white patients with RA who had
established disease, the susceptible PAD4 haplotype was
 CHAPTER 56    Autoantibodies in Rheumatoid Arthritis 839
strikingly associated with PAD4 autoantibodies, even
though the association of the susceptible haplotype with
RA development has not been observed in white popula-
tions.163 The sensitivity of PAD4 antibodies for RA is in the
30% to 40% range, with a specificity of greater than 95%.
PAD4 autoantibodies are associated with anti-CCP anti-
bodies and are independently associated with more severe,
erosive RA that persists despite treatment with TNF inhibi-
tors.166,167 Recently, a subset of anti-PAD4 antibodies identi-
fied by their cross-reactivity to the related protein PAD3
has been identified and appears to be the major driver
of the previously seen associations with erosive disease
severity and progression despite treatment.136 These anti-
bodies were also associated with anti-CCP antibodies, as
well as the SE alleles. Furthermore, patients with PAD3/
PAD4 cross-reactive antibodies are at the highest risk for
the development of RA-associated ILD, an effect signifi-
cantly augmented in patients who were past or present
cigarette smokers (OR, 61.4).168 The causal relationship
between PAD3/PAD4 antibody development and cigarette
smoking is unknown but suggests that environmental factors
such as cigarette smoke may trigger the development of
RA-associated autoantibodies in susceptible persons (see
later section Potential Environmental Factors in Rheuma-
toid Arthritis Pathogenesis).
MECHANISMS OF CITRULLINATED
AUTOANTIGEN PRODUCTION IN
RHEUMATOID ARTHRITIS
Two potential mechanisms have been implicated in aber-
rant PAD activation and the production of citrullinated
autoantigens in the rheumatoid joint. One mechanism
involves the hyperactivation of PADs intra-cellularly, and
the second relies on extra-cellular autoantigen citrullina-
tion. Importantly, these mechanisms are not mutually
exclusive but are complementary. Initial immunohisto-
chemical studies of synovial tissue focused attention on
synoviocytes and inflammatory cells as potential sources of
PADs and citrullinated antigens in the RA joint.132,133 How-
ever, whereas functional studies to address PAD expression,
activity, and production of citrullinated autoantigens in iso-
lated synoviocytes are limited and inconclusive,169 the study
of neutrophils has provided important insights into mecha-
nisms of autoantigen citrullination in RA.64,75,170 These cells
represent one of the most abundant cell types in RA syno-
vial fluid and constitutively express several PADs.64 Further-
more, they can hypercitrullinate and generate citrullinated
autoantigens when activated with calcium ionophores,64
and they are highly enriched in citrullinated autoantigens
when freshly isolated from the RA joint.75 Two mechanisms
have been proposed to trigger PAD activation and citrulli-
nated autoantigen production in neutrophils in RA: (1)
formation of neutrophil extra-cellular traps (NETs), an anti-
microbial system in which granule proteins and chromatin
are extruded from the cell and form extra-cellular fibers that
bind pathogens,170 and (2) membranolytic damage mediated
by complement or perforin.75
During NET formation, PAD4-mediated histone citrul-
lination is important for chromatin unfolding.171 Activation
of PAD4 during this process is believed to result in citrul-
lination of other molecules, resulting in autoantigen genera-
tion. Alternatively, it has been proposed that PADs may be
released during NET formation, contributing to extra-
cellular citrullination. Although NETs may be an important
source of inflammatory signals and some citrullinated auto-
antigens, the contribution of this process to generating the
whole set of citrullinated molecules found in the rheuma-
toid joint (i.e., the RA citrullinome) is unclear.170 Instead,
recent studies have found that immune pathways that form
pores in cell membranes and that are active in the RA joint
(e.g., the complement system’s membrane attack complex
[MAC] and perforin released from cytotoxic cells) are
potent activators of PADs and efficient inducers of the RA
joint citrullinome.75 The membranolytic damage induced by
these pathways is associated with high intra-cellular calcium
flux,172 which likely explains the hyperactivation of PADs
in the damaged cell. It is also possible that other forms of
cell death may play a role in mediating enhanced citrullina-
tion in the RA joint, particularly if these death pathways
(e.g., pyroptosis or necroptosis) result in extra-cellular
leakage of PADs. The discovery that fibrinogen is heavily
citrullinated in the RA joint provided initial evidence that
citrullination can indeed occur extra-cellularly.59 Further-
more, soluble PAD2 and PAD4 have been detected in the
synovial fluid from patients with RA,60,173 suggesting that
these enzymes can gain access to the extra-cellular space
during inflammation.
Although the extra-cellular calcium concentration in
synovial fluid (0.5 to 1 mM) is more than 1000-fold higher
than intra-cellular levels, it is still not optimal for maximal
PAD activity in vitro. Recent studies have demonstrated
that anti-PAD3/PAD4 cross-reactive autoantibodies are
able to enhance PAD4 activity at calcium concentrations
found extra-cellularly.136 These anti-PAD3/PAD4 autoanti-
bodies decrease the enzymatic requirement for calcium into
the physiologic range and greatly augment PAD4 activity.
Thus PAD4 released by neutrophils during NET formation
or other forms of cell deaths may encounter anti-PAD4
antibodies in the synovial fluid with the capacity to activate
enzymatic function, contributing to extra-cellular citrulli-
nated autoantigen generation in RA.
NOVEL POST-TRANSLATIONAL
MODIFICATIONS TARGETED BY
ANTIBODIES IN RHEUMATOID ARTHRITIS
Anti–Carbamylated Protein Antibodies
Proteins that contain homocitrulline (i.e., carbamylated
proteins) recently have been recognized as an important
target of autoantibodies in RA, with IgG and IgA antibod-
ies recognizing carbamylated antigens identified in more
than 40% of patients with RA.174 In contrast to citrul­
lination, carbamylation is a nonenzymatic PTM in which
homocitrulline is generated by the reaction of cyanate with
the primary amine of lysine residues (Figure 56-3B). Carba-
mylation therefore accumulates in conditions that enhance
cyanate levels, such as uremia, inflammation, and cigarette
smoking.175 In RA, inflammation is likely the most impor-
tant source of carbamylated proteins. Here, myeloperoxi-
dase (MPO) released from neutrophils promotes the
conversion of thiocyanate to cyanate, further enhancing
carbamylation.175,176 Although homocitrulline structurally
 840 PART 7    DIAGNOSTIC TESTS AND PROCEDURES IN RHEUMATIC DIEASES
resembles citrulline (homocitrulline is one methylene group
longer), the finding that anti–carbamylated protein (anti-
CarP) antibodies are present in approximately 35% of
ACPA− patients and that these antibodies are predictive of
a more severe disease course independent of ACPA status
has suggested that these antibodies are not cross-reactive
between the two PTMs.174 Moreover, anti-CarP antibodies
appear many years before the diagnosis of RA and predict
the development of RA independent of anti-CCP antibod-
ies in patients with arthralgia.177-179 Despite the growing
interest in anti-CarP antibodies, a major caveat is that their
primary protein targets in RA are still unknown. Indeed,
carbamylated–fetal calf serum and carbamylated-fibrinogen
are used as surrogate antigens for anti-CarP detection. Fur-
thermore, carbamylated proteins have not yet been described
in the RA joint.
Anti–Malondialdehyde-Acetaldehyde
Adducts Antibodies
Malondialdehyde-acetaldehyde (MAA) adducts are the
most recently described target of anti-PTM antibodies in
RA.180 Similar to carbamylation, the generation of MAA
protein adducts is nonenzymatic and involves the preferen-
tial modification of the primary amine of lysine residues.
During oxidative stress caused by a variety of stimuli, includ-
ing inflammation and exposure to alcohol, reactive oxygen
species (ROS) trigger peroxidation of membrane lipids,
resulting in the formation of malondialdehyde (MDA) and
acetaldehyde (AA),181-184 which react with proteins, result-
ing in the formation of stable MDA-AA protein adducts,
referred to as MAA (Figure 56-3C).185,186 These adducts
have been implicated in contributing to inflammation and
cellular injury associated with cardiovascular and alcoholic
liver disease.181-183 Interestingly, RA synovial tissue, but not
tissue from patients with osteoarthritis, is enriched in MAA-
modified proteins, and patients with RA have antibodies
targeting this modification when they are detected using
MAA-modified albumin as a surrogate antigen.180 Whereas
anti-MAA antibodies are strongly associated with anti-CCP
and RF, it is interesting that anti-MAA antibodies of the
IgG isotype are found in 88% of anti-CCP− patients, sug-
gesting that these may be useful biomarkers, particularly in
persons with ACPA− RA. However, whether this antibody
system is clinically relevant in RA remains to be defined.
In this regard, it is important to highlight that anti-MAA
adduct antibodies are not specific for RA but have also been
found in patients with alcohol-induced liver disease, diabe-
tes, and CVD.187-189 Therefore these antibodies may not
exclusively be markers of joint damage but may be generated
as a result of comorbidities associated with RA. Similar to
carbamylation, the primary targets of anti-MAA adduct
antibodies in RA are not yet known.
Anti-citrullinated Protein Antibodies, Anti–
Carbamylated Protein, and Anti–
Malondialdehyde-Acetaldehyde Antibodies
in Rheumatoid Arthritis: Insights
into Pathogenesis
The finding that persons with RA have antibodies targeting
different PTMs is intriguing and unlikely to be accidental.
Because neutrophils produce all components (i.e., PADs,
MPO, and ROS) required to generate the current set of
modified autoantigens in RA (i.e., ACPAs, anti-CarP, and
anti-MAA), it has been suggested recently that these dis-
tinct antibody systems may indeed represent markers of a
common pathogenic event driven by neutrophils, poten-
tially placing these cells at the center of autoantigen
production in RA pathogenesis.190 Confirming this hypoth-
esis would have important implications for future anti-
neutrophil therapies in this disease.
OTHER AUTOANTIBODY SPECIFICITIES IN
RHEUMATOID ARTHRITIS RECOGNIZING
NONMODIFIED AUTOANTIGENS
Anti-RA33 antibodies were discovered in 1989 as a novel
autoantibody specificity recognizing a nuclear protein with
an apparent molecular mass of 33 kDa.191 These autoanti-
bodies initially were detected in 35% of patients with RA
but also were detected in a small number of patients who
had other autoimmune or degenerative rheumatic disorders.
The antigen was termed RA33, and it was the first descrip-
tion of a nuclear antigen apparently specific for RA. Later,
the antigen was identified as the A2 protein of the hetero-
geneous nuclear ribonucleoprotein complex (hnRNP-
A2).192 Further characterization of this antibody showed
that it was not strictly specific for RA but was also found
in approximately 20% of patients with SLE and in 40%
to 60% of patients with mixed connective tissue disease
(MCTD).193,194 However, in SLE and MCTD, they usually
occur together with antibodies to U1–small nuclear ribo­
nucleoproteins (snRNPs) or Smith (Sm) antigen. Therefore
anti-RA33 without concomitant anti–U1 snRNP autoanti-
bodies were found to have specificity of 96% for RA.193
Additionally, anti-RA33 antibodies in RA, SLE, and
MCTD are distinguished by their recognition of different
conformation-dependent epitopes in hnRNP-A2.195 Inter-
estingly, despite their limited specificity, in the absence of
RF and ACPAs, anti-RA33 antibodies in patients with very
early arthritis (<3 months’ duration) are associated with a
relatively mild nonerosive disease course, potentially iden-
tifying patients with a good prognosis who will respond well
to treatment with disease-modifying anti-rheumatic drugs
(DMARDs).196 In studies in human samples, stimulation
assays using hnRNP-A2–induced T cell proliferation in
almost 60% of the patients with RA, but only 20% of the
control subjects, with substantially stronger responses
observed in patients with RA.197 Interestingly, immunohis-
tochemical analyses revealed pronounced overexpression of
hnRNP-A2 in synovial tissue of patients with RA, placing
the antigen at sites of disease in RA.
Another autoantigen of potential interest in RA is
glucose-6-phosphate isomerase (G6PI). Arthritis in the K/
BxN mouse model results from pathogenic immunoglobu-
lins that recognize G6PI,198-200 a glycolytic enzyme residing
in the cytoplasm of all cells. Moreover, antibodies directed
against G6PI can, alone, transfer arthritis to healthy recipi-
ents.201 The pathogenic potential of this antibody prompted
its study in human RA. However, although initial studies
reported a high frequency of such antibodies in sera of
patients with RA,202 these results have been the subject of
debate.203-205 Overall, anti-G6PI antibodies in patients with
 CHAPTER 56    Autoantibodies in Rheumatoid Arthritis 841
RA range between 12% to 29% in different cohorts, with a
higher prevalence in patients with active disease. Patients
with psoriatic arthritis, undifferentiated arthritis, and
spondylarthropathy also displayed anti-G6PI antibodies at
similar frequencies (12% to 25%), and similar titers are
detected in a proportion (5% to 10%) of control subjects or
patients with Crohn’s disease or sarcoidosis.205 The potential
relevance of these antibodies in RA is still uncertain.
ANTI-CITRULLINATED PROTEIN
ANTIBODIES IN RHEUMATOID ARTHRITIS:
INSIGHTS INTO DISEASE MECHANISM
Although our understanding of the pathogenesis of RA
remains incomplete, current models employ a similar con-
struct to other autoimmune rheumatic diseases—that is, in
the setting of a complex genetic predisposition, environ-
mental and stochastic events function to initiate an autoim-
mune process recognizing specific self-antigens, where the
immune response itself participates in immune amplifica-
tion and tissue injury (Figure 56-4). The striking association
of RA (and particularly ACPAs) with particular class II
major histocompatibility complex (MHC) alleles under-
scores at least one component of this framework. Whether
RA autoantibodies are directly pathogenic or whether they
are markers of inflammation and/or damage remains uncer-
tain, particularly for RFs. However, ACPAs have several
features that suggest direct involvement in RA pathogenesis
(Figure 56-3):
1. The targets of these antibodies (i.e., citrullinated pro-
teins) are abnormally expressed and highly enriched in
synovial tissue and fluid of patients with RA.60,68,133
2. The antibody response directed against these citrulli-
nated antigens precedes RA onset17,104,110,206 and is
highly specific for RA and predictive of disease
progression.83-87,207
3. ACPA positivity is closely linked with the best-known
predisposing genetic risk factors for the development of
RA: the HLA-DR SE alleles,116,120,208,209 in particular,
HLA-DRB1*04,118,210,211 linking genetic predisposition
and autoantibody production.
4. Peptidylcitrulline contained in peptides derived from
aggrecan and vimentin is preferred to arginine in the
electropositive P4 pocket of the RA-susceptible HLA-
DRB1*04:01/04, and HLA-II tetramers containing these
peptides identified circulating citrullinated epitope–
specific CD4+ T cells.212
5. Plasma cells producing antibodies against citrullinated
proteins are present in RA synovial tissue.46
6. About one-half of the anti-CCP+ RA patients have
circulating immune complexes containing citrullinated
fibrinogen, and immunohistochemical staining has
shown co-localization of the fibrinogen, immunoglobu-
lin, and complement component C3 in RA pannus
tissue.213
7. Immune complexes containing citrullinated fibrinogen
can also activate monocyte-derived macrophages to
produced TNF via engagement of Fcγ receptors and
Toll-like receptor 4.214,215 Similar observations imply-
ing overlapping pathogenic properties are likely also
operative in the case of RFs in RA. Recent evidence
also indicates the that distinct glycosylation patterns
in ACPAs may influence pathogenicity of these
antibodies.216,217
Potential Environmental Factors in
Rheumatoid Arthritis Pathogenesis
Although abundant citrullination is found in RA synovial
tissue once disease is established, no studies have addressed
whether abnormal synovial citrullination precedes the
onset of clinical RA (a requisite to explain the presence of
antibodies against citrullinated proteins long before clinical
disease). Such evidence will be very challenging to obtain.
At this stage, it is therefore important to be aware that the
joint is not the only possible site of abnormal citrullination
that might initiate the RA-specific immune responses to
citrullinated autoantigens. It is possible that the joint
becomes targeted secondarily after the ACPA immune
response has been initiated at another site, as a consequence
of an inflammatory event triggered by a common environ-
mental exposure, such as periodontal infection/inflammation
and/or smoking (Figure 56-4). Although no evidence exists
that viral proteins are citrullinated in vivo, a subset of
ACPA+ RA sera has been demonstrated to cross-react with
in vitro citrullinated peptides from Epstein-Barr virus
EBNA-1 and human papilloma virus (HPV)-47 E2345–362
proteins.218,219 Defining the kinetics of appearance of these
antibodies during disease development, and demonstrating
directly that these modified proteins are generated during
natural infection, will be important to determine if this
cross-reactivity is relevant to RA pathogenesis.
Tantalizing similarities and epidemiologic associations
exist between periodontitis and RA, including the presence
of bone erosions, association with similar class II MHC
alleles and smoking, and enrichment of severe periodontal
disease in patients with RA.220-224 These connections have
focused attention on the study of oral pathogens that express
enzymes with PAD activity, specifically Porphyromonas gin-
givalis,225 a Gram-negative bacterium commonly present as
a biofilm in the gingival crevice and intra-cellularly in oral
epithelial cells. P. gingivalis expresses its own citrullinating
enzyme named PPAD, which produces ammonia that likely
has negative effects on neutrophil function.226 PPAD is not
an ortholog of mammalian PADs, having no sequence
homology with mammalian PADs, and showing significant
differences in citrullination activity: (1) PPAD can citrul-
linate free L-arginine in addition to protein-bound arginine,
(2) the enzyme does not require calcium or any other metal
ion for activity, and (3) it has preference for citrullination
of carboxy-terminal arginines. P. gingivalis also produces
arginine gingipains, endopeptidases that cleave substrates
after arginine residues, a process that generates C-terminal
arginine residues in substrates, which are then citrullinated
by PPAD.226 Two mechanisms have been proposed by
which PPAD may be relevant to RA pathogenesis: (1) by
generating citrullinated bacterial and host proteins and
(2) through autocitrullination. The initial finding that
antibodies to CEP-1 (the immunodominant peptide from
citrullinated human α-enolase) cross-react with P. gingivalis
enolase after citrullination with rabbit PAD opened the
possibility that ACPA development might be initially
driven by citrullinated bacterial products, which subse-
quently cross-react with citrullinated endogenous proteins
 ASYMPTOMATIC PHASE
A. Initiation Smoking
Viral infection
Environmental factors *
* Bacterial infection
activate the immune system Pg-PAD
*
(e.g., P. gingivalis)
* **
*
P. gingivalis other undefined factors

*
*
PADs + Ca 2+ * Membranolytic cell damage
Citrullination is activated in * (e.g., perforin and MAC), NETs,
immune cells expressing PADs *
Citrullination and others undefined.

PTPN22 (1858 C/T)

Genetic susceptibility YES NO PADI4 polymorphisms
HLA-DR shared epitope alleles
Other undefined genes
Initial ACPA and anti-PAD4
autoantibody response

B. Transition
Augmenting factors drive progression
The ACPA response is expanded over to overt disease phenotype
years and precedes the elevation (e.g., infection, injury, epitope
of inflammatory cytokines Cytokines spreading, isotype switching, etc.)

