Peptidylarginine deiminases and the pathogenesis of rheumatoid arthritis

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European Journal of Internal Medicine xxx (2009) xxxndashxxx

EJINME-01802 No of Pages 7

Contents lists available at ScienceDirect

European Journal of Internal Medicine

j ourna l homepage wwwe lsev ie rcom locate e j im

ARTICLE IN PRESS

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^Review article

Peptidylarginine deiminases and the pathogenesis of rheumatoid arthritisA reflection of the involvement of transglutaminase in coeliac disease

Paringl Stenberg a Bodil Roth a Frank A Wollheim b

a Lund University Department of Clinical Sciences Malmouml Clinical Coagulation Research Unit Malmouml University Hospital S-205 02 Malmouml Swedenb Department of Rheumatology Lund University Hospital S-221 85 Lund Sweden

UAbbreviations ACPA anti-citrullinated peptide antibdithiothreitol EDTA ethylenediaminetetraacetic acidFXIII factor XIII GAD glutamic acid decarboxylaspeptidylarginine deiminase pSS primary Sjoumlgrens syndrRF rheumatoid factor SE shared epitopes SLE systemi Corresponding author Tel +46 40 33 37 85 fax +

E-mail address palstenbergteliacom (P Stenberg

0953-6205$ ndash see front matter copy 2009 European Fededoi101016jejim200908007

Please cite this article as Stenberg P et a(2009) doi101016jejim200908007

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a r t i c l e i n f o

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Article historyReceived 26 June 2009Received in revised form 17 August 2009Accepted 20 August 2009Available online xxxx

Keywords

^Alcohol

^Autoimmune

^Coeliac disease

^Pathogenesis

^Peptidylarginine deiminase

^Post-translational

^Rheumatoid arthritis

^Smoking

^Transglutaminase

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OPost-translational modifications are associated with certain autoimmune diseases For example in the initialsteps of coeliac disease (CD) transglutaminase type 2 (TG2) catalyzes a post-translational deamidation ofspecific glutamine residues in dietary gluten resulting in antibodies against both modified gliadin andagainst TG2 Anti-TG2 has become a specific biomarker for CD In rheumatoid arthritis (RA) the presence ofantibodies against citrullinated peptides (ACPA) characterizes a distinct subset of this inflammatory disorderMoreover antibodies against the enzyme that catalyzes the citrullination (peptidylarginine deiminase PAD)are found in RA Their relation to disease severity indicates a possible pathogenetic role Thus in twomajor autoimmune diseases (CD and RA) antibodies are present against a post-translationally modifiedsubstrate and against the calcium-dependent thiol-enzyme (TG2 and PAD respectively