Asymptomatic Asymptomatic/symptomatic threshold:

Symptomatic When autoantibodies and cytokines
overcome potential compensatory
pathways the disease becomes
clinically evident
CLINICAL PHENOTYPE Autoantibodies bind citrullinated
C. Propagation/damage autoantigens in the joint and activate
inflammatory/damage pathways
(e.g., cellular and humoral immunity, complement, etc.)
*
*

Damaged cells release PADs and

*

PAD4
*
citrullinated autoantigens. * *
* *
PADs continue citrullination *
*
extra-cellularly, which is enhanced by *
activating anti-PAD4 antibodies. * Antigen-presenting cells
*

recognize immune complexes

*

and present citrullinated

*

* peptides to T cells
*
*

Neutrophils and monocytes express

*

PADs, and citrullination is induced

during cell activation, NETs, and
membranolylitic damage

TNF

T cells mediate tissue destruction via the granule pathway, secrete

cytokines, and recruit/activate inflammatory cells
Figure 56-4  A feed-forward model of rheumatoid arthritis (RA) pathogenesis: a pivotal role for protein citrullination. A, During disease initiation,
immune pathways activated against environmental factors (e.g., perforin-induced cytotoxicity, neutrophil extra-cellular traps (NETs), and complement
activation) generate the production of citrullinated proteins in peptidylarginine deiminase (PAD)-expressing cells. Although this process is likely normal
and self-limited in the majority of the cases, some genetically susceptible persons may have the risk of developing autoantibodies against components
involved in the process of citrullination (e.g., PAD4 and citrullinated proteins). This phase is likely asymptomatic because the autoantibodies are in low
titer, they may not be pathogenic, and they target a limited number of citrullinated antigens.78,217 The presence of autoantibodies may persist for
several months to decades, during which a transition phase may occur. In some cases, autoantibody positivity may never progress to RA (i.e., anti-
citrullinated protein antibody [ACPA]+, RA− persons).99,100 B, Depending on the exposure to augmenting factors that promote citrullination (e.g., infec-
tions, smoking, and injury), a transition phase occurs in which the ACPA response is amplified. This phase is characterized by an accumulation of
multiple ACPA specificities, reflecting the process of epitope spreading.78 The expansion of the autoantibody response closely correlates with the
appearance of pre-clinical inflammation that includes the elevation of inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin (IL)-6,
IL-12p70, and interferon γ.78 Moreover, ACPAs acquire a pro-inflammatory Fc glycosylation phenotype prior to the onset of RA.217 Together, the changes
in the autoantibody response and the elevation in cytokines predict the imminent onset of clinical RA.78,217 It is likely that compensatory pathways
might be able to sustain a “healthy state” during the process of transition. However, when autoantibodies and cytokines overcome the threshold of
compensation, the clinical phase is likely initiated. C, Once initiated, the immune response drives a feed-forward loop of target tissue destruction and
disease propagation, resulting in the clinical phenotype. Although the citrullinating events that initiate the ACPA response may occur outside the joint,
during disease propagation, citrullinated autoantigens are generated in the synovium by inflammatory cell PADs, leading to amplification of the
immune response within the target tissue. Citrullinated antigens are indicated (*). HLA-DR, Human leukocyte antigen–DR region; MAC, membrane
attack complex.
 CHAPTER 56    Autoantibodies in Rheumatoid Arthritis 843
in the microenvironment of the inflamed joint.227 Subse-
quently, it was found that arginine gingipain and PPAD,
respectively, cleave and citrullinate human fibrinogen and
α-enolase in vitro, extending this hypothesis that host pro-
teins citrullinated by the bacterial enzyme may also play a
role in initiating the ACPA response in RA.228 Although
this model is intriguing, the citrullination of bacterial
enolase by PPAD or by a human host PAD during bacterial
lysis of phagocytic cells has not been demonstrated. In addi-
tion, it is unclear whether the C-terminal citrullinated
human fibrinogen and α-enolase fragments generated by
gingipains and PPAD are indeed recognized by ACPAs
in RA. An alternative mechanism that has been proposed
for the involvement of PPAD in RA pathogenesis is that
autocitrullinated PPAD may act as the inciting citrullinated
antigen that may trigger the ACPA response in RA.229
Although this theory is provocative, it remains controver-
sial because PPAD autocitrullination appears to be an arti-
fact generated when the protein is artificially expressed in
Escherichia coli.230,231
Another environmental factor with potential relevance
in initiating production of ACPAs is cigarette smoking.
Interestingly, recent evidence has demonstrated (at least in
some populations) a strong interaction between smoking
and SE alleles in the development of ACPA+ RA.119,232-234
However, whereas primarily HLA-DRB1*04 alleles have
been associated with ACPA production,118,210,211 in smokers,
HLA-DRB1*0101, *0102, and *1001 alleles showed the
closest association with positive anti-CCP.119 The observa-
tion that cigarette smoking induces increased expression of
PAD2 and protein citrullination in lungs,51 that anti-CCP
antibodies of the IgA class are found in about one-third of
patients with recent-onset RA,235 and that anti-CCP anti-
bodies can be found in patients with ILD who do not have
RA99,100 has suggested that the lung or other mucosal sur-
faces may play a role in generating or sustaining autoanti-
bodies to citrullinated proteins. These gene-environment
interactions, playing out in microenvironments outside the
joint, add important dimensions to the framework within
which RA pathogenesis must be investigated and under-
stood. Although it is possible that none of the detailed
mechanisms currently defined represent the unifying
mechanism of pathogenesis in RA, the broader view of
potential immunizing environments and the search for a
frequent worldwide environmental exposure is of great
importance.
The full references for this chapter can be found on
ExpertConsult.com.
SELECTED REFERENCES
1. Waaler E: On the occurrence of a factor in human serum activating
the specific agglutintion of sheep blood corpuscles. 1939. APMIS
115:422–438, 2007.
2. Rose HM, Ragan C, et al: Differential agglutination of normal and
sensitized sheep erythrocytes by sera of patients with rheumatoid
arthritis. Proc Soc Exp Biol Med 68:1–6, 1948.
3. Henney CS, Stanworth DR: Reaction of rheumatoid factor with the
isolated polypeptide chains of human 7S gamma-globulin. Nature
201:511–512, 1964.
6. Shmerling RH, Delbanco TL: The rheumatoid factor: an analysis of
clinical utility. Am J Med 91:528–534, 1991.
7. Shmerling RH, Delbanco TL: How useful is the rheumatoid factor?
An analysis of sensitivity, specificity, and predictive value. Arch Intern
Med 152:2417–2420, 1992.
8. Dorner T, Egerer K, Feist E, et al: Rheumatoid factor revisited. Curr
Opin Rheumatol 16:246–253, 2004.
11. Randen I, et al: Rheumatoid factor V genes from patients with rheu-
matoid arthritis are diverse and show evidence of an antigen-driven
response. Immunol Rev 128:49–71, 1992.
14. Nishimura K, et al: Meta-analysis: diagnostic accuracy of anti-cyclic
citrullinated peptide antibody and rheumatoid factor for rheumatoid
arthritis. Ann Intern Med 146:797–808, 2007.
17. Nielen MM, et al: Specific autoantibodies precede the symptoms of
rheumatoid arthritis: a study of serial measurements in blood donors.
Arthritis Rheum 50:380–386, 2004.
18. Halldorsdottir HD, Jonsson T, Thorsteinsson J, et al: A prospective
study on the incidence of rheumatoid arthritis among people with
persistent increase of rheumatoid factor. Ann Rheum Dis 59:149–151,
2000.
19. Jansen LM, van der Horst-Bruinsma IE, van Schaardenburg D, et al:
Predictors of radiographic joint damage in patients with early rheu-
matoid arthritis. Ann Rheum Dis 60:924–927, 2001.
21. Nienhuis RL, Mandema E: A new serum factor in patients with
rheumatoid arthritis; the antiperinuclear factor. Ann Rheum Dis
23:302–305, 1964.
22. Sondag-Tschroots IR, Aaij C, Smit JW, et al: The antiperinuclear
factor. 1. The diagnostic significance of the antiperinuclear factor for
rheumatoid arthritis. Ann Rheum Dis 38:248–251, 1979.
23. Hoet RM, Boerbooms AM, Arends M, et al: Antiperinuclear factor,
a marker autoantibody for rheumatoid arthritis: colocalisation of the
perinuclear factor and profilaggrin. Ann Rheum Dis 50:611–618,
1991.
31. Aggarwal R, Liao K, Nair R, et al: Anti-citrullinated peptide anti-
body assays and their role in the diagnosis of rheumatoid arthritis.
Arthritis Rheum 61:1472–1483, 2009.
32. Young BJ, Mallya RK, Leslie RD, et al: Anti-keratin antibodies in
rheumatoid arthritis. Br Med J 2:97–99, 1979.
37. Paimela L, Gripenberg M, Kurki P, et al: Antikeratin antibodies:
diagnostic and prognostic markers for early rheumatoid arthritis. Ann
Rheum Dis 51:743–746, 1992.
38. Simon M, et al: The cytokeratin filament-aggregating protein filla-
grin is the target of the co-called “antikeratin antibodies,” autoanti-
bodies specific for rheumatoid arthritis. J Clin Invest 92:1387–1393,
1993.
39. Sebbag M, et al: The antiperinuclear factor and the so-called antik-
eratin antibodies are the same rheumatoid arthritis-specific autoan-
tibodies. J Clin Invest 95:2672–2679, 1995.
43. Vossenaar ER, Zendman AJ, van Venrooij WJ, et al: PAD, a growing
family of citrullinating enzymes: genes, features and involvement in
disease. Bioessays 25:1106–1118, 2003.
44. Schellekens GA, de Jong BA, van den Hoogen FH, et al: Citrulline
is an essential constituent of antigenic determinants recognized by
rheumatoid arthritis-specific autoantibodies. J Clin Invest 101:273–
281, 1998.
45. Baeten D, et al: Specific presence of intracellular citrullinated pro-
teins in rheumatoid arthritis synovium: relevance to antifilaggrin
autoantibodies. Arthritis Rheum 44:2255–2262, 2001.
46. Masson-Bessiere C, et al: In the rheumatoid pannus, anti-filaggrin
autoantibodies are produced by local plasma cells and constitute a
higher proportion of IgG than in synovial fluid and serum. Clin Exp
Immunol 119:544–552, 2000.
49. Chapuy-Regaud S, et al: Fibrin deimination in synovial tissue is not
specific for rheumatoid arthritis but commonly occurs during syno-
vitides. J Immunol 174:5057–5064, 2005.
50. Makrygiannakis D, et al: Citrullination is an inflammation-dependent
process. Ann Rheum Dis 65:1219–1222, 2006.
54. Schellekens GA, et al: The diagnostic properties of rheumatoid
arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis
Rheum 43:155–163, 2000.
55. van Venrooij WJ, Zendman AJ: Anti-CCP2 antibodies: an overview
and perspective of the diagnostic abilities of this serological marker
for early rheumatoid arthritis. Clin Rev Allergy Immunol 34:36–39,
2008.
56. van Gaalen FA, Visser H, Huizinga TW: A comparison of the diag-
nostic accuracy and prognostic value of the first and second anti-
cyclic citrullinated peptides (CCP1 and CCP2) autoantibody tests
for rheumatoid arthritis. Ann Rheum Dis 64:1510–1512, 2005.
 844 PART 7    DIAGNOSTIC TESTS AND PROCEDURES IN RHEUMATIC DIEASES
57. Lutteri L, Malaise M, Chapelle JP: Comparison of second- and third-
generation anti-cyclic citrullinated peptide antibodies assays for
detecting rheumatoid arthritis. Clin Chim Acta 386:76–81, 2007.
58. dos Anjos LM, et al: A comparative study of IgG second- and third-
generation anti-cyclic citrullinated peptide (CCP) ELISAs and their
combination with IgA third-generation CCP ELISA for the diagnosis
of rheumatoid arthritis. Clin Rheumatol 28:153–158, 2009.
59. Masson-Bessière C, et al: The major synovial targets of the rheuma-
toid arthritis-specific antifilaggrin autoantibodies are deiminated
forms of the alpha- and beta-chains of fibrin. J Immunol 166:4177–
4184, 2001.
62. Vossenaar ER, et al: Rheumatoid arthritis specific anti-Sa antibodies
target citrullinated vimentin. Arthritis Res Ther 6:R142–R150, 2004.
63. van Beers JJ, et al: Anti-citrullinated fibronectin antibodies in rheu-
matoid arthritis are associated with human leukocyte antigen-DRB1
shared epitope alleles. Arthritis Res Ther 14:R35, 2012.
64. Darrah E, Rosen A, Giles JT, et al: Peptidylarginine deiminase 2, 3
and 4 have distinct specificities against cellular substrates: novel
insights into autoantigen selection in rheumatoid arthritis. Ann
Rheum Dis 71:92–98, 2012.
67. Kinloch A, et al: Identification of citrullinated alpha-enolase as a
candidate autoantigen in rheumatoid arthritis. Arthritis Res Ther
7:R1421–R1429, 2005.
73. van Beers JJ, et al: The rheumatoid arthritis synovial fluid citrulli-
nome reveals novel citrullinated epitopes in apolipoprotein E,
myeloid nuclear differentiation antigen, and beta-actin. Arthritis
Rheum 65:69–80, 2013.
74. Saulot V, et al: Presence of autoantibodies to the glycolytic enzyme
alpha-enolase in sera from patients with early rheumatoid arthritis.
Arthritis Rheum 46:1196–1201, 2002.
75. Romero V, et al: Immune-mediated pore-forming pathways induce
cellular hypercitrullination and generate citrullinated autoantigens
in rheumatoid arthritis. Sci Transl Med 5:209ra150, 2013.
76. Tutturen AE, Fleckenstein B, de Souza GA: Assessing the citrulli-
nome in rheumatoid arthritis synovial fluid with and without enrich-
ment of citrullinated peptides. J Proteome Res 13:2867–2873, 2014.
77. Bang H, et al: Mutation and citrullination modifies vimentin to a
novel autoantigen for rheumatoid arthritis. Arthritis Rheum 56:2503–
2511, 2007.
78. Sokolove J, et al: Autoantibody epitope spreading in the pre-clinical
phase predicts progression to rheumatoid arthritis. PLoS One
7:e35296, 2012.
79. Wagner CA, et al: Identification of anticitrullinated protein anti-
body reactivities in a subset of anti-CCP-negative rheumatoid arthri-
tis: association with cigarette smoking and HLA-DRB1 “shared
epitope” alleles. Ann Rheum Dis 74:579–586, 2015.
80. van Gaalen FA, et al: Autoantibodies to cyclic citrullinated peptides
predict progression to rheumatoid arthritis in patients with undif-
ferentiated arthritis: a prospective cohort study. Arthritis Rheum
50:709–715, 2004.
81. Jansen LM, et al: The predictive value of anti-cyclic citrullinated
peptide antibodies in early arthritis. J Rheumatol 30:1691–1695, 2003.
82. Nell VP, et al: Autoantibody profiling as early diagnostic and prog-
nostic tool for rheumatoid arthritis. Ann Rheum Dis 64:1731–1736,
2005.
83. Vencovsky J, et al: Autoantibodies can be prognostic markers of an
erosive disease in early rheumatoid arthritis. Ann Rheum Dis 62:427–
430, 2003.
84. Mewar D, et al: Independent associations of anti-cyclic citrullinated
peptide antibodies and rheumatoid factor with radiographic severity
of rheumatoid arthritis. Arthritis Res Ther 8:R128, 2006.
85. Syversen SW, et al: High anti-cyclic citrullinated peptide levels and
an algorithm of four variables predict radiographic progression in
patients with rheumatoid arthritis: results from a 10-year longitudinal
study. Ann Rheum Dis 67:212–217, 2008.
86. De Rycke L, et al: Rheumatoid factor and anticitrullinated protein
antibodies in rheumatoid arthritis: diagnostic value, associations with
radiological progression rate, and extra-articular manifestations. Ann
Rheum Dis 63:1587–1593, 2004.
87. Turesson C, et al: Rheumatoid factor and antibodies to cyclic citrul-
linated peptides are associated with severe extra-articular manifesta-
tions in rheumatoid arthritis. Ann Rheum Dis 66:59–64, 2007.
88. Quinn MA, et al: Anti-CCP antibodies measured at disease onset
help identify seronegative rheumatoid arthritis and predict radiologi-
cal and functional outcome. Rheumatology (Oxford) 45:478–480,
2006.
89. Kelly CA, et al: Rheumatoid arthritis-related interstitial lung disease:
associations, prognostic factors and physiological and radiological
characteristics—a large multicentre UK study. Rheumatology (Oxford)
53:1676–1682, 2014.
90. Giles JT, et al: Association of fine specificity and repertoire expan-
sion of anticitrullinated peptide antibodies with rheumatoid arthritis
associated interstitial lung disease. Ann Rheum Dis 73:1487–1494,
2014.
91. Aubart F, et al: High levels of anti-cyclic citrullinated peptide auto-
antibodies are associated with co-occurrence of pulmonary diseases
with rheumatoid arthritis. J Rheumatol 38:979–982, 2011.
92. Zhu J, Zhou Y, Chen X, et al: A metaanalysis of the increased risk of
rheumatoid arthritis-related pulmonary disease as a result of serum
anticitrullinated protein antibody positivity. J Rheumatol 41:1282–
1289, 2014.
94. Marasovic-Krstulovic D, Martinovic-Kaliterna D, Fabijanic D, et al:
Are the anti-cyclic citrullinated peptide antibodies independent pre-
dictors of myocardial involvement in patients with active rheumatoid
arthritis? Rheumatology (Oxford) 50:1505–1512, 2011.
97. Bongartz T, et al: Citrullination in extra-articular manifestations of
rheumatoid arthritis. Rheumatology (Oxford) 46:70–75, 2007.
99. Gizinski AM, et al: Rheumatoid arthritis (RA)-specific autoantibod-
ies in patients with interstitial lung disease and absence of clinically
apparent articular RA. Clin Rheumatol 28:611–613, 2009.
101. Visser K, et al: Pretreatment serum levels of anti-cyclic citrullinated
peptide antibodies are associated with the response to methotrexate
in recent-onset arthritis. Ann Rheum Dis 67:1194–1195, 2008.
102. Farragher TM, et al: Benefit of early treatment in inflammatory poly-
arthritis patients with anti-cyclic citrullinated peptide antibodies
versus those without antibodies. Arthritis Care Res (Hoboken) 62:
664–675, 2010.
103. Braun-Moscovici Y, et al: Anti-cyclic citrullinated protein antibodies
as a predictor of response to anti-tumor necrosis factor-alpha therapy
in patients with rheumatoid arthritis. J Rheumatol 33:497–500, 2006.
107. Kolfenbach JR, et al: Autoimmunity to peptidyl arginine deiminase
type 4 precedes clinical onset of rheumatoid arthritis. Arthritis Rheum
62:2633–2639, 2010.
109. Kokkonen H, et al: Antibodies of IgG, IgA and IgM isotypes against
cyclic citrullinated peptide precede the development of rheumatoid
arthritis. Arthritis Res Ther 13:R13, 2011.
110. Rantapaa-Dahlqvist S, et al: Antibodies against cyclic citrullinated
peptide and IgA rheumatoid factor predict the development of rheu-
matoid arthritis. Arthritis Rheum 48:2741–2749, 2003.
111. Kolfenbach JR, et al: A prospective approach to investigating the
natural history of preclinical rheumatoid arthritis (RA) using first-
degree relatives of probands with RA. Arthritis Rheum 61:1735–1742,
2009.
112. Berglin E, et al: Radiological outcome in rheumatoid arthritis is pre-
dicted by presence of antibodies against cyclic citrullinated peptide
before and at disease onset, and by IgA-RF at disease onset. Ann
Rheum Dis 65:453–458, 2006.
113. Majka DS, et al: Duration of preclinical rheumatoid arthritis-related
autoantibody positivity increases in subjects with older age at time
of disease diagnosis. Ann Rheum Dis 67:801–807, 2008.
114. Gregersen PK, Silver J, Winchester RJ: The shared epitope hypoth-
esis. An approach to understanding the molecular genetics of suscep-
tibility to rheumatoid arthritis. Arthritis Rheum 30:1205–1213, 1987.
115. van der Helm-van Mil AH, Huizinga TW: Advances in the genetics
of rheumatoid arthritis point to subclassification into distinct disease
subsets. Arthritis Res Ther 10:205, 2008.
116. Huizinga TW, et al: Refining the complex rheumatoid arthritis phe-
notype based on specificity of the HLA-DRB1 shared epitope for
antibodies to citrullinated proteins. Arthritis Rheum 52:3433–3438,
2005.
117. Irigoyen P, et al: Regulation of anti-cyclic citrullinated peptide anti-
bodies in rheumatoid arthritis: contrasting effects of HLA-DR3 and
the shared epitope alleles. Arthritis Rheum 52:3813–3818, 2005.
118. Snir O, et al: Multiple antibody reactivities to citrullinated antigens
in sera from patients with rheumatoid arthritis: association with
HLA-DRB1 alleles. Ann Rheum Dis 68:736–743, 2009.
119. van der Helm-van Mil AH, et al: The HLA-DRB1 shared epitope
alleles differ in the interaction with smoking and predisposition to
antibodies to cyclic citrullinated peptide. Arthritis Rheum 56:425–
432, 2007.
120. Johansson M, Arlestig L, Hallmans G, et al: PTPN22 polymorphism
and anti-cyclic citrullinated peptide antibodies in combination
 CHAPTER 56    Autoantibodies in Rheumatoid Arthritis 845
strongly predicts future onset of rheumatoid arthritis and has a speci-
ficity of 100% for the disease. Arthritis Res Ther 8:R19, 2006.
121. Stanford SM, Mustelin TM, Bottini N: Lymphoid tyrosine phospha-
tase and autoimmunity: human genetics rediscovers tyrosine phos-
phatases. Semin Immunopathol 32:127–136, 2010.
122. Shirai H, Blundell TL, Mizuguchi K: A novel superfamily of enzymes
that catalyze the modification of guanidino groups. Trends Biochem
Sci 26:465–468, 2001.
128. Arita K, et al: Structural basis for Ca(2+)-induced activation of
human PAD4. Nat Struct Mol Biol 11:777–783, 2004.
132. Foulquier C, et al: Peptidyl arginine deiminase type 2 (PAD-2) and
PAD-4 but not PAD-1, PAD-3, and PAD-6 are expressed in rheuma-
toid arthritis synovium in close association with tissue inflammation.
Arthritis Rheum 56:3541–3553, 2007.
133. Chang X, et al: Localization of peptidylarginine deiminase 4 (PADI4)
and citrullinated protein in synovial tissue of rheumatoid arthritis.
Rheumatology (Oxford) 44:40–50, 2005.
136. Darrah E, et al: Erosive rheumatoid arthritis is associated with anti-
bodies that activate PAD4 by increasing calcium sensitivity. Sci Transl
Med 5:186ra65, 2013.
141. Andrade F, et al: Autocitrullination of human peptidyl arginine
deiminase type 4 regulates protein citrullination during cell activa-
tion. Arthritis Rheum 62:1630–1640, 2010.
142. Mechin MC, et al: Deimination is regulated at multiple levels includ-
ing auto-deimination of peptidylarginine deiminases. Cell Mol Life Sci
67:1491–1503, 2010.
147. Loos T, et al: Citrullination of CXCL10 and CXCL11 by peptidylar-
ginine deiminase: a naturally occurring posttranslational modifica-
tion of chemokines and new dimension of immunoregulation. Blood
112:2648–2656, 2008.
155. Li P, et al: PAD4 is essential for antibacterial innate immunity medi-
ated by neutrophil extracellular traps. J Exp Med 207:1853–1862,
2010.
157. Suzuki A, et al: Functional haplotypes of PADI4, encoding citrulli-
nating enzyme peptidylarginine deiminase 4, are associated with
rheumatoid arthritis. Nat Genet 34:395–402, 2003.
163. Harris ML, et al: Association of autoimmunity to peptidyl arginine
deiminase type 4 with genotype and disease severity in rheumatoid
arthritis. Arthritis Rheum 58:1958–1967, 2008.
164. Bang SY, et al: Peptidyl arginine deiminase type IV (PADI4) haplo-
types interact with shared epitope regardless of anti-cyclic citrulli-
nated peptide antibody or erosive joint status in rheumatoid arthritis:
a case control study. Arthritis Res Ther 12:R115, 2010.
165. Suzuki T, et al: PADI4 and HLA-DRB1 are genetic risks for radio-
graphic progression in RA patients, independent of ACPA status:
results from the IORRA cohort study. PLoS One 8:e61045, 2013.
166. Halvorsen EH, et al: Serum IgG antibodies to peptidylarginine deim-
inase 4 in rheumatoid arthritis and associations with disease severity.
Ann Rheum Dis 67:414–417, 2008.
167. Halvorsen EH, et al: Serum IgG antibodies to peptidylarginine deim-
inase 4 predict radiographic progression in patients with rheumatoid
arthritis treated with tumour necrosis factor-alpha blocking agents.
Ann Rheum Dis 68:249–252, 2009.
168. Giles JT, et al: Association of cross-reactive antibodies targeting
peptidyl-arginine deiminase 3 and 4 with rheumatoid arthritis-
associated interstitial lung disease. PLoS One 9:e98794, 2014.
170. Khandpur R, et al: NETs are a source of citrullinated autoantigens
and stimulate inflammatory responses in rheumatoid arthritis. Sci
Transl Med 5:178ra40, 2013.
173. Damgaard D, Senolt L, Nielsen MF, et al: Demonstration of extracel-
lular peptidylarginine deiminase (PAD) activity in synovial fluid of
patients with rheumatoid arthritis using a novel assay for citrullina-
tion of fibrinogen. Arthritis Res Ther 16:498, 2014.
174. Shi J, et al: Autoantibodies recognizing carbamylated proteins are
present in sera of patients with rheumatoid arthritis and predict joint
damage. Proc Natl Acad Sci U S A 108:17372–17377, 2011.
177. Shi J, et al: Anti-carbamylated protein (anti-CarP) antibodies
precede the onset of rheumatoid arthritis. Ann Rheum Dis 73:780–
783, 2014.
180. Thiele GM, et al: Malondialdehyde-acetaldehyde adducts and anti-
malondialdehyde-acetaldehyde antibodies in rheumatoid arthritis.
Arthritis Rheumatol 67:645–655, 2015.
190. Darrah E, Andrade F: Editorial: citrullination, and carbamylation,
and malondialdehyde-acetaldehyde! Oh my! Entering the forest of
autoantigen modifications in rheumatoid arthritis. Arthritis Rheuma-
tol. 67:604–608, 2015.
191. Hassfeld W, et al: Demonstration of a new antinuclear antibody
(anti-RA33) that is highly specific for rheumatoid arthritis. Arthritis
Rheum 32:1515–1520, 1989.
196. Nell-Duxneuner V, et al: Autoantibody profiling in patients with
very early rheumatoid arthritis: a follow-up study. Ann Rheum Dis
69:169–174, 2010.
198. Kouskoff V, et al: Organ-specific disease provoked by systemic auto-
immunity. Cell 87:811–822, 1996.
203. Kassahn D, Kolb C, Solomon S, et al: Few human autoimmune sera
detect GPI. Nat Immunol 3:411–412, 2002.
205. Matsumoto I, et al: Low prevalence of antibodies to glucose-6-
phosphate isomerase in patients with rheumatoid arthritis and a
spectrum of other chronic autoimmune disorders. Arthritis Rheum
48:944–954, 2003.
207. Zendman AJ, Vossenaar ER, van Venrooij WJ: Autoantibodies to
citrullinated (poly)peptides: a key diagnostic and prognostic marker
for rheumatoid arthritis. Autoimmunity 37:295–299, 2004.
208. Kokkonen H, Johansson M, Innala L, et al: The PTPN22 1858C/T
polymorphism is associated with anti-cyclic citrullinated peptide
antibody-positive early rheumatoid arthritis in northern Sweden.
Arthritis Res Ther 9:R56, 2007.
209. van der Helm-van Mil AH, et al: The HLA-DRB1 shared epitope
alleles are primarily a risk factor for anti-cyclic citrullinated peptide
antibodies and are not an independent risk factor for development
of rheumatoid arthritis. Arthritis Rheum 54:1117–1121, 2006.
210. van der Helm-van Mil AH, Wesoly JZ, Huizinga TW: Understanding
the genetic contribution to rheumatoid arthritis. Curr Opin Rheuma-
tol 17:299–304, 2005.
212. Scally SW, et al: A molecular basis for the association of the HLA-
DRB1 locus, citrullination, and rheumatoid arthritis. J Exp Med
210:2569–2582, 2013.
213. Zhao X, et al: Circulating immune complexes contain citrullinated
fibrinogen in rheumatoid arthritis. Arthritis Res Ther 10:R94, 2008.
214. Clavel C, et al: Induction of macrophage secretion of tumor necrosis
factor alpha through Fcgamma receptor IIa engagement by rheuma-
toid arthritis-specific autoantibodies to citrullinated proteins com-
plexed with fibrinogen. Arthritis Rheum 58:678–688, 2008.
215. Sokolove J, Zhao X, Chandra PE, et al: Immune complexes contain-
ing citrullinated fibrinogen costimulate macrophages via Toll-like
receptor 4 and Fc gamma receptor. Arthritis Rheum 63:53–62, 2011.
217. Rombouts Y, et al: Anti-citrullinated protein antibodies acquire a
pro-inflammatory Fc glycosylation phenotype prior to the onset of
rheumatoid arthritis. Ann Rheum Dis 74:234–241, 2015.
221. Kasser UR, et al: Risk for periodontal disease in patients with
longstanding rheumatoid arthritis. Arthritis Rheum 40:2248–2251,
1997.
223. de Pablo P, Chapple IL, Buckley CD, et al: Periodontitis in systemic
rheumatic diseases. Nat Rev Rheumatol 5:218–224, 2009.
224. Lundberg K, Wegner N, Yucel-Lindberg T, et al: Periodontitis in
RA—the citrullinated enolase connection. Nat Rev Rheumatol
6:727–730, 2010.
227. Lundberg K, et al: Antibodies to citrullinated alpha-enolase peptide
1 are specific for rheumatoid arthritis and cross-react with bacterial
enolase. Arthritis Rheum 58:3009–3019, 2008.
228. Wegner N, et al: Peptidylarginine deiminase from Porphyromonas
gingivalis citrullinates human fibrinogen and alpha-enolase: implica-
tions for autoimmunity in rheumatoid arthritis. Arthritis Rheum 62:
2662–2672, 2010.
230. Konig MF, Paracha AS, Moni M, et al: Defining the role of Porphy-
romonas gingivalis peptidylarginine deiminase (PPAD) in rheumatoid
arthritis through the study of PPAD biology. Ann Rheum Dis pii:ann
rheumdis-2014-205385, May 26, 2014. 74:2054–61, 2015.
232. Klareskog L, et al: A new model for an etiology of rheumatoid
arthritis: smoking may trigger HLA-DR (shared epitope)-restricted
immune reactions to autoantigens modified by citrullination. Arthritis
Rheum 54:38–46, 2006.
233. Linn-Rasker SP, et al: Smoking is a risk factor for anti-CCP antibod-
ies only in rheumatoid arthritis patients who carry HLA-DRB1
shared epitope alleles. Ann Rheum Dis 65:366–371, 2006.
234. Klareskog L, Malmstrom V, Lundberg K, et al: Smoking, citrullina-
tion and genetic variability in the immunopathogenesis of rheuma-
toid arthritis. Semin Immunol 23:92–98, 2011.
 CHAPTER 56    Autoantibodies in Rheumatoid Arthritis 845.e1
REFERENCES
1. Waaler E: On the occurrence of a factor in human serum activating
the specific agglutintion of sheep blood corpuscles. 1939. APMIS
115:422–438, 2007.
2. Rose HM, Ragan C, et al: Differential agglutination of normal and
sensitized sheep erythrocytes by sera of patients with rheumatoid
arthritis. Proc Soc Exp Biol Med 68:1–6, 1948.
3. Henney CS, Stanworth DR: Reaction of rheumatoid factor with the
isolated polypeptide chains of human 7S gamma-globulin. Nature
201:511–512, 1964.
4. Plotz CM, Singer JM: The latex fixation test. I. Application to the
serologic diagnosis of rheumatoid arthritis. Am J Med 21:888–892,
1956.
5. Plotz CM, Singer JM: The latex fixation test. II. Results in rheuma-
toid arthritis. Am J Med 21:893–896, 1956.
6. Shmerling RH, Delbanco TL: The rheumatoid factor: an analysis of
clinical utility. Am J Med 91:528–534, 1991.
7. Shmerling RH, Delbanco TL: How useful is the rheumatoid factor?
An analysis of sensitivity, specificity, and predictive value. Arch Intern
Med 152:2417–2420, 1992.
8. Dorner T, Egerer K, Feist E, et al: Rheumatoid factor revisited. Curr
Opin Rheumatol 16:246–253, 2004.
9. Prentice AG, Hickling P, Wiseman IC, et al: Prospective comparison
of laser nephelometry with standard agglutination techniques for
detection of rheumatoid factor. J Clin Pathol 40:216–220, 1987.
10. Anuradha V, Chopra A: In the era of nephelometry, latex agglutina-
tion is still good enough to detect rheumatoid factor. J Rheumatol
32:2343–2344, 2005.
11. Randen I, et al: Rheumatoid factor V genes from patients with rheu-
matoid arthritis are diverse and show evidence of an antigen-driven
response. Immunol Rev 128:49–71, 1992.
12. Randen I, et al: Synovial IgG rheumatoid factors show evidence of
an antigen-driven immune response and a shift in the V gene reper-
toire compared to IgM rheumatoid factors. Eur J Immunol 23:1220–
1225, 1993.
13. Jonsson T, et al: Combined elevation of IgM and IgA rheumatoid
factor has high diagnostic specificity for rheumatoid arthritis. Rheu-
matol Int 18:119–122, 1998.
14. Nishimura K, et al: Meta-analysis: diagnostic accuracy of anti-cyclic
citrullinated peptide antibody and rheumatoid factor for rheumatoid
arthritis. Ann Intern Med 146:797–808, 2007.
15. Aho K, et al: When does rheumatoid disease start? Arthritis Rheum
28:485–489, 1985.
16. Aho K, Heliovaara M, Maatela J, et al: Rheumatoid factors antedat-
ing clinical rheumatoid arthritis. J Rheumatol 18:1282–1284, 1991.
17. Nielen MM, et al: Specific autoantibodies precede the symptoms of
rheumatoid arthritis: a study of serial measurements in blood donors.
Arthritis Rheum 50:380–386, 2004.
18. Halldorsdottir HD, Jonsson T, Thorsteinsson J, et al: A prospective
study on the incidence of rheumatoid arthritis among people with
persistent increase of rheumatoid factor. Ann Rheum Dis 59:149–151,
2000.
19. Jansen LM, van der Horst-Bruinsma IE, van Schaardenburg D, et al:
Predictors of radiographic joint damage in patients with early rheu-
matoid arthritis. Ann Rheum Dis 60:924–927, 2001.
20. Panayi GS: B cells: a fundamental role in the pathogenesis of rheu-
matoid arthritis? Rheumatology (Oxford) 44(Suppl 2):ii3–ii7, 2005.
21. Nienhuis RL, Mandema E: A new serum factor in patients with
rheumatoid arthritis; the antiperinuclear factor. Ann Rheum Dis
23:302–305, 1964.
22. Sondag-Tschroots IR, Aaij C, Smit JW, et al: The antiperinuclear
factor. 1. The diagnostic significance of the antiperinuclear factor for
rheumatoid arthritis. Ann Rheum Dis 38:248–251, 1979.
23. Hoet RM, Boerbooms AM, Arends M, et al: Antiperinuclear factor,
a marker autoantibody for rheumatoid arthritis: colocalisation of the
perinuclear factor and profilaggrin. Ann Rheum Dis 50:611–618,
1991.
24. Janssens X, Veys EM, Verbruggen G, et al: The diagnostic signifi-
cance of the antiperinuclear factor for rheumatoid arthritis. J Rheu-
matol 15:1346–1350, 1988.
25. Cassani F, et al: Antiperinuclear factor in an Italian series of patients
with rheumatoid arthritis. Ric Clin Lab 13:347–352, 1983.
26. Berthelot JM, Maugars Y, Audrain M, et al: Specificity of antiperi-
nuclear factor for rheumatoid arthritis in rheumatoid factor-positive
sera. Br J Rheumatol 34:716–720, 1995.
27. Manera C, Franceschini F, Cretti L, et al: Clinical heterogeneity of
rheumatoid arthritis and the antiperinuclear factor. J Rheumatol 21:
2021–2025, 1994.
28. Youinou P, et al: The antiperinuclear factor. I. Clinical and serologic
associations. Clin Exp Rheumatol 8:259–264, 1990.
29. Youinou P, et al: The antiperinuclear factor. II. Variability of the
perinuclear antigen. Clin Exp Rheumatol 8:265–269, 1990.
30. Youinou P, Le GP: The reliability of the antiperinuclear factor test
despite the inconstancy of the targeted antigens. J Rheumatol 21:
1990–1991, 1994.
31. Aggarwal R, Liao K, Nair R, et al: Anti-citrullinated peptide anti-
body assays and their role in the diagnosis of rheumatoid arthritis.
Arthritis Rheum 61:1472–1483, 2009.
32. Young BJ, Mallya RK, Leslie RD, et al: Anti-keratin antibodies in
rheumatoid arthritis. Br Med J 2:97–99, 1979.
33. Scott DL, Delamere JP, Jones LJ, et al: Significance of laminar anti-
keratin antibodies to rat oesophagus in rheumatoid arthritis. Ann
Rheum Dis 40:267–271, 1981.
34. Miossec P, Youinou P, Le GP, et al: Clinical relevance of antikera-
tin antibodies in rheumatoid arthritis. Clin Rheumatol 1:185–189,
1982.
35. Ordeig J, Guardia J: Diagnostic value of antikeratin antibodies in
rheumatoid arthritis. J Rheumatol 11:602–604, 1984.
36. Vincent C, et al: High diagnostic value in rheumatoid arthritis of
antibodies to the stratum corneum of rat oesophagus epithelium,
so-called “antikeratin antibodies.” Ann Rheum Dis 48:712–722, 1989.
37. Paimela L, Gripenberg M, Kurki P, et al: Antikeratin antibodies:
diagnostic and prognostic markers for early rheumatoid arthritis. Ann
Rheum Dis 51:743–746, 1992.
38. Simon M, et al: The cytokeratin filament-aggregating protein filla-
grin is the target of the co-called “antikeratin antibodies,” autoanti-
bodies specific for rheumatoid arthritis. J Clin Invest 92:1387–1393,
1993.
39. Sebbag M, et al: The antiperinuclear factor and the so-called antik-
eratin antibodies are the same rheumatoid arthritis-specific autoan-
tibodies. J Clin Invest 95:2672–2679, 1995.
40. Markova NG, et al: Profilaggrin is a major epidermal calcium-binding
protein. Mol Cell Biol 13:613–625, 1993.
41. Senshu T, et al: Detection of deiminated proteins in rat skin: probing
with a monospecific antibody after modification of citrulline residues.
J Invest Dermatol 105:163–169, 1995.
42. Senshu T, Kan S, Ogawa H, et al: Preferential deimination of keratin
K1 and filaggrin during the terminal differentiation of human epider-
mis. Biochem Biophys Res Commun 225:712–719, 1996.
43. Vossenaar ER, Zendman AJ, van Venrooij WJ, et al: PAD, a growing
family of citrullinating enzymes: genes, features and involvement in
disease. Bioessays 25:1106–1118, 2003.
44. Schellekens GA, de Jong BA, van den Hoogen FH, et al: Citrulline
is an essential constituent of antigenic determinants recognized by
rheumatoid arthritis-specific autoantibodies. J Clin Invest 101:273–
281, 1998.
45. Baeten D, et al: Specific presence of intracellular citrullinated pro-
teins in rheumatoid arthritis synovium: relevance to antifilaggrin
autoantibodies. Arthritis Rheum 44:2255–2262, 2001.
46. Masson-Bessière C, et al: In the rheumatoid pannus, anti-filaggrin
autoantibodies are produced by local plasma cells and constitute a
higher proportion of IgG than in synovial fluid and serum. Clin Exp
Immunol 119:544–552, 2000.
47. Wood DD, et al: Myelin localization of peptidylarginine deiminases
2 and 4: comparison of PAD2 and PAD4 activities. Lab Invest 88:354–
364, 2008.
48. Chavanas S, et al: Peptidylarginine deiminases and deimination in
biology and pathology: relevance to skin homeostasis. J Dermatol Sci
44:63–72, 2006.
49. Chapuy-Regaud S, et al: Fibrin deimination in synovial tissue is not
specific for rheumatoid arthritis but commonly occurs during syno-
vitides. J Immunol 174:5057–5064, 2005.
50. Makrygiannakis D, et al: Citrullination is an inflammation-dependent
process. Ann Rheum Dis 65:1219–1222, 2006.
51. Makrygiannakis D, et al: Smoking increases peptidylarginine deimi-
nase 2 enzyme expression in human lungs and increases citrullination
in BAL cells. Ann Rheum Dis 67:1488–1492, 2008.
52. Dyson HJ, Wright PE: Antigenic peptides. FASEB J 9:37–42, 1995.
53. Dorow DS, et al: Two large immunogenic and antigenic myoglobin
peptides and the effects of cyclisation. Mol Immunol 22:1255–1264,
1985.
 845.e2 PART 7    DIAGNOSTIC TESTS AND PROCEDURES IN RHEUMATIC DIEASES
54. Schellekens GA, et al: The diagnostic properties of rheumatoid
arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis
Rheum 43:155–163, 2000.
55. van Venrooij WJ, Zendman AJ: Anti-CCP2 antibodies: an overview
and perspective of the diagnostic abilities of this serological marker
for early rheumatoid arthritis. Clin Rev Allergy Immunol 34:36–39,
2008.
56. van Gaalen FA, Visser H, Huizinga TW: A comparison of the diag-
nostic accuracy and prognostic value of the first and second anti-
cyclic citrullinated peptides (CCP1 and CCP2) autoantibody tests
for rheumatoid arthritis. Ann Rheum Dis 64:1510–1512, 2005.
57. Lutteri L, Malaise M, Chapelle JP: Comparison of second- and third-
generation anti-cyclic citrullinated peptide antibodies assays for
detecting rheumatoid arthritis. Clin Chim Acta 386:76–81, 2007.
58. dos Anjos LM, et al: A comparative study of IgG second- and third-
generation anti-cyclic citrullinated peptide (CCP) ELISAs and their
combination with IgA third-generation CCP ELISA for the diagnosis
of rheumatoid arthritis. Clin Rheumatol 28:153–158, 2009.
59. Masson-Bessière C, et al: The major synovial targets of the rheuma-
toid arthritis-specific antifilaggrin autoantibodies are deiminated
forms of the alpha- and beta-chains of fibrin. J Immunol 166:4177–
4184, 2001.
60. Kinloch A, et al: Synovial fluid is a site of citrullination of autoan-
tigens in inflammatory arthritis. Arthritis Rheum 58:2287–2295, 2008.
61. Vossenaar ER, et al: Expression and activity of citrullinating pepti-
dylarginine deiminase enzymes in monocytes and macrophages. Ann
Rheum Dis 63:373–381, 2004.
62. Vossenaar ER, et al: Rheumatoid arthritis specific anti-Sa antibodies
target citrullinated vimentin. Arthritis Res Ther 6:R142–R150, 2004.
63. van Beers JJ, et al: Anti-citrullinated fibronectin antibodies in rheu-
matoid arthritis are associated with human leukocyte antigen-DRB1
shared epitope alleles. Arthritis Res Ther 14:R35, 2012.
64. Darrah E, Rosen A, Giles JT, et al: Peptidylarginine deiminase 2, 3
and 4 have distinct specificities against cellular substrates: novel
insights into autoantigen selection in rheumatoid arthritis. Ann
Rheum Dis 71:92–98, 2012.
65. Harlow L, et al: Identification of citrullinated hsp90 isoforms as novel
autoantigens in rheumatoid arthritis-associated interstitial lung
disease. Arthritis Rheum 65:869–879, 2013.
66. Dwivedi N, et al: Felty’s syndrome autoantibodies bind to deiminated
histones and neutrophil extracellular chromatin traps. Arthritis
Rheum 64:982–992, 2012.
67. Kinloch A, et al: Identification of citrullinated alpha-enolase as a
candidate autoantigen in rheumatoid arthritis. Arthritis Res Ther
7:R1421–R1429, 2005.
68. Matsuo K, et al: Identification of novel citrullinated autoantigens of
synovium in rheumatoid arthritis using a proteomic approach. Arthri-
tis Res Ther 8:R175, 2006.
69. Suzuki A, et al: Anti-citrullinated collagen type I antibody is a target
of autoimmunity in rheumatoid arthritis. Biochem Biophys Res
Commun 333:418–426, 2005.
70. Burkhardt H, et al: Humoral immune response to citrullinated col-
lagen type II determinants in early rheumatoid arthritis. Eur J
Immunol 35:1643–1652, 2005.
71. Okazaki Y, et al: Identification of citrullinated eukaryotic translation
initiation factor 4G1 as novel autoantigen in rheumatoid arthritis.
Biochem Biophys Res Commun 341:94–100, 2006.
72. Goeb V, et al: Candidate autoantigens identified by mass spectrom-
etry in early rheumatoid arthritis are chaperones and citrullinated
glycolytic enzymes. Arthritis Res Ther 11:R38, 2009.
73. van Beers JJ, et al: The rheumatoid arthritis synovial fluid citrulli-
nome reveals novel citrullinated epitopes in apolipoprotein E,
myeloid nuclear differentiation antigen, and beta-actin. Arthritis
Rheum 65:69–80, 2013.
74. Saulot V, et al: Presence of autoantibodies to the glycolytic enzyme
alpha-enolase in sera from patients with early rheumatoid arthritis.
Arthritis Rheum 46:1196–1201, 2002.
75. Romero V, et al: Immune-mediated pore-forming pathways induce
cellular hypercitrullination and generate citrullinated autoantigens
in rheumatoid arthritis. Sci Transl Med 5:209ra150, 2013.
76. Tutturen AE, Fleckenstein B, de Souza GA: Assessing the citrulli-
nome in rheumatoid arthritis synovial fluid with and without enrich-
ment of citrullinated peptides. J Proteome Res 13:2867–2873, 2014.
77. Bang H, et al: Mutation and citrullination modifies vimentin to a
novel autoantigen for rheumatoid arthritis. Arthritis Rheum 56:2503–
2511, 2007.
78. Sokolove J, et al: Autoantibody epitope spreading in the pre-clinical
phase predicts progression to rheumatoid arthritis. PLoS One 7:
e35296, 2012.
79. Wagner CA, et al: Identification of anticitrullinated protein anti-
body reactivities in a subset of anti-CCP-negative rheumatoid arthri-
tis: association with cigarette smoking and HLA-DRB1 “shared
epitope” alleles. Ann Rheum Dis 74:579–586, 2015.
80. van Gaalen FA, et al: Autoantibodies to cyclic citrullinated peptides
predict progression to rheumatoid arthritis in patients with undif-
ferentiated arthritis: a prospective cohort study. Arthritis Rheum 50:
709–715, 2004.
81. Jansen LM, et al: The predictive value of anti-cyclic citrullinated
peptide antibodies in early arthritis. J Rheumatol 30:1691–1695, 2003.
82. Nell VP, et al: Autoantibody profiling as early diagnostic and prog-
nostic tool for rheumatoid arthritis. Ann Rheum Dis 64:1731–1736,
2005.
83. Vencovsky J, et al: Autoantibodies can be prognostic markers of an
erosive disease in early rheumatoid arthritis. Ann Rheum Dis 62:427–
430, 2003.
84. Mewar D, et al: Independent associations of anti-cyclic citrullinated
peptide antibodies and rheumatoid factor with radiographic severity
of rheumatoid arthritis. Arthritis Res Ther 8:R128, 2006.
85. Syversen SW, et al: High anti-cyclic citrullinated peptide levels and
an algorithm of four variables predict radiographic progression in
patients with rheumatoid arthritis: results from a 10-year longitudinal
study. Ann Rheum Dis 67:212–217, 2008.
86. De Rycke L, et al: Rheumatoid factor and anticitrullinated protein
antibodies in rheumatoid arthritis: diagnostic value, associations with
radiological progression rate, and extra-articular manifestations. Ann
Rheum Dis 63:1587–1593, 2004.
87. Turesson C, et al: Rheumatoid factor and antibodies to cyclic citrul-
linated peptides are associated with severe extra-articular manifesta-
tions in rheumatoid arthritis. Ann Rheum Dis 66:59–64, 2007.
88. Quinn MA, et al: Anti-CCP antibodies measured at disease onset
help identify seronegative rheumatoid arthritis and predict radiologi-
cal and functional outcome. Rheumatology (Oxford) 45:478–480,
2006.
89. Kelly CA, et al: Rheumatoid arthritis-related interstitial lung disease:
associations, prognostic factors and physiological and radiological
characteristics—a large multicentre UK study. Rheumatology (Oxford)
53:1676–1682, 2014.
90. Giles JT, et al: Association of fine specificity and repertoire expan-
sion of anticitrullinated peptide antibodies with rheumatoid arthritis
associated interstitial lung disease. Ann Rheum Dis 73:1487–1494,
2014.
91. Aubart F, et al: High levels of anti-cyclic citrullinated peptide auto-
antibodies are associated with co-occurrence of pulmonary diseases
with rheumatoid arthritis. J Rheumatol 38:979–982, 2011.
92. Zhu J, Zhou Y, Chen X, et al: A metaanalysis of the increased risk of
rheumatoid arthritis-related pulmonary disease as a result of serum
anticitrullinated protein antibody positivity. J Rheumatol 41:1282–
1289, 2014.
93. Giles JT, et al: Left ventricular structure and function in patients with
rheumatoid arthritis, as assessed by cardiac magnetic resonance
imaging. Arthritis Rheum 62:940–951, 2010.
94. Marasovic-Krstulovic D, Martinovic-Kaliterna D, Fabijanic D, et al:
Are the anti-cyclic citrullinated peptide antibodies independent pre-
dictors of myocardial involvement in patients with active rheumatoid
arthritis? Rheumatology (Oxford) 50:1505–1512, 2011.
95. Lopez-Longo FJ, et al: Association between anti-cyclic citrullinated
peptide antibodies and ischemic heart disease in patients with rheu-
matoid arthritis. Arthritis Rheum 61:419–424, 2009.
96. Giles JT, et al: Myocardial citrullination in rheumatoid arthritis: a
correlative histopathologic study. Arthritis Res Ther 14:R39, 2012.
97. Bongartz T, et al: Citrullination in extra-articular manifestations of
rheumatoid arthritis. Rheumatology (Oxford) 46:70–75, 2007.
98. Sokolove J, et al: Brief report: citrullination within the atheroscle-
rotic plaque: a potential target for the anti-citrullinated protein anti-
body response in rheumatoid arthritis. Arthritis Rheum 65:1719–1724,
2013.
99. Gizinski AM, et al: Rheumatoid arthritis (RA)-specific autoantibod-
ies in patients with interstitial lung disease and absence of clinically
apparent articular RA. Clin Rheumatol 28:611–613, 2009.
100. Fischer A, et al: Lung disease with anti-CCP antibodies but not
rheumatoid arthritis or connective tissue disease. Respir Med 106:
1040–1047, 2012.
 CHAPTER 56    Autoantibodies in Rheumatoid Arthritis 845.e3
101. Visser K, et al: Pretreatment serum levels of anti-cyclic citrullinated
peptide antibodies are associated with the response to methotrexate
in recent-onset arthritis. Ann Rheum Dis 67:1194–1195, 2008.
102. Farragher TM, et al: Benefit of early treatment in inflammatory poly-
arthritis patients with anti-cyclic citrullinated peptide antibodies
versus those without antibodies. Arthritis Care Res (Hoboken) 62:
664–675, 2010.
103. Braun-Moscovici Y, et al: Anti-cyclic citrullinated protein antibodies
as a predictor of response to anti-tumor necrosis factor-alpha therapy
in patients with rheumatoid arthritis. J Rheumatol 33:497–500, 2006.
104. Kurki P, Aho K, Palosuo T, et al: Immunopathology of rheumatoid
arthritis. Antikeratin antibodies precede the clinical disease. Arthritis
Rheum 35:914–917, 1992.
105. Arbuckle MR, et al: Development of anti-dsDNA autoantibodies
prior to clinical diagnosis of systemic lupus erythematosus. Scand J
Immunol 54:211–219, 2001.
106. Arbuckle MR, et al: Development of autoantibodies before the clini-
cal onset of systemic lupus erythematosus. N Engl J Med 349:1526–
1533, 2003.
107. Kolfenbach JR, et al: Autoimmunity to peptidyl arginine deiminase
type 4 precedes clinical onset of rheumatoid arthritis. Arthritis Rheum
62:2633–2639, 2010.
108. Eriksson C, et al: Autoantibodies predate the onset of systemic lupus
erythematosus in northern Sweden. Arthritis Res Ther 13:R30, 2011.
109. Kokkonen H, et al: Antibodies of IgG, IgA and IgM isotypes against
cyclic citrullinated peptide precede the development of rheumatoid
arthritis. Arthritis Res Ther 13:R13, 2011.
110. Rantapaa-Dahlqvist S, et al: Antibodies against cyclic citrullinated
peptide and IgA rheumatoid factor predict the development of rheu-
matoid arthritis. Arthritis Rheum 48:2741–2749, 2003.
111. Kolfenbach JR, et al: A prospective approach to investigating the
natural history of preclinical rheumatoid arthritis (RA) using first-
degree relatives of probands with RA. Arthritis Rheum 61:1735–1742,
2009.
112. Berglin E, et al: Radiological outcome in rheumatoid arthritis is pre-
dicted by presence of antibodies against cyclic citrullinated peptide
before and at disease onset, and by IgA-RF at disease onset. Ann
Rheum Dis 65:453–458, 2006.
113. Majka DS, et al: Duration of preclinical rheumatoid arthritis-related
autoantibody positivity increases in subjects with older age at time
of disease diagnosis. Ann Rheum Dis 67:801–807, 2008.
114. Gregersen PK, Silver J, Winchester RJ: The shared epitope hypoth-
esis. An approach to understanding the molecular genetics of suscep-
tibility to rheumatoid arthritis. Arthritis Rheum 30:1205–1213, 1987.
115. van der Helm-van Mil AH, Huizinga TW: Advances in the genetics
of rheumatoid arthritis point to subclassification into distinct disease
subsets. Arthritis Res Ther 10:205, 2008.
116. Huizinga TW, et al: Refining the complex rheumatoid arthritis phe-
notype based on specificity of the HLA-DRB1 shared epitope for
antibodies to citrullinated proteins. Arthritis Rheum 52:3433–3438,
2005.
117. Irigoyen P, et al: Regulation of anti-cyclic citrullinated peptide anti-
bodies in rheumatoid arthritis: contrasting effects of HLA-DR3 and
the shared epitope alleles. Arthritis Rheum 52:3813–3818, 2005.
118. Snir O, et al: Multiple antibody reactivities to citrullinated antigens
in sera from patients with rheumatoid arthritis: association with
HLA-DRB1 alleles. Ann Rheum Dis 68:736–743, 2009.
119. van der Helm-van Mil AH, et al: The HLA-DRB1 shared epitope
alleles differ in the interaction with smoking and predisposition to
antibodies to cyclic citrullinated peptide. Arthritis Rheum 56:425–
432, 2007.
120. Johansson M, Arlestig L, Hallmans G, et al: PTPN22 polymorphism
and anti-cyclic citrullinated peptide antibodies in combination
strongly predicts future onset of rheumatoid arthritis and has a speci-
ficity of 100% for the disease. Arthritis Res Ther 8:R19, 2006.
121. Stanford SM, Mustelin TM, Bottini N: Lymphoid tyrosine phospha-
tase and autoimmunity: human genetics rediscovers tyrosine phos-
phatases. Semin Immunopathol 32:127–136, 2010.
122. Shirai H, Blundell TL, Mizuguchi K: A novel superfamily of enzymes
that catalyze the modification of guanidino groups. Trends Biochem
Sci 26:465–468, 2001.
123. Thompson PR, Fast W: Histone citrullination by protein arginine
deiminase: is arginine methylation a green light or a roadblock? ACS
Chem Biol 1:433–441, 2006.
124. Chavanas S, et al: Comparative analysis of the mouse and human
peptidylarginine deiminase gene clusters reveals highly conserved
non-coding segments and a new human gene, PADI6. Gene 330:19–
27, 2004.
125. Nakashima K, et al: Molecular characterization of peptidylarginine
deiminase in HL-60 cells induced by retinoic acid and 1alpha,25-
dihydroxyvitamin D(3). J Biol Chem 274:27786–27792, 1999.
126. Nakashima K, Hagiwara T, Yamada M: Nuclear localization of pep-
tidylarginine deiminase V and histone deimination in granulocytes.
J Biol Chem 277:49562–49568, 2002.
127. Cherrington BD, Morency E, Struble AM, et al: Potential role for
peptidylarginine deiminase 2 (PAD2) in citrullination of canine
mammary epithelial cell histones. PLoS One 5:e11768, 2010.
128. Arita K, et al: Structural basis for Ca(2+)-induced activation of
human PAD4. Nat Struct Mol Biol 11:777–783, 2004.
129. Asaga H, Nakashima K, Senshu T, et al: Immunocytochemical local-
ization of peptidylarginine deiminase in human eosinophils and neu-
trophils. J Leukoc Biol 70:46–51, 2001.
130. Nachat R, et al: Peptidylarginine deiminase isoforms 1-3 are expressed
in the epidermis and involved in the deimination of K1 and filaggrin.
J Invest Dermatol 124:384–393, 2005.
131. Esposito G, et al: Peptidylarginine deiminase (PAD) 6 is essential for
oocyte cytoskeletal sheet formation and female fertility. Mol Cell
Endocrinol 273:25–31, 2007.
132. Foulquier C, et al: Peptidyl arginine deiminase type 2 (PAD-2) and
PAD-4 but not PAD-1, PAD-3, and PAD-6 are expressed in rheuma-
toid arthritis synovium in close association with tissue inflammation.
Arthritis Rheum 56:3541–3553, 2007.
133. Chang X, et al: Localization of peptidylarginine deiminase 4 (PADI4)
and citrullinated protein in synovial tissue of rheumatoid arthritis.
Rheumatology (Oxford) 44:40–50, 2005.
134. Arita K, et al: Structural basis for histone N-terminal recognition by
human peptidylarginine deiminase 4. Proc Natl Acad Sci U S A 103:
5291–5296, 2006.
135. Slade DJ, et al: Protein arginine deiminase 2 binds calcium in an
ordered fashion: implications for inhibitor design. ACS Chem Biol
10:1043–1053, 2015.
136. Darrah E, et al: erosive rheumatoid arthritis is associated with anti-
bodies that activate PAD4 by increasing calcium sensitivity. Sci Transl
Med 5:186ra65, 2013.
137. Hill HR, Augustine NH, Jaffe HS: Human recombinant interferon
gamma enhances neonatal polymorphonuclear leukocyte activation
and movement, and increases free intracellular calcium. J Exp Med
173:767–770, 1991.
138. Pozzan T, Lew DP, Wollheim CB, et al: Is cytosolic ionized cal-
cium regulating neutrophil activation? Science 221:1413–1415,
1983.
139. Brandolini L, et al: IL-1 beta primes IL-8-activated human neutro-
phils for elastase release, phospholipase D activity, and calcium flux.
J Leukoc Biol 59:427–434, 1996.
140. Wong K, Kwan-Yeung L: Sphingosine mobilizes intracellular calcium
in human neutrophils. Cell Calcium 14:493–505, 1993.
141. Andrade F, et al: Autocitrullination of human peptidyl arginine
deiminase type 4 regulates protein citrullination during cell activa-
tion. Arthritis Rheum 62:1630–1640, 2010.
142. Mechin MC, et al: Deimination is regulated at multiple levels includ-
ing auto-deimination of peptidylarginine deiminases. Cell Mol Life Sci
67:1491–1503, 2010.
143. Borders CL Jr, et al: A structural role for arginine in proteins: mul-
tiple hydrogen bonds to backbone carbonyl oxygens. Protein Sci 3:
541–548, 1994.
144. Tarcsa E, et al: Protein unfolding by peptidylarginine deiminase. Sub-
strate specificity and structural relationships of the natural substrates
trichohyalin and filaggrin. J Biol Chem 271:30709–30716, 1996.
145. Mechin MC, et al: The peptidylarginine deiminases expressed in
human epidermis differ in their substrate specificities and subcellular
locations. Cell Mol Life Sci 62:1984–1995, 2005.
146. Raijmakers R, et al: Experimental autoimmune encephalomyelitis
induction in peptidylarginine deiminase 2 knockout mice. J Comp
Neurol 498:217–226, 2006.
147. Loos T, et al: Citrullination of CXCL10 and CXCL11 by peptidylar-
ginine deiminase: a naturally occurring posttranslational modifica-
tion of chemokines and new dimension of immunoregulation. Blood
112:2648–2656, 2008.
148. Mortier A, et al: Posttranslational modification of the NH2-terminal
region of CXCL5 by proteases or peptidylarginine deiminases (PAD)
differently affects its biological activity. J Biol Chem 285:29750–
29759, 2010.
 845.e4 PART 7    DIAGNOSTIC TESTS AND PROCEDURES IN RHEUMATIC DIEASES
149. Proost P, et al: Citrullination of CXCL8 by peptidylarginine deimi-
nase alters receptor usage, prevents proteolysis, and dampens tissue
inflammation. J Exp Med 205:2085–2097, 2008.
150. Struyf S, et al: Citrullination of CXCL12 differentially reduces
CXCR4 and CXCR7 binding with loss of inflammatory and anti-
HIV-1 activity via CXCR4. J Immunol 182:666–674, 2009.
151. Hagiwara T, Nakashima K, Hirano H, et al: Deimination of arginine
residues in nucleophosmin/B23 and histones in HL-60 granulocytes.
Biochem Biophys Res Commun 290:979–983, 2002.
152. Cuthbert GL, et al: Histone deimination antagonizes arginine meth-
ylation. Cell 118:545–553, 2004.
153. Hagiwara T, Hidaka Y, Yamada M: Deimination of histone H2A and
H4 at arginine 3 in HL-60 granulocytes. Biochemistry 44:5827–5834,
2005.
154. Wang Y, et al: Human PAD4 regulates histone arginine methylation
levels via demethylimination. Science 306:279–283, 2004.
155. Li P, et al: PAD4 is essential for antibacterial innate immunity medi-
ated by neutrophil extracellular traps. J Exp Med 207:1853–1862,
2010.
156. Wright PW, et al: ePAD, an oocyte and early embryo-abundant pep-
tidylarginine deiminase-like protein that localizes to egg cytoplasmic
sheets. Dev Biol 256:73–88, 2003.
157. Suzuki A, et al: Functional haplotypes of PADI4, encoding citrulli-
nating enzyme peptidylarginine deiminase 4, are associated with
rheumatoid arthritis. Nat Genet 34:395–402, 2003.
158. Kang CP, et al: A functional haplotype of the PADI4 gene associated
with increased rheumatoid arthritis susceptibility in Koreans. Arthritis
Rheum 54:90–96, 2006.
159. Barton A, et al: A functional haplotype of the PADI4 gene associated
with rheumatoid arthritis in a Japanese population is not associated
in a United Kingdom population. Arthritis Rheum 50:1117–1121,
2004.
160. Burr ML, et al: PADI4 genotype is not associated with rheumatoid
arthritis in a large UK Caucasian population. Ann Rheum Dis 69:666–
670, 2010.
161. Horikoshi N, et al: Structural and biochemical analyses of the human
PAD4 variant encoded by a functional haplotype gene. Acta Crystal-
logr D Biol Crystallogr 67:112–118, 2011.
162. Zhao J, Zhao Y, He J, et al: Prevalence and significance of anti-
peptidylarginine deiminase 4 antibodies in rheumatoid arthritis.
J Rheumatol 35:969–974, 2008.
163. Harris ML, et al: Association of autoimmunity to peptidyl arginine
deiminase type 4 with genotype and disease severity in rheumatoid
arthritis. Arthritis Rheum 58:1958–1967, 2008.
164. Bang SY, et al: Peptidyl arginine deiminase type IV (PADI4) haplo-
types interact with shared epitope regardless of anti-cyclic citrulli-
nated peptide antibody or erosive joint status in rheumatoid arthritis:
a case control study. Arthritis Res Ther 12:R115, 2010.
165. Suzuki T, et al: PADI4 and HLA-DRB1 are genetic risks for
radiographic progression in RA patients, independent of ACPA
status: results from the IORRA cohort study. PLoS One 8:e61045,
2013.
166. Halvorsen EH, et al: Serum IgG antibodies to peptidylarginine deim-
inase 4 in rheumatoid arthritis and associations with disease severity.
Ann Rheum Dis 67:414–417, 2008.
167. Halvorsen EH, et al: Serum IgG antibodies to peptidylarginine deim-
inase 4 predict radiographic progression in patients with rheumatoid
arthritis treated with tumour necrosis factor-alpha blocking agents.
Ann Rheum Dis 68:249–252, 2009.
168. Giles JT, et al: Association of cross-reactive antibodies targeting
peptidyl-arginine deiminase 3 and 4 with rheumatoid arthritis-
associated interstitial lung disease. PLoS One 9:e98794, 2014.
169. Ferrari-Lacraz S, et al: Contact with stimulated T cells up-regulates
expression of peptidylarginine deiminase 2 and 4 by human mono-
cytes. Eur Cytokine Netw 23:36–44, 2012.
170. Khandpur R, et al: NETs are a source of citrullinated autoantigens
and stimulate inflammatory responses in rheumatoid arthritis. Sci
Transl Med 5:178ra40, 2013.
171. Wang Y, et al: Histone hypercitrullination mediates chromatin
decondensation and neutrophil extracellular trap formation. J Cell
Biol 184:205–213, 2009.
172. Keefe D, et al: Perforin triggers a plasma membrane-repair response
that facilitates CTL induction of apoptosis. Immunity 23:249–262,
2005.
173. Damgaard D, Senolt L, Nielsen MF, et al: Demonstration of extracel-
lular peptidylarginine deiminase (PAD) activity in synovial fluid of
patients with rheumatoid arthritis using a novel assay for citrullina-
tion of fibrinogen. Arthritis Res Ther 16:498, 2014.
174. Shi J, et al: Autoantibodies recognizing carbamylated proteins are
present in sera of patients with rheumatoid arthritis and predict joint
damage. Proc Natl Acad Sci U S A 108:17372–17377, 2011.
175. Wang Z, et al: Protein carbamylation links inflammation, smoking,
uremia and atherogenesis. Nat Med 13:1176–1184, 2007.
176. Holzer M, et al: Myeloperoxidase-derived chlorinating species induce
protein carbamylation through decomposition of thiocyanate and
urea: novel pathways generating dysfunctional high-density lipopro-
tein. Antioxid Redox Signal 17:1043–1052, 2012.
177. Shi J, et al: Anti-carbamylated protein (anti-CarP) antibodies pre-
cede the onset of rheumatoid arthritis. Ann Rheum Dis 73:780–783,
2014.
178. Shi J, et al: Anti-carbamylated protein antibodies are present in
arthralgia patients and predict the development of rheumatoid arthri-
tis. Arthritis Rheum 65:911–915, 2013.
179. Gan RW, et al: Anti-carbamylated protein antibodies are present
prior to rheumatoid arthritis and are associated with its future diag-
nosis. J Rheumatol 42:572–579, 2015.
180. Thiele GM, et al: Malondialdehyde-acetaldehyde adducts and anti-
malondialdehyde-acetaldehyde antibodies in rheumatoid arthritis.
Arthritis Rheumatol 67:645–655, 2015.
181. Hill GE, et al: Association of malondialdehyde-acetaldehyde (MAA)
adducted proteins with atherosclerotic-induced vascular inflamma-
tory injury. Atherosclerosis 141:107–116, 1998.
182. Tuma DJ: Role of malondialdehyde-acetaldehyde adducts in liver
injury. Free Radic Biol Med 32:303–308, 2002.
183. Niemela O: Aldehyde-protein adducts in the liver as a result
of ethanol-induced oxidative stress. Front Biosci 4:D506–D513,
1999.
184. Ayala A, Munoz MF, Arguelles S: Lipid peroxidation: production,
metabolism, and signaling mechanisms of malondialdehyde and
4-hydroxy-2-nonenal. Oxid Med Cell Longev 2014:360438, 2014.
185. Tuma DJ, Thiele GM, Xu D, et al: Acetaldehyde and malondialde-
hyde react together to generate distinct protein adducts in the liver
during long-term ethanol administration. Hepatology 23:872–880,
1996.
186. Tuma DJ, Newman MR, Donohue TM Jr, et al: Covalent binding of
acetaldehyde to proteins: participation of lysine residues. Alcohol Clin
Exp Res 11:579–584, 1987.
187. Anderson DR, et al: Unique antibody responses to malondialdehyde-
acetaldehyde (MAA)-protein adducts predict coronary artery disease.
PLoS One 9:e107440, 2014.
188. Rolla R, et al: Detection of circulating antibodies against
malondialdehyde-acetaldehyde adducts in patients with alcohol-
induced liver disease. Hepatology 31:878–884, 2000.
189. Vehkala L, et al: Plasma IgA antibody levels to malondialdehyde
acetaldehyde-adducts are associated with inflammatory mediators,
obesity and type 2 diabetes. Ann Med 45:501–510, 2013.
190. Darrah E, Andrade F: Editorial: citrullination, and carbamylation,
and malondialdehyde-acetaldehyde! Oh my! Entering the forest of
autoantigen modifications in rheumatoid arthritis. Arthritis Rheuma-
tol. 67:604–608, 2015.
191. Hassfeld W, et al: Demonstration of a new antinuclear antibody
(anti-RA33) that is highly specific for rheumatoid arthritis. Arthritis
Rheum 32:1515–1520, 1989.
192. Steiner G, et al: Purification and partial sequencing of the nuclear
autoantigen RA33 shows that it is indistinguishable from the A2
protein of the heterogeneous nuclear ribonucleoprotein complex.
J Clin Invest 90:1061–1066, 1992.
193. Steiner G, Skriner K, Hassfeld W, et al: Clinical and immunological
aspects of autoantibodies to RA33/hnRNP-A/B proteins—a link
between RA, SLE and MCTD. Mol Biol Rep 23:167–171, 1996.
194. Meyer O, et al: Anti-RA 33 antinuclear autoantibody in rheumatoid
arthritis and mixed connective tissue disease: comparison with anti-
keratin and antiperinuclear antibodies. Clin Exp Rheumatol 11:473–
478, 1993.
195. Skriner K, et al: Anti-A2/RA33 autoantibodies are directed to the
RNA binding region of the A2 protein of the heterogeneous nuclear
ribonucleoprotein complex—differential epitope recognition in
rheumatoid arthritis, systemic lupus erythematosus, and mixed con-
nective tissue disease. J Clin Invest 100:127–135, 1997.
196. Nell-Duxneuner V, et al: Autoantibody profiling in patients with
very early rheumatoid arthritis: a follow-up study. Ann Rheum Dis
69:169–174, 2010.
 CHAPTER 56    Autoantibodies in Rheumatoid Arthritis 845.e5
197. Fritsch R, et al: Characterization of autoreactive T cells to the auto-
antigens heterogeneous nuclear ribonucleoprotein A2 (RA33) and
filaggrin in patients with rheumatoid arthritis. J Immunol 169:1068–
1076, 2002.
198. Kouskoff V, et al: Organ-specific disease provoked by systemic auto-
immunity. Cell 87:811–822, 1996.
199. Kouskoff V, et al: A new mouse model of rheumatoid arthritis: organ-
specific disease provoked by systemic autoimmunity. Ryumachi 37:
147, 1997.
200. Korganow AS, et al: From systemic T cell self-reactivity to organ-
specific autoimmune disease via immunoglobulins. Immunity 10:451–
461, 1999.
201. Maccioni M, et al: Arthritogenic monoclonal antibodies from K/
BxN mice. J Exp Med 195:1071–1077, 2002.
202. Schaller M, Burton DR, Ditzel HJ: Autoantibodies to GPI in rheu-
matoid arthritis: linkage between an animal model and human
disease. Nat Immunol 2:746–753, 2001.
203. Kassahn D, Kolb C, Solomon S, et al: Few human autoimmune sera
detect GPI. Nat Immunol 3:411–412, 2002.
204. Schubert D, Schmidt M, Zaiss D, et al: Autoantibodies to GPI and
creatine kinase in RA. Nat Immunol 3:411–413, 2002.
205. Matsumoto I, et al: Low prevalence of antibodies to glucose-6-
phosphate isomerase in patients with rheumatoid arthritis and a
spectrum of other chronic autoimmune disorders. Arthritis Rheum
48:944–954, 2003.
206. Aho K, et al: Antifilaggrin antibodies within “normal” range predict
rheumatoid arthritis in a linear fashion. J Rheumatol 27:2743–2746,
2000.
207. Zendman AJ, Vossenaar ER, van Venrooij WJ: Autoantibodies to
citrullinated (poly)peptides: a key diagnostic and prognostic marker
for rheumatoid arthritis. Autoimmunity 37:295–299, 2004.
208. Kokkonen H, Johansson M, Innala L, et al: The PTPN22 1858C/T
polymorphism is associated with anti-cyclic citrullinated peptide
antibody-positive early rheumatoid arthritis in northern Sweden.
Arthritis Res Ther 9:R56, 2007.
209. van der Helm-van Mil AH, et al: The HLA-DRB1 shared epitope
alleles are primarily a risk factor for anti-cyclic citrullinated peptide
antibodies and are not an independent risk factor for development
of rheumatoid arthritis. Arthritis Rheum 54:1117–1121, 2006.
210. van der Helm-van Mil AH, Wesoly JZ, Huizinga TW: Understanding
the genetic contribution to rheumatoid arthritis. Curr Opin Rheuma-
tol 17:299–304, 2005.
211. Kapitany A, et al: Associations between serum anti-CCP antibody,
rheumatoid factor levels and HLA-DR4 expression in Hungarian
patients with rheumatoid arthritis. Isr Med Assoc J 10:32–36, 2008.
212. Scally SW, et al: A molecular basis for the association of the HLA-
DRB1 locus, citrullination, and rheumatoid arthritis. J Exp Med
210:2569–2582, 2013.
213. Zhao X, et al: Circulating immune complexes contain citrulli-
nated fibrinogen in rheumatoid arthritis. Arthritis Res Ther 10:R94,
2008.
214. Clavel C, et al: Induction of macrophage secretion of tumor necrosis
factor alpha through Fcgamma receptor IIa engagement by rheuma-
toid arthritis-specific autoantibodies to citrullinated proteins com-
plexed with fibrinogen. Arthritis Rheum 58:678–688, 2008.
215. Sokolove J, Zhao X, Chandra PE, et al: Immune complexes contain-
ing citrullinated fibrinogen costimulate macrophages via Toll-like
receptor 4 and Fcgamma receptor. Arthritis Rheum 63:53–62, 2011.
216. Rombouts Y, et al: Extensive glycosylation of ACPA-IgG variable
domains modulates binding to citrullinated antigens in rheumatoid
arthritis. Ann Rheum Dis pii:annrheumdis-2014-206598, Jan 13,
2015. [Epub ahead of print].
217. Rombouts Y, et al: Anti-citrullinated protein antibodies acquire a
pro-inflammatory Fc glycosylation phenotype prior to the onset of
rheumatoid arthritis. Ann Rheum Dis 74:234–241, 2015.
218. Pratesi F, Tommasi C, Anzilotti C, et al: Deiminated Epstein-Barr
virus nuclear antigen 1 is a target of anti-citrullinated protein anti-
bodies in rheumatoid arthritis. Arthritis Rheum 54:733–741, 2006.
219. Shi J, Sun X, Zhao Y, et al: Prevalence and significance of antibodies
to citrullinated human papilloma virus-47 E2345-362 in rheumatoid
arthritis. J Autoimmun 31:131–135, 2008.
220. Sjostrom L, Laurell L, Hugoson A, et al: Periodontal conditions in
adults with rheumatoid arthritis. Community Dent Oral Epidemiol
17:234–236, 1989.
221. Kasser UR, et al: Risk for periodontal disease in patients with long-
standing rheumatoid arthritis. Arthritis Rheum 40:2248–2251, 1997.
222. Mercado FB, Marshall RI, Klestov AC, et al: Relationship between
rheumatoid arthritis and periodontitis. J Periodontol 72:779–787,
2001.
223. de Pablo P, Chapple IL, Buckley CD, et al: Periodontitis in systemic
rheumatic diseases. Nat Rev Rheumatol 5:218–224, 2009.
224. Lundberg K, Wegner N, Yucel-Lindberg T, et al: Periodontitis in
RA-the citrullinated enolase connection. Nat Rev Rheumatol 6:
727–730, 2010.
225. Rosenstein ED, Greenwald RA, Kushner LJ, et al: Hypothesis: the
humoral immune response to oral bacteria provides a stimulus for the
development of rheumatoid arthritis. Inflammation 28:311–318,
2004.
226. McGraw WT, Potempa J, Farley D, et al: Purification, characteriza-
tion, and sequence analysis of a potential virulence factor from Por-
phyromonas gingivalis, peptidylarginine deiminase. Infect Immun 67:
3248–3256, 1999.
227. Lundberg K, et al: Antibodies to citrullinated alpha-enolase peptide
1 are specific for rheumatoid arthritis and cross-react with bacterial
enolase. Arthritis Rheum 58:3009–3019, 2008.
228. Wegner N, et al: Peptidylarginine deiminase from Porphyromonas
gingivalis citrullinates human fibrinogen and alpha-enolase: implica-
tions for autoimmunity in rheumatoid arthritis. Arthritis Rheum
62:2662–2672, 2010.
229. Quirke AM, et al: Heightened immune response to autocitrullinated
Porphyromonas gingivalis peptidylarginine deiminase: a potential
mechanism for breaching immunologic tolerance in rheumatoid
arthritis. Ann Rheum Dis 73:263–269, 2014.
230. Konig MF, Paracha AS, Moni M, et al: Defining the role of Porphy-
romonas gingivalis peptidylarginine deiminase (PPAD) in rheumatoid
arthritis through the study of PPAD biology. Ann Rheum Dis pii:ann
rheumdis-2014-205385, May 26, 2014. [Epub ahead of print].
231. Konig MF, Bingham CO III, Andrade F: PPAD is not targeted as a
citrullinated protein in rheumatoid arthritis, but remains a candidate
for inducing autoimmunity. Ann Rheum Dis 74:e8, 2015.
232. Klareskog L, et al: A new model for an etiology of rheumatoid
arthritis: smoking may trigger HLA-DR (shared epitope)-restricted
immune reactions to autoantigens modified by citrullination. Arthritis
Rheum 54:38–46, 2006.
233. Linn-Rasker SP, et al: Smoking is a risk factor for anti-CCP antibod-
ies only in rheumatoid arthritis patients who carry HLA-DRB1
shared epitope alleles. Ann Rheum Dis 65:366–371, 2006.
234. Klareskog L, Malmstrom V, Lundberg K, et al: Smoking, citrullina-
tion and genetic variability in the immunopathogenesis of rheuma-
toid arthritis. Semin Immunol 23:92–98, 2011.
235. Svard A, Kastbom A, Reckner-Olsson A, et al: Presence and utility
of IgA-class antibodies to cyclic citrullinated peptides in early rheu-
matoid arthritis: the Swedish TIRA project. Arthritis Res Ther 10:R75,
2008.
 CHAPTER 57 
Acute Phase Reactants and the
Concept of Inflammation
César E. Fors Nieves • Bruce N. Cronstein • Amit Saxena
KEY POINTS
Inflammation comprises a complex and highly variable set of
processes that represent a response to tissue damage from
infection or injury.
The acute phase response, a major accompaniment of
inflammation, is induced by inflammation-associated
cytokines and includes a reorchestration of acute phase
protein synthesis by the liver.
The erythrocyte sedimentation rate (ESR), the most
commonly used clinical measure of inflammation, depends
on numerous physical and chemical characteristics of blood,
many of which are not related to inflammation.
The quintessential acute phase protein, C-reactive protein
(CRP), not only offers biomarker utility in the clinic, but also
plays a role in host defense by recognition of biologic
substrates, activation of the complement pathway, and by
binding to leukocytes.
Cytokines, chemokines, adhesion molecules, and other
products of activated inflammatory cells are secreted during
inflammation and play roles in the inflammatory response,
but several problems limit the clinical usefulness of their
quantitation for routine clinical purposes.
CRP and ESR may reflect disease activity and correlate with
disease prognosis in rheumatoid arthritis but generally are
not helpful for differential diagnosis.
Although CRP and ESR correlate with clinical activity in many
inflammatory rheumatic diseases, the absent or modest CRP
response seen in some patients with active systemic lupus
erythematosus remains unexplained.
CRP or ESR is elevated in more than 80% of patients with
polymyalgia rheumatica and in approximately 95% of
patients with giant cell arteritis. Both are useful for disease
follow-up, but their imperfect correlation with disease
activity indicates that these measures cannot supplant sound
clinical judgment.
Minor elevations of CRP, within the normal population
reference range, are associated with increased risk of
myocardial infarction but are nonspecific (especially in
patients with rheumatic disease). Minor CRP elevation is
associated with well-recognized risk factors for cardiovascular
disease, which may explain its predictive value.
The inflammatory response is the body’s natural defense
against unchecked tissue damage from infection or injury.
It occurs during the acute phase of an inciting event and,
if the stimulus is not eliminated, in a chronic, putatively
healing stage. When excessive or uncontrolled, these
846
responses have the potential to cause significant harm to
the host through processes such as autoimmune diseases,
allergic reactions, and septic shock.
The concept of inflammation itself has evolved over
thousands of years. Celsus first described the cardinal signs
of redness, swelling, heat, and pain in the first century ad.
The fifth sign, loss of function, is often attributed to Galen’s
work in second century.1 Since that time, countless advances
have been made in the understanding of inflammation, from
histologic findings of inflammatory cells within tissues, to
the discovery of hematologic and soluble mediators such as
cytokines and complement. Also, the molecular signaling
pathways that drive both its protective effects and the inap-
propriate injurious responses have been recently elucidated.
With increasing insight into mechanisms of the inflam-
matory response has come an appreciation of its significant
complexity. Molecular and microscopic processes are differ-
ent during acute and chronic stages, and diverse responses
are induced by various types of exogenous and endogenous
stimuli (e.g., bacteria, viruses, parasites, crystals, allergens,
and ischemia). More recently, the role of the milieu of the
inflammatory response, especially at the level of the vascu-
lar endothelium, has taken on considerable importance.2
Furthermore, inherent redundancies of functions have been
noted, as have interactions between the mediators of inflam-
mation that allow for a broad, effective response, but these
redundancies make it difficult to understand the pathways
of inflammation in a linear fashion. These intricacies have
resulted in a vague definition and on continued reliance on
the final downstream macroscopic cardinal signs to tie
together all of the ongoing processes. It is these same intri-
cacies that have made evaluation of inflammation through
laboratory tests imprecise.
Basic hematologic abnormalities may give clues to the
presence of inflammation, but different patterns are often
associated with underlying causes and are not universally
found. Leukocytosis can be seen in infections, acute crystal
diseases, and some autoimmune disorders such as adult
Still’s disease. Anemia is often associated with certain dis-
eases that cause chronic inflammation, such as rheumatoid
arthritis (RA). Reactive thrombocytosis occurs secondary
to the release of cytokines after an inciting infectious or
inflammatory event, and the role of platelets and platelet-
derived mediators in stimulating inflammation has been
described at the molecular level.3
Laboratory tests most commonly used by physicians to
obtain information about the extent of inflammation are
those that measure the acute phase reaction. In response
to injury, local inflammatory cells secrete cytokines that
 CHAPTER 57    Acute Phase Reactants and the Concept of Inflammation 847

influence the liver to increase or decrease production of TABLE 57-1  Human Acute Phase Proteins
various proteins. The erythrocyte sedimentation rate (ESR) Plasma Proteins that Increase in Inflammation
has been the classic marker of inflammation, with serum Complement System
C-reactive protein (CRP) taking on an increasingly promi-   C3
nent role. Other novel inflammatory markers such as pro-   C4
  C9
calcitonin have been recognized, but their clinical utility  Factor B
has yet to be fully determined.   C1 inhibitor
CRP elevations, especially minor ones, have been noted   C4b-binding protein
in numerous conditions that traditionally have not been  Mannose-binding lectin
considered inflammatory, most significantly involving the Coagulation and fibrinolytic system
cardiovascular system. This has shed light on subclinical  Fibrinogen
inflammation as a possible factor in the pathogenesis of a   Plasminogen
  Tissue plasminogen activator
great number of diseases.  Urokinase
  Protein S
  Vitronectin
ACUTE PHASE RESPONSE   Plasminogen-activator inhibitor 1
Within minutes of tissue injury, activation of the innate Anti-proteases
immune system induces cytokine production that results in   α-Protease inhibitor
a multisystem acute phase response involving the liver, vas-   α-Anti-chymotrypsin
  Pancreatic secretory trypsin inhibitor
cular system, bone marrow, and central nervous system.4,5   Inter-α-trypsin inhibitors
Many elements of the reaction can be regarded as part of
the innate response and are defensive or adaptive in nature.6 Transport proteins
  Ceruloplasmin
Mouse studies have shown that as much as 7% of the regula-   Haptoglobin
tory gene pool undergoes significant changes in expression   Hemopexin
during inflammation, and that induction of liver acute Participants in inflammatory responses
phase genes is mediated by the transcription factor signal  Secreted phospholipase A2
transducer and activator of transcription 3 (STAT3).7-9  Lipopolysaccharide-binding protein
Although the acute phase response can trigger numerous   Interleukin-1–receptor antagonist
neuroendocrine, hematopoietic, and metabolic effects, the   Granulocyte colony-stimulating factor
changes in plasma proteins synthesized by hepatocytes are Others
monitored as signs of underlying inflammation (Tables 57-1   C-reactive protein
 Serum amyloid A
and 57-2). An acute phase protein is one in which the   α-Acid glycoprotein
plasma concentration changes from baseline by at least 25%  Fibronectin
during inflammation; responses vary in terms of concentra-  Ferritin
tion and kinetics (Figure 57-1).10 CRP and serum amyloid   Angiotensinogen
A (SAA) levels increase more than 1000-fold during acute Plasma Proteins that Decrease in Inflammation
infection and peak at 2 to 3 days. Concentrations of other Albumin
proteins peak at longer periods and can range from a 50% Transferrin
Transthyretin
increase in complement and ceruloplasmin, to a several-fold α-HS glycoprotein
amplification in haptoglobin, fibrinogen, α1-proteinase Alpha-fetoprotein
inhibitor, and α1-acid glycoprotein. Other proteins are Thyroxine-binding globulin
negative acute phase proteins, the concentrations of which Insulin-like growth factor I
Factor XII
fall during the inflammatory response. These include anti-
thrombin III, protein S, prealbumin, albumin, transferrin, HS, Heremans-Schmid.
and apolipoprotein A-I.4,5 From Gabay C, Kushner I: Acute-phase proteins and other systemic
Hepatic stimulation of acute phase proteins is induced responses to inflammation. N Engl J Med 340:448–454, 1999.
by cytokines released by activated monocytes, macrophages,
neutrophils, natural killer (NK) cells, and endothelial cells
acting at the front lines of the inflammatory response. The and interactions of cytokines.5 The roles of the acute phase
main cytokine influencing the liver is IL-6, once called the proteins themselves will be discussed throughout the chapter
“hepatocyte-stimulating factor.” It likely mediates protein but have been found to include direct involvement in
expression via the Janus kinase (JAK) and STAT3 path- host defense by activation of the complement, proteinase
ways, as well as C/EBP family members and Rel proteins inhibition, and antioxidant activity.14 However, some of the
(nuclear factor-κB [NF-κB]).9,11 During initial stages, IL-1 described in vitro effects of proteins may not be relevant
and TNF synergize with IL-6 and trigger further IL-6 pro- in vivo.
duction, but their roles are limited.12 The soluble IL-6
receptor amplifies IL-6 effects both locally and systemically. Erythrocyte Sedimentation Rate
IL-6 also performs a protective role during disease, inducing
the expression of an IL-1 receptor antagonist.13 Although ESR is an indirect screen for elevated concentra-
Acute phase protein levels are not uniform in their tions of acute phase proteins, it has been the most widely
expression; this is likely related to the underlying patho- used marker of inflammation for almost a century. Measure-
physiologic state and is regulated by different combinations ment of ESR is performed when blood is placed in a vertical
 848 PART 7    DIAGNOSTIC TESTS AND PROCEDURES IN RHEUMATIC DISEASES

TABLE 57-2 Other Acute Phase Phenomena tube and the rate of fall of erythrocytes is measured. The
Neuroendocrine Changes ancient Greeks recognized increased red blood cell (RBC)
Fever, somnolence, and anorexia sedimentation as a way to detect “bad bodily humors,” but
Increased secretion of corticotropin-releasing hormone, our modern understanding and use of RBC sedimentation
corticotropin, and cortisol as a test date back to the German scholar Fahraeus in
Increased secretion of arginine vasopressin
Decreased production of insulin-like growth factor I 1918.15 He determined that certain plasma proteins, espe-
Increased adrenal secretion of catecholamines cially fibrinogen, are able to lower the electrostatic charge
Hematopoeitic Changes
on RBC surfaces so they can aggregate, form rouleaux, and
Anemia of chronic disease fall faster.
Leukocytosis Several factors are involved in acceleration of ESR.
Thrombocytosis Asymmetric plasma proteins, such as fibrinogen and, to a
Metabolic Changes lesser extent, alpha2, beta, and gamma globulins decrease
Loss of muscle and negative nitrogen balance the negative charge of erythrocytes (zeta potential) that
Decreased gluconeogenesis prevents rouleaux formation. Red cell factors also play a role
Osteoporosis
Increased hepatic lipogenesis
in that changes in plasma ratios in anemic states also favor
Increased lipolysis in adipose tissue rouleaux. However, microcytosis, polycythemia, and abnor-
Decreased lipoprotein lipase activity in muscle and adipose tissue mally shaped RBCs (e.g., sickle cells and spherocytes)
Cachexia hinder aggregation and lower the ESR.16 Conditions that
Hepatic Changes elevate fibrinogen, even if they are not necessarily consid-
Increased metallothionein, inducible nitric oxide synthase, heme ered inflammatory, can raise ESR. These include pregnancy,
oxygenase, manganese superoxide dismutase, and tissue inhibitor diabetes, end-stage renal disease, and heart disease. Major
of metalloproteinase-1
Decreased phosphoenolpyruvate carboxykinase activity
increases in the concentration of a single molecular species,
such as a monoclonal immunoglobulin in multiple myeloma,
Changes in Nonprotein Plasma Constituents
Hypozincemia, hypoferremia, and hypercupremia
also cause increased sedimentation.17 The ESR is elevated
Increased plasma retinol and glutathione concentrations in obesity, as is CRP, presumably as a result of IL-6 secretion
by adipocytes.18 Factors such as glucocorticoids, cryoglobu-
From Gabay C, Kushner I: Acute-phase proteins and other systemic lins, hypofibrinogenemia, and hyperviscosity lower the
responses to inflammation. N Engl J Med 340:448–454, 1999. value.4 The physiochemical dynamics that allow for sedi-
mentation has been a continued source of debate, with
disparate models presented to explain how proteins on cell
surfaces interact to cause RBC aggregation.19
Although novel and rapid tests for ESR have proven
30,100 promising, the International Committee for Standardiza-
tion in Hematology continues to recommend the Wester-
30,000
gren technique of testing anti-coagulated blood.20,21 The
700 usual accepted upper limits of normal are 15 mm/hr for
males and 20 mm/hr for females; however, the ESR increases
600 C-reactive protein with age and varies by race, which calls the reliability of the
Plasma concentration

test into question. A simple formula for calculating the

500 upper limit of normal ESR at any age has been used regu-
(% change)

Serum amyloid A larly. In men, age in years divided by 2; in women, 10 plus

400 age in years divided by 2. Despite the ability to control for
300
age, other limitations of the test have been noted and are
Haptoglobin listed in Table 57-3. The relative virtues of CRP determina-
Fibrinogen tion have diminished some of the importance of ESR, but
200
it remains an easy, inexpensive test with a wealth of back-
100 C3 ground literature. Therefore the sedimentation rate contin-
ues to play a prominent role in clinical practice.
0
Albumin Transferrin
C-Reactive Protein
0 7 14 21
C-reactive protein is an acute phase protein, the serum
Inflammatory Days concentration of which reflects ongoing inflammation
stimulus
better than other tests in most, but not all, diseases.22 CRP
Figure 57-1  Typical plasma acute phase protein changes after a mod- was identified in 1930, when sera obtained from patients
erate inflammatory stimulus. Several patterns of response are seen:
major acute phase protein, increase 100-fold (e.g., C-reactive protein, with Streptococcus pneumonia infection were found to
serum amyloid A); moderate acute phase protein, increase twofold to contain a protein that could bind to the “C” polysaccharide
fourfold (e.g., fibrinogen and haptoglobin); minor acute phase protein, of the bacterial cell wall. This protein circulates as a 115 kDa
increase 50% to 100% (e.g., complement C3); and negative acute phase pentamer of noncovalently linked 23 kDa subunits, which
protein, decrease (e.g., albumin, transferrin). (Modified from Gitlin JD,
Colten HR: Molecular biology of the acute-phase plasma proteins. In Pick E,
has been highly conserved during hundreds of millions of
Landy M, editors: Lymphokines, vol 14, San Diego, 1987, Academic Press, years of evolution. In contrast to immunoglobulins and
pp 123–153.) complement components, CRP deficiency in humans has
 CHAPTER 57    Acute Phase Reactants and the Concept of Inflammation 849
TABLE 57-3  Comparison of Erythrocyte Sedimentation Rate and C-Reactive Protein
Erythrocyte Sedimentation Rate
Advantages Much clinical information in the literature
May reflect overall health status
Disadvantages Affected by red blood cell morphology
Affected by anemia and polycythemia
Reflects levels of many plasma proteins, not all of which are
acute phase proteins
Responds slowly to inflammatory stimuli
Requires fresh sample
May be affected by drugs
not been described. Genome-wide associated studies per-
formed recently have shown that at least seven distinct loci
are involved in the basal expression of CRP,23-25 which is
upregulated upon stimulation by the transcription factors C/
EBP and Rel.26 It is present in trace concentrations in the
plasma of all humans (roughly 1 mg/L, with higher concen-
trations in women and the elderly). Plasma C-reactive
protein is synthesized by hepatocytes, although other sites
of local production and possibly minimal secretion have
been suggested.
The precise function of CRP is unknown and may be
varied, but it exhibits important recognition and activation
capabilities, and it binds to numerous ligands.27 CRP recog-
nizes phosphocholine, phospholipids, fibronectin, chroma-
tin, and histones, all of which are exposed at sites of tissue
damage and by apoptotic cells; CRP may target them for
clearance.28 C-reactive protein bridges the gap between
innate and adaptive immunity by activating the classic
complement pathway and interacting with cells of the
immune system through binding of Fcγ receptors.29,30 CRP
induces inflammatory cytokines, tissue factors, and shedding
of the IL-6 receptor, all of which result in a complement-
dependent increase in tissue damage.28 Other CRP func-
tions are anti-inflammatory, including promotion of the
non-inflammatory clearance of apoptotic cells and preven-
tion of neutrophil adhesion to the endothelium.31,32 Thus
CRP may play many pathophysiologic roles during the
course of the inflammatory process.14,33
After an acute inflammatory stimulus, CRP concentra-
tion increases rapidly and peaks at 2 to 3 days at levels that
reflect the extent of tissue injury. If the stimulus has been
removed, serum CRP levels drop rapidly, with a half-life of
roughly 19 hours.34 Persistent elevations in CRP are seen in
chronic inflammatory states such as active RA, pulmonary
tuberculosis, or extensive malignant disease.
Immunoassays and laser nephelometry are used at modest
cost to quantify serum CRP levels. Most healthy adults have
levels less than 0.3 mg/dL. The significance of minor eleva-
tions in CRP is a subject of debate and will be discussed
subsequently. However, usual methods of CRP determina-
tion are less precise at concentrations in the range of 0.3 to
1 mg/dL, so high-sensitivity (hs) CRP methods are used to
accurately measure these levels. Generally, concentrations
greater than 1 mg/dL reflect clinically significant inflamma-
tory disease.35,36 Concentrations of 1 to 10 mg/dL can be
considered to represent moderate increases, and concentra-
C-Reactive Protein
Rapid response to inflammatory stimuli
Wide range of clinically relevant values are detectable
Unaffected by age and gender
Reflects value of a single acute phase protein
Can be measured on stored sera
Quantitation is precise and reproducible
Not sensitive to changes in SLE disease activity
TABLE 57-4  Conditions Associated with Elevated C-Reactive
Protein Levels
Normal or Minor Elevation (<1 mg/dL)
Vigorous exercise
Common cold
Pregnancy
Gingivitis
Seizures
Depression
Insulin resistance and diabetes
Several genetic polymorphisms
Obesity
Moderate Elevation (1 to 10 mg/dL)
Myocardial infarction
Malignancies
Pancreatitis
Mucosal infection (bronchitis, cystitis)
Most systemic autoimune diseases
Rheumatoid arthritis
Marked Elevation (>10 mg/dL)
Acute bacterial infection (80% to 85%)
Major trauma
Systemic vasculitis
tions greater than 10 mg/dL show marked increases. Most
patients with extremely high levels (e.g., >15 mg/dL) have
bacterial infection. One study found that in patients with
CRP concentrations greater than 50 mg/dL, infection was
present in 88% of participants.37 Clinical conditions associ-
ated with varying degrees of elevation of CRP are listed in
Table 57-4, and the range of CRP concentrations in many
rheumatologic diseases is shown in Figure 57-2.
Several limitations associated with the use of C-reactive
protein measurement must be acknowledged. No unifor-
mity in reporting concentrations has been noted between
laboratories, and values can be conveyed in mg/L, µg/mL,
or mg/dL. Similar to ESR, population studies show a skewed,
rather than gaussian, distribution, which renders parametric
statistical tests inappropriate for interpretation of CRP
data. Population differences in CRP levels in the United
States have been reported between sexes and among
racial groups. This is presumably also prevalent as an issue
in practice in other populations globally. Elevation of
C-reactive protein in the elderly may represent age-related
disorders, the pathogenesis of which may involve low-grade
inflammation, which complicates the issue of what levels
are considered normal.38
 850 PART 7    DIAGNOSTIC TESTS AND PROCEDURES IN RHEUMATIC DISEASES
Serum CRP Levels (mg/dL)
0.1 0.2 0.5
Normal
Osteoarthritis
Rheumatoid
arthritis
Gout
Lupus without
serositis
Lupus with
serositis
Spondylitis
Vasculitis
Adult Still’s
disease
Figure 57-2  Range of C-reactive protein (CRP) levels in rheumatic disease. Authors’ estimates of expected levels of CRP (mg/dL) in certain rheumatic
diseases.
Procalcitonin
Procalcitonin (PCT), a propeptide of calcitonin, has
recently received increased attention as a useful measure to
differentiate between acute bacterial infection and other
inflammatory and febrile syndromes. Studies have shown
that elevated serum levels of PCT have a significantly
higher specificity for infection than ESR and CRP.39-42 PCT
has been studied extensively as a clinical tool to facilitate
the decision as to when to start and stop anti-bacterial
agents in patients with pneumonia,43,44 and it has also shown
promise in differentiating between infectious and noninfec-
tious causes of fever after orthopedic surgery, in which ESR
and CRP levels can be difficult to interpret.45,46 For all of
these reasons, it remains an intriguing clinical tool for use
in patients with fevers in the setting of autoimmune disease
who are commonly on immunosuppressive medications and
at higher risk of infection.
Normally produced in the C-cells of the thyroid gland,
PCT is usually cleaved into calcitonin, katacalcin, and an
N-terminal residue. Under normal circumstances, PCT is
not released into the bloodstream; however, during severe
infection plasma, levels can increase dramatically.47,48 Inter-
estingly, this does not lead to an increase in plasma calcito-
nin levels or activity. At this time, the exact site of PCT
production, as well as its pathophysiologic role during sepsis,
remains uncertain.49
In healthy humans, PCT levels are undetectable or very
low (<0.1 ng/mL). During systemic bacterial infection,
PCT levels can increase to higher than 100 ng/mL. Cur-
rently available diagnostic tests measure the calcitonin/N-
ProCT part of the protein and therefore only a fragment of
the 114 to 116 amino-acid chain of the prohormone. A
useful reference range to exclude sepsis and systemic inflam-
mation is less than or equal to 0.2 ng/mL. Plasma levels of
greater than or equal to 0.5 ng/mL should be interpreted as
abnormal and suggest sepsis. After reaching peak levels,
circulating PCT has a half-life of approximately 22 to 35
hours in serum, and its concentration declines with a 50%
plasma-disappearance rate of roughly 1 to 1½ days. It is
1.0 2.0 5.0 10 20 50 100
worth noting that in patients with severe renal dysfunction
these rates may be prolonged, but accumulation does not
occur.50
As a biomarker for infection in autoimmune diseases, the
role of PCT has not yet been fully established. In contrast
to CRP, PCT levels are not elevated in in the majority of
cases of noninfectious inflammation or nonbacterial infec-
tion. However, exceptions to this rule include some vascu-
litis syndromes such as Kawasaki’s disease, Goodpasture’s
syndrome, adult-onset Still’s disease51,52 and granulomatosis
with polyangiitis,53 for which observational studies have
demonstrated elevated levels of PCT in patients without
evidence of bacterial infection. In systemic lupus erythema-
tosus, data are mixed, and no definitive statement regarding
the usefulness of PCT can be made at this time.54-56
Other Acute Phase Proteins
Measurement of other acute phase proteins has been of
limited value clinically because their responses to tissue
injury are often slower, and the magnitude of concentration
change is smaller than with CRP. Serum amyloid A (SAA),
a circulating family of proteins produced by hepatocytes,
adipocytes, macrophages, and fibroblast-like synoviocytes
has been correlated with disease activity in a number
of inflammatory disorders.57 However, reliable testing for
acute phase SAA is not widely available, and data about
levels expected in disease are limited. Serum ferritin is mod-
erately increased, triggered by cytokines such as IL-1, IL-6,
IL-18, and TNF. Levels are frequently high in adult-onset
Still’s disease and systemic lupus erythematosus (SLE), and
they correlate with disease activity.58,59 Hepcidin, a liver-
derived anti-microbial peptide and regulator of iron homeo-
stasis, is induced by inflammation, and by IL-6 in particular.60
Its levels rise in parallel with ferritin, and it is important in
the development of anemia of chronic disease, acting as a
negative regulator of iron absorption and macrophage iron
release teleologically, to deprive microbes of iron.61 Trans-
ferrin, which binds and transports iron, is a negative acute
phase protein.
 CHAPTER 57    Acute Phase Reactants and the Concept of Inflammation 851
Apolipoprotein A-I, the principal protein constituent of
high-density lipoprotein (HDL), is another negative acute
phase protein. In chronic inflammatory diseases such as RA
and SLE, decreased levels may contribute to increased risk
of thrombotic events.62,63 Serum albumin and prealbumin
(transthyretin) are also negative acute phase proteins,
although their measurement is not more helpful in diagnosis
or prognosis than standard tests.64 Serum complement frac-
tions become depressed when the system is activated in
certain autoimmune disorders but otherwise rise during the
acute phase response.
Cytokines
Although not acute phase proteins in the classic sense,
cytokines display the most striking acute phase behavior of
any circulating proteins. IL-6 responds dramatically to tissue
injury, with concentration changes that are faster and
greater than those of CRP or SAA. Acute inflammation and
chronic inflammation have been associated with increases
in IL-6, and serum levels of this cytokine have been cor-
related with the severity and course of disease in RA,
juvenile arthritis, ankylosing spondylitis, and polymyalgia
rheumatica (PMR).65,66 It is also more sensitive than ESR
for detecting disease activity in giant cell arteritis (GCA).67
IL-6 levels may be useful in monitoring inflammation if
hepatocytes are damaged to the point that they are not able
to synthesize acute phase proteins.4 Knowledge of soluble
IL-6 receptor levels may also be helpful in this regard. The
importance of cytokines such as TNF and IL-1 has been
inferred by the successful reduction of inflammation by their
therapeutic inhibitors. Certain diseases, such as the TNF
receptor–associated periodic syndrome (TRAPS) and the
autoinflammatory syndromes that involve mutations of the
inflammasome controlling IL-1, point to the importance of
these cytokines.
Increased levels of several other cytokines and circulat-
ing cytokine receptors have been associated with inflamma-
tion or disease activity as well (Table 57-5).67,68 Different
patterns of cytokine responses have been reported in differ-
ent diseases, suggesting that cytokine determinations are
potentially useful clinically.69,70 However, their quantitation
presents several problems related to their short plasma half-
TABLE 57-5  Products of Inflammatory, Endothelial, and
Resident Target Tissue Cells/Matrix
Cytokines and Related Molecules
Cytokines:
  IL-1
  IL-6
  IL-12
  IFN-α
  TNF
Granulocyte-macrophage colony-stimulating factor
IL-1 receptor antagonist
Products of Inflammatory and Endothelial Cells
Calprotectin
von Willebrand factor
Soluble adhesion molecules (e.g., sVCAM and sE-selectin)
Hyaluronic acid
Collagen and aggrecan degradation products
Osteocalcin
sVCAM, Soluble vascular cell adhesion molecule.
lives, the presence of blocking factors and natural inhibi-
tors, and other technical considerations.71 At present, high
costs, limited availability, and the absence of standardiza-
tion discourage the measurement of plasma cytokines and
their receptors in clinical practice.
ACUTE PHASE REACTANTS IN THE
MANAGEMENT OF RHEUMATIC DISEASES
Measurement of ESR and CRP has no role in the diagnosis
of any particular disease, including RA, osteoarthritis, SLE,
PMR, GCA, or other inflammatory arthropathies. However,
these measurements can be clinically helpful in three ways:
(1) in evaluating the extent or severity of inflammation, (2)
in monitoring changes in disease activity over time, and (3)
in assessing prognosis.
Rheumatoid Arthritis
ESR and CRP cannot be used in the diagnosis of RA,
because 45% of patients may have normal serum levels at
presentation,72 although these values represent part of the
diagnostic syndrome or classification criteria sets. These
tests are more appropriately applied in RA for monitoring
disease activity and response to therapy. Although ESR
traditionally has been more widely used for these purposes,
many studies have suggested that CRP levels correlate
better with disease activity.73 Some recent reports state that
CRP levels may overestimate disease response compared
with ESR; others claim that differences between the two are
minimal.73-75 The existence of patients with depressed CRP
concentrations caused by carrying low-CRP–associated
genetic variants must be taken into account when this test
is used universally.76 Matrix metalloproteinase (MMP)-3,
pro-MMP-3, and soluble E-selectin have also been proposed
as markers for RA disease activity. Their measurements cor-
relate with CRP levels but do not provide more information
than is provided by standard tests.77-79
CRP levels average 2 to 3 mg/dL in adult patients with
RA who have moderate disease activity.80 However, varia-
tion is considerable. At least 5% to 10% of patients have
values in the normal range, whereas a few patients with
severe disease activity have levels greater than 10 mg/dL.
ESR values have been found to remain stable over the
years.81 ESR and CRP have long been used to follow the
response to therapy; in general, effective disease-modifying
anti-rheumatic drug therapy decreases CRP levels by
approximately 40%. Inhibition of joint damage by these
agents usually is accompanied by marked improvement in
acute phase reactants. Progression of joint damage can
occur while the patient is on therapy, however, despite
decreases in ESR and CRP.82 Even more striking improve-
ment has been seen with biologic agents introduced since
the 1990s, providing objective laboratory support for the
encouraging clinical responses observed. In early reports of
anti-TNF therapy, CRP and SAA levels declined by 75%
and 85%, respectively, in approximately 1 week.83 Treat-
ment with abatacept, the T cell CD80/CD86:CD28
co-stimulation modulator, resulted in significant decreases
in CRP at both 90 and 360 days of therapy.84 Tofacitinib, a
JAK inhibitor, also appeared to effectively decrease DAS28-
ESR scores in RA patients in clinical trials; therefore it may
 852 PART 7    DIAGNOSTIC TESTS AND PROCEDURES IN RHEUMATIC DISEASES
be a useful tool to monitor disease activity.85 In one study,
failure to suppress CRP levels 2 weeks after initiation of
infliximab therapy identified most patients who would prove
to be clinical nonresponders after 12 weeks.86 In contrast to
traditional disease-modifying anti-rheumatic drugs, TNF
inhibitors have been found to inhibit joint damage even
while clinical activity, reflected by CRP levels, remains
high.87 Tocilizumab, a human IL-6 receptor antibody,
improves RA by inhibiting effects of the cytokine. However,
because of its mechanism of action, inflammatory markers
such as ESR and CRP drop to negative values, so that track-
ing them may not reflect the actual effect of the drug; care
must be taken when monitoring disease activity during
tocilizumab therapy.88,89
ESR and CRP also have value as prognostic indicators in
RA. Elevated acute phase reactant levels are associated with
early synovitis and erosions as detected by magnetic reso-
nance imaging, with inflammatory cellular infiltrates in
synovium, and with osteoclastic activation and reduced
bone mineral density.90-92 CRP predicts radiographic pro-
gression, as do ESR and the matrix metalloproteinases
MMP-3 and MMP-1.77,78,93-95 Finally, and perhaps most im-
portant, acute phase reactants correlate with work disability
on long-term follow-up and predict progression to major
joint replacement.96,97 As in the normal population, CRP
levels are associated with death from cardiovascular disease.98
In patients with RA in whom heart failure developed, ESR
was higher during the 6-month period immediately preced-
ing the onset of heart failure than earlier in tits course.99
Serum or synovial fluid levels of many other tissue prod-
ucts (see Table 57-5) have been correlated with clinical
measures of disease activity, severity, and radiographic
damage.
Newer quantitative and objective assays that incorporate
some of the measures previously mentioned as well as other
novel ones, have recently come into the market to aid in
the management and earlier diagnosis of patients with RA.
Multibiomarker disease activity (MBDA) tests are increas-
ingly popular and could play a significant role the future
management and study of RA; however, more studies are
required to determine their clinical usefulness across differ-
ent mechanistic interventions.
Systemic Lupus Erythematosus
Although serum levels of CRP often parallel disease activity
in autoimmune disorders, it has been recognized that SLE
is an exception.100 Although marked CRP responses are
seen in subsets of patients, such as those with serositis or
chronic synovitis, many (e.g., patients with nephritis) show
mild or no elevation during periods of activity.101-103 Serum
levels of SAA are also relatively low in comparison with
those of patients with RA, which may explain why rates of
secondary amyloidosis are decreased in these patients104 In
contrast, ESR correlates with disease activity and accrued
tissue damage in SLE.105 Fibrinogen levels increased over
time in patients, regardless of disease activity.106 Data are
insufficient to evaluate the potential use of some of the
other newer markers described previously, including PCT,
but many SLE patients with normal CRP levels show ele-
vated IL-6 concentrations.107 Therefore a deficiency in IL-6
does not explain the muted CRP response in SLE.
Although the concomitant decrease in SAA may point
against it, several investigations have raised the possibility
that low CRP levels may be related to the pathogenesis of
SLE: (1) An association has been noted between SLE and
a genetic polymorphism associated with low CRP levels;
(2) it has been observed that low CRP levels may contrib-
ute to defective clearance of autoantigens during apopto-
sis; and (3) the therapeutic efficacy of CRP has been
reported in mouse models of SLE.101,108-111 Recent studies
have also raised the possibility that type I interferon (IFN),
which is expressed significantly in SLE, may inhibit CRP
expression.112,113
Substantial CRP elevation in SLE patients is more likely
to result from superimposed infection than from activation
of lupus. CRP levels greater than 6 mg/dL in these patients
should serve as an impetus to exclude the possibility of
infection, just as they should in other diseases.10 Such levels
should not be regarded as proof of infection, however; as
indicated earlier, marked CRP elevation related to active
SLE can be seen in the absence of infection.
Carotid plaque and intima-media wall thickness, corre-
lates of atherosclerotic vascular disease, have been found in
association with minor CRP elevation in women with SLE,
as they have in patients with RA.114,115
Polymyalgia Rheumatica and Giant
Cell Arteritis
The diagnosis of PMR or GCA is supported by an elevated
ESR, often greater than 100 mm/hr. However, such eleva-
tion is no longer regarded as a sine qua non of these disor-
ders; continuing reports suggest that 10% to 20% of patients
with PMR can have “normal” ESRs, depending on which
value is taken as the limit of normal. Such patients tend to
have fewer systemic symptoms and less severe, less frequent
anemia.116 They have the same frequency of positive tem-
poral artery biopsy results, however, as patients with ele-
vated ESR.117,118
Only approximately 5% of patients with GCA had ESR
values less than 40 mm/hr; these patients had fewer visual
and systemic symptoms than patients with high ESR
values.119 In contrast to these findings, ESR and CRP were
found to be significantly lower in patients with ocular
involvement, most commonly in the range of 70 mm/hr to
100 mm/hr. Patients with ESR greater than 100 mm/hr had
decreased incidence of visual ischemic events.120-122
In PMR and GCA, CRP and ESR have been regarded
in the past as equally valuable in assessing disease activity.
However, recent reports suggest that CRP is more sensitive
for both conditions and should be included routinely in the
diagnostic workup.118,123,124 The report that IL-6 is more
sensitive than ESR for indicating disease activity in GCA
is of particular interest.125 Subsets of patients with PMR
who have persistently elevated levels of CRP and IL-6
despite corticosteroid treatment have a higher risk of
relapse.126 A polymorphism at the IL-6 gene promoter has
characterized these PMR patients with persistently elevated
levels of the cytokine.127 Clinical manifestations of disease,
even in the presence of a normal ESR or CRP level, should
not be ignored. Extreme elevation of the ESR in the absence
of symptoms of PMR or GCA should raise suspicion of
other disorders, such as infection, malignancy, or renal
 CHAPTER 57    Acute Phase Reactants and the Concept of Inflammation 853
disease. PCT levels in patients with PMR or GCA are
usually normal according to a French prospective study.
Elevated PCT levels in these patients with a normal tem-
poral artery biopsy would be suggestive of an alternate
diagnosis.128
Numerous markers of endothelial perturbation, although
not acute phase reactants in the strict sense, are elevated in
plasma in various inflammatory disorders of vessels, particu-
larly PMR, GCA, and other vasculitides.129 These molecules
include von Willebrand factor, thrombomodulin, some
vasoactive prostanoids, and a variety of adhesion molecules,
such as vascular cell adhesion molecule-1.
Adult-Onset Still’s Disease
Markedly elevated concentrations of ferritin, dispropor-
tionately high compared with those of other acute phase
reactants, have long been noted in adult-onset Still’s disease
but are not specific.130 Only a small percentage, commonly
less than 20%, of ferritin is glycosylated in adult-onset
Still’s disease, a criterion included in recently proposed
classification criteria for this condition.131,132 Extremely
elevated ferritin levels have been found in macrophage
activation syndrome, and 40% of individuals with this con-
dition meet the criteria for adult-onset Still’s disease, sug-
gesting to some that the two disorders are not distinct
entities.133-135 Concentrations of serum IL-18 were extremely
elevated in patients with active adult-onset Still’s disease
compared with those of patients with other connective
tissue diseases or of healthy individuals and were correlated
with serum ferritin values and disease severity.136 The cyto-
kine profile in the sera of patients suggests a type 1 T helper
(Th) cell response, with significantly higher levels of TNF,
IL-6, and IL-8, in addition to IL-18.137 The role of IL-1
has been inferred from significant improvement in disease
activity by the IL-1 receptor antagonist, anakinra.138,139 It
has been suggested that IFN-α may be responsible for the
hyperferritinemia of adult-onset Still’s disease.130 CRP
levels are usually markedly elevated in this disease, and
PCT levels have also been found to be disproportionately
elevated without evidence of acute bacterial infection in
these patients.51,52
Ankylosing Spondylitis
Ankylosing spondylitis ordinarily does not lead to a substan-
tial increase in ESR or CRP. Median ESR and CRP levels
are 13 mm/hr and 1.6 mg/dL, respectively, in patients with
only spinal involvement, and 21 mm/hr and 2.5 mg/dL in
patients with peripheral involvement or associated inflam-
matory bowel disease.140 Treatment with infliximab led to
an average decrease of 75% in CRP concentration after
12 weeks. Patients with lower CRP levels showed little
improvement, however, raising the possibility that patients
with higher CRP values show better responses to anti-TNF
treatment than patients with lower CRP levels.141-143 High-
sensitivity CRP may better correlate with clinical disease
activity compared with standard CRP testing.144 It has been
reported that levels of IL-8, IL-17, and IL-23 are elevated
in the serum of patients with active ankylosing spondylitis,
and polymorphisms in the IL-23 receptor gene are associ-
ated with the disease.145-147
Osteoarthritis
Minor CRP elevations of 0.3 to 1.0 mg/dL have been
reported in patients with osteoarthritis, particularly those
with progressive joint damage.148 However, no evidence
supports this association independent of body mass index
(BMI), because obesity is a common accompaniment of
osteoarthritis.149 Although local inflammation likely plays a
role in the pathogenesis of osteoarthritis, systemic inflam-
mation likely does not. However, CRP levels are higher in
patients with erosive osteoarthritis of the hand than in
those with nonerosive osteoarthritis.150
Other Rheumatic Diseases
Acute phase markers are elevated in numerous rheumatic
diseases while the inflammatory cascade commences. Ele-
vated ESR and CRP can be found in systemic vasculitides,
crystal arthropathies, psoriatic and reactive arthritides, and
infectious joint diseases.151-154 Monitoring for elevated SAA
levels has been proposed as a tool for diagnosis and medica-
tion adjustment in familial Mediterranean fever,155although
this test is not widely available, limiting its utility. CRP is
often normal in patients with primary Sjögren’s syndrome,
and those with elevated responses do not differ clinically
from patients with normal levels.156 Oligoarticular-onset
juvenile idiopathic arthritis is classically considered as not
associated with elevated inflammatory markers, although
they are present in patients most at risk for systemic
disease.157
PRACTICAL USE OF ACUTE PHASE
REACTANTS
In the past, rheumatologists were found to use the ESR more
than twice as frequently as they did CRP levels,158 although
ESR reflects many complex, poorly understood changes in
the physical and chemical characteristics of blood not asso-
ciated with inflammation. As indicated earlier, the refer-
ence normal values for ESR are unclear. It is well established
that mean ESR values increase substantially with age and
differ between men and women. Difficulties associated with
interpretation of the ESR and an increasingly positive clini-
cal experience with CRP suggest that rheumatologists may
benefit from relying more on CRP than ESR testing.159 No
single ideal test can be used to evaluate the acute phase
response, however. The relative virtues of these tests in fol-
lowing patients with RA have been discussed.74,160
Discrepancies between ESR and CRP may result from
effects of blood constituents that are not related to inflam-
mation, but that can influence the ESR. In addition,
patterns of acute phase protein changes differ in different
conditions.5 ESR may be markedly elevated in many patients
with active SLE, whereas the CRP is normal. Undoubtedly,
numerous other clinical situations exist in which similar
discrepancies occur. Although a falsely high ESR has many
non-inflammatory physicochemical causes (some known,
and many unknown), CRP values greater than 1 mg/dL
almost invariably reflect a clinically significant inflamma-
tory process. In light of these considerations, many authors
believe that several tests, rather than a single test, should
be performed and interpreted in their clinical context. It
 854 PART 7    DIAGNOSTIC TESTS AND PROCEDURES IN RHEUMATIC DISEASES
has been suggested that ESR, which is associated with
anemia and immunoglobulin levels, may reflect general
severity in RA, whereas CRP is a better test of active inflam-
mation per se.161
C-REACTIVE PROTEIN AND
HEALTH: ASSOCIATIONS WITH
NONRHEUMATOLOGIC CONDITIONS
Although most ostensibly healthy individuals have CRP
concentrations of 0.3 mg/dL or less, some have concentra-
tions greater than 1 mg/dL. Such minor CRP elevation has
long been attributed to trivial tissue injury or to minimal
inflammatory processes, such as gingivitis. However, recent
data indicate that CRP concentrations between 0.3 and
1 mg/dL have clinical relevance. This finding has led to an
explosion of published literature measuring CRP levels in
cardiac, neurologic, neoplastic, pulmonary, and even psy-
chiatric disease.162-166 The foundation of these investigations
is the well-established notion that when a high-sensitivity
CRP assay is used, serum CRP levels greater than 0.3 mg/
dL indicate increased the relative risk of atherogenesis and
future myocardial infarction.167 The statistical strength of
this is as robust as, but not more robust than, established
risk factors such as hypertension, diabetes, and hyper­
cholesterolemia.168-170 The primary unanswered questions
remain: Why does CRP predict myocardial infarction? Is it
a pathogenic mediator itself?
The observation that CRP is associated with many non-
inflammatory conditions (e.g., low levels of physical activ-
ity, low intake of fruits and vegetables, a variety of other
“unhealthy” diets, smoking, hypertension, obesity, sleep
deprivation, and low alcohol intake) indicates that classic
inflammation does not invariably underlie a CRP response.171
Many of these CRP-associated conditions are known to be
risk factors for cardiovascular disease, confounding the situ-
ation and suggesting that CRP predicts myocardial infarc-
tion because of its association with these risk factors. In
addition, elevated CRP may not reflect inflammation, but
rather a response to the presence of distressed, metabolically
disturbed cells.171 This reverse causation offers a potential
explanation of the laboratory result, because atherosclerosis
might trigger an elevation in CRP.
Nevertheless, CRP has been known for many years to
bind to low-density lipoprotein (LDL) and to activate
complement—a potentially pro-inflammatory response; it
has also been detected in atherosclerotic plaques.29,172-174
These observations have raised the possibility that CRP
may play a direct causal role in coronary heart disease.
Proatherogenic effects secondary to CRP injections given
to mice, however, may have been caused by contaminants
in commercial CRP preparations, not by CRP itself.175
Because statin drugs lower CRP and LDL, some consider
their effects to provide indirect evidence of a causal role of
CRP. In the justification for the use of statins in prevention:
an intervention trial evaluating rosuvastatin (JUPITER)
trial, patients with normal LDL and elevated CRP were
prescribed rosuvastatin, 20 mg daily, or placebo, and were
followed prospectively. Those in the treatment arm had
significant reductions in major cardiovascular events.176
However, whether the effects of the study can be solely
dependent on CRP reduction remains unclear; alternative
explanations have been suggested, including the question
of the importance of lowering LDL even among patients
with healthy levels.177 Whether to target CRP levels in
cardiovascular disease is an ongoing topic of intense debate.
Because no CRP inhibitor drugs are available to directly
measure the effect of lowering the protein, recent studies
have centered on observing patients with genetic variations
that result in different baseline levels of CRP. To date,
results have been conflicting with regard to the role
of genetically determined CRP levels in coronary heart
disease.178-182
Epidemiologic studies that describe an association of
CRP levels with morbidity and mortality in many chronic
diseases, and even in normal aging, have become a cottage
industry. It is important to remember that these are obser-
vational population studies. Although such associations
may have broad and intriguing implications, particularly at
a societal level, they reflect probabilities. This limits their
clinical value when they are applied to individual patients.
Full references for this chapter can be found on
ExpertConsult.com.
SELECTED REFERENCES
2. Biedermann B: Vascular endothelium: checkpoint for inflammation
and immunity. News Physiol Sci 16:84–88, 2001.
3. Gawaz M, Langer H, May A: Platelets in inflammation and athero-
genesis. J Clin Invest 115:3378–3384, 2005.
4. Dayer E, Dayer JM, Roux-Lombard P: Primer: the practical use of
biologic markers of rheumatic and systemic inflammatory diseases.
Nat Clin Pract Rheum 3:512–520, 2007.
5. Gabay C, Kushner I: Acute-phase proteins and other systemic
responses to inflammation. N Engl J Med 340:448–454, 1999.
6. Yoo JY, Desiderio S: Innate and acquired immunity intersect in a
global view of the acute-phase response. Proc Natl Acad Sci U S A
100:1157–1162, 2003.
8. Alonzi T, Maritano D, Gorgoni B, et al: Essential role of STAT3 in
the control of the acute-phase response as revealed by inducible gene
activation in the liver. Mol Cell Biol 21:1621–1632, 2001.
10. Morley JJ, Kushner I: Serum C-reactive protein levels in disease. Ann
N Y Acad Sci 389:406–418, 1982.
12. Xing Z, Gauldie J, Cox G, et al: IL-6 is an anti-inflammatory cytokine
required for controlling local or systemic acute inflammatory
responses. J Clin Invest 101:311–320, 1998.
13. Jones SA, Horiuchi S, Topley N, et al: The soluble interleukin 6
receptor: mechanisms of production and implications in disease.
FASEB J 15:43–58, 2001.
14. Volanakis JE: Human C-reactive protein: expression, structure, and
function. Mol Immunol 38:189–197, 2001.
17. Sox HC Jr, Liang MH: The erythrocyte sedimentation rate: guide-
lines for rational use. Ann Intern Med 104:515–523, 1986.
18. Bastard JP, Maachi M, Van Nhieu JT, et al: Adipose tissue IL-6
content correlates with resistance to insulin activation of glucose
uptake both in vivo and in vitro. J Clin Endocrinol Metab 87:2084–
2089, 2002.
20. Cha CH, Park CJ, Cha YJ, et al: Erythrocyte sedimentation rate
measurements by TEST 1 better reflect inflammation than do those
by the Westergren method in patients with malignancy, autoimmune
disease, or infection. Am J Clin Pathol 131:189–194, 2009.
22. Pepys MB, Hirschfield GM: C-reactive protein: a critical update.
J Clin Invest 111:1805–1812, 2003.
23. Ridker PM, Pare G, Parker A, et al: Loci related to metabolic-
syndrome pathways including LEPR, HNF1A, IL6R, and GCKR
associate with plasma C-reactive protein: the Women’s Genome
Health Study. Am J Hum Genet 82:1185–1192, 2008.
26. Cha-Molstad H, Young DP, Kushner I, et al: The interaction of C-rel
with C/EBPbeta enhances C/EBPbeta binding to the C-reactive
protein gene promoter. Mol Immunol 44:2933–2942, 2007.
 CHAPTER 57    Acute Phase Reactants and the Concept of Inflammation 855
28. Griselli M, Herbert J, Hutchinson WL, et al: C-reactive protein and
complement are important mediators of tissue damage in acute myo-
cardial infarction. J Exp Med 190:1733–1740, 1999.
29. Marnell L, Mold C, Du Clos TW: C-reactive protein: an activator of
innate immunity and a modulator of adaptive immunity. Immunol Res
30:261–277, 2004.
31. Gershov D: C-reactive protein binds to apoptotic cells, protects the
cells from assembly of the terminal complement components, and
sustains an anti-inflammatory innate immune response: implications
for systemic autoimmunity. J Exp Med 192:1353–1364, 2000.
32. Zouki C, Beauchamp M, Baron C, et al: Prevention of in vitro neu-
trophil adhesion to endothelial cells through shedding of L-selectin
by C-reactive protein and peptides derived from C-reactive protein.
J Clin Invest 100:522–529, 1997.
34. Vigushin DM, Pepys MB, Hawkins PN: Metabolic and scintigraphic
studies of radioiodinated human C-reactive protein in health and
disease. J Clin Invest 91:1351–1357, 1993.
35. Morley JJ, Kushner I: Serum C-reactive protein levels in disease. Ann
N Y Acad Sci 389:406–418, 1982.
36. Macy EM, Hayes TE, Tracy RP: Variability in the measurement of
C-reactive protein in healthy subjects: implications for reference
intervals and epidemiological applications. Clin Chem 43:52–58,
1997.
37. Vanderschueren S, Deeren D, Knockaert DC, et al: Extremely ele-
vated C-reactive protein. Eur J Intern Med 17:430–433, 2006.
38. Woloshin S, Schwartz LM: Distribution of C-reactive protein values
in the United States. N Engl J Med 352:1611–1613, 2005.
39. Wu JY, Lee SH, Chang SS, et al: Use of serum procalcitonin to
detect bacterial infection in patients with autoimmune diseases: a
systemic review and meta-analysis. Arthritis Rheum 64:3034–3042,
2012.
41. Wacker C, Prkno A, Schlattmann P, et al: Procalcitonin as a diag-
nostic marker for sepsis: a systematic review and meta-analysis. Lancet
Infect Dis 13:426–435, 2013.
43. Christ-Crain M, Jaccard-Stolz D, Bingisser R, et al: Effect of
procalcitonin-guided treatment on antibiotic use and outcome in
lower respiratory tract infections: cluster-randomised, single-blinded
intervention trial. Lancet 363:600–607, 2004.
44. Christ-Crain M, Stolz D, Bissinger R, et al: Procalcitonin guidance
of antibiotic therapy in community-acquired pneumonia: a random-
ized trial. Am J Respir Crit Care Med 174:84–93, 2006.
47. Snider RH, Nylen ES, Becker KL: Procalcitonin and its component
peptides in systemic inflammation: immunochemical characteriza-
tion. J Investig Med 45:552–560, 1997.
49. Assicot M, Gendrel D, Carsin H, et al: High serum procalcitonin
concentrations in patients with sepsis and infection. Lancet 341:515–
518, 1993.
51. Chen DY, Chen YM, Lan JL, et al: Diagnostic value of procalcitonin
for differentiation between bacterial infection and non-infectious
inflammation in febrile patients with active adult-onset Still’s disease.
Ann Rheum Dis 68:1074–1075, 2009.
52. Scire CA, Cavagna L, Montecucco C, et al: Diagnostic value of
procalcitonin measurement in febrile patients with systemic autoim-
mune diseases. Clin Exp Rheumatol 24:123–128, 2006.
54. Lanoix JP, Bourgeois AM, Schmidt J, et al: Serum procalcitonin does
not differentiate between infection and disease flare in patients with
systemic lupus erythematosus. Lupus 20:125–130, 2011.
55. Kim HA, Jeon JY, An JM, et al: C-reactive protein is a more sensitive
and specific marker for diagnosing bacterial infections in systemic
lupus erythematosus compared to S100A8/A9 and procalcitonin.
J Rheumatol 39:728–734, 2012.
56. Bador KM, Intan S, Hussin S, et al: Serum procalcitonin has negative
predictive value for bacterial infection in active systemic lupus ery-
thematosus. Lupus 21:1172–1177, 2011.
58. Efthimiou P, Paik PK, Bielory L: Diagnosis and management of adult
onset Still’s disease. Ann Rheum Dis 65:564–572, 2006.
59. Nishiya K, Hashimoto K: Elevation of serum ferritin levels as a
marker for active systemic lupus erythematosus. Clin Exp Rheumatol
15:39–44, 1997.
60. Nemeth E, Valore EV, Territo M, et al: Hepcidin, a putative mediator
of anemia of inflammation, is a type II acute-phase protein. Blood
101:2461–2463, 2003.
62. Lahita RG, Rivkin E, Cavanagh I, et al: Low levels of total choles-
terol, high-density lipoprotein, and apolipoprotein A1 in association
with anticardiolipin antibodies in patients with systemic lupus ery-
thematosus. Arthritis Rheum 36:1566–1574, 1993.
63. Park YB, Lee SK, Lee WK, et al: Lipid profiles in untreated patients
with rheumatoid arthritis. J Rheumatol 26:1701–1704, 1999.
64. Johnson AM, Merlini G, Sheldon J, et al: Clinical indications for
plasma proteins assays: transthyretin (prealbumin) in inflammation
and malnutrition—International Federation of Clinical Chemistry
and Laboratory Medicine (IFCC), IFCC Scientific Division Commit-
tee on Plasma Proteins (C-PP). Clin Chem Lab Med 45:419–426,
2007.
65. Tutuncu ZN, Bilgie A, Kennedy LG, et al: Interleukin-6, acute phase
reactants and clinical status in anklyosing spondylitis. Ann Rheum
Dis 53:425–426, 1994.
66. Uddhammar A, Sundqvist KG, Ellis B, et al: Cytokines and adhesion
molecules in patients with polymyalgia rheumatica. Br J Rheumatol
37:766–769, 1998.
67. Luqmani R, Sheeran T, Robinson M, et al: Systemic cytokine mea-
surements: their role in monitoring the response to therapy in
patients with rheumatoid arthritis. Clin Exp Rheumatol 12:503–508,
1994.
68. Pountain G, Hazleman B, Cawston TE: Circulating levels of IL-1beta,
IL-6 and soluble IL-2 receptor in polymyalgia rheumatica and giant
cell arteritis and rheumatoid arthritis. Br J Rheumatol 37:797–798,
1998.
69. Gabay C, Cakir N, Moral F, et al: Circulating levels of tumor necrosis
factor soluble receptors in systemic lupus erythematosus are signifi-
cantly higher than in other rheumatic diseases and correlate with
disease activity. J Rheumatol 24:303–308, 1997.
70. Gabay C, Gay-Croisier F, Roux-Lombard P, et al: Elevated serum
levels of interleukin-1 receptor antagonist in polymyositis/
dermatomyositis: a biologic marker of disease activity with a possible
role in the lack of acute-phase protein response. Arthritis Rheum
37:1744–1751, 1994.
72. Sokka T, Pincus T: Erythrocyte sedimentation rate, C-reactive
protein, or rheumatoid factor is normal at presentation in 35-45% of
patients with rheumatoid arthritis seen between 1980 and 2004:
analysis from Finland and the United States. J Rheumatol 36:1387–
1390, 2009.
73. Matsui T, Kuga Y, Kaneko A, et al: Disease Activity Score 28
(DAS28) using C-reactive protein underestimates disease activity
and overestimates EULAR response criteria compared with DAS28
using erythrocyte sedimentation rate in a large observational cohort
of rheumatoid arthritis patients in Japan. Ann Rheum Dis 66:1221–
1226, 2007.
76. Rhodes B, Merriman ME, Harrison A, et al: A genetic association
study of serum acute-phase C-reactive protein levels in rheumatoid
arthritis: implications for clinical interpretation. PLoS Med 7:
e1000341, 2010.
78. Posthumus MD, Limburg PC, Westra J, et al: Serum matrix metal-
loproteinase 3 levels in comparison to C-reactive protein in periods
with and without progression of radiological damage in patients with
early rheumatoid arthritis. Clin Exp Rheumatol 21:465–472, 2003.
79. Kuuliala A, Eberhardt K, Takala A, et al: Circulating soluble
E-selectin in early rheumatoid arthritis: a prospective five year study.
Ann Rheum Dis 61:242–246, 2002.
80. Aletaha D, Smolen JS: The rheumatoid arthritis patient in the clinic:
comparing more than 1300 consecutive DMARD courses. Rheumatol-
ogy (Oxford) 41:1367–1374, 2002.
81. Wolfe F, Pincus T: The level of inflammation in rheumatoid arthritis
is determined early and remains stable over the longterm course of
the illness. J Rheumatol 28:1817–1824, 2001.
82. Sanmarti R, Gomez A, Ercilla G, et al: Radiological progression in
early rheumatoid arthritis after DMARDS: a one-year follow-up study
in a clinical setting. Rheumatology (Oxford) 42:1044–1049, 2003.
83. Charles P, Elliott MJ, Davis D, et al: Regulation of cytokines, cyto-
kine inhibitors, and acute-phase proteins following anti-TNF alpha
therapy in rheumatoid arthritis. J Immunol 163:1521–1528, 1999.
84. Weisman MH, Durez P, Hallegua D, et al: Reduction of inflammatory
biomarker response by abatacept in treatment of rheumatoid arthritis.
J Rheumatol 33:2162–2166, 2006.
85. Lee EB, Fleischmann R, Hall S, et al: Tofacitinib versus methotrexate
in rheumatoid arthritis. N Engl J Med 370:2377–2386, 2014.
86. Buch MH, Seto Y, Bingham SJ, et al: C-reactive protein as a predic-
tor of infliximab treatment outcome in patients with rheumatoid
arthritis: defining subtypes of nonresponse and subsequent response
to etanercept. Arthritis Rheum 52:42–48, 2005.
87. Smolen JS, Van Der Heijde DM, St Clair EW, et al: Predictors of
joint damage in patients with early rheumatoid arthritis treated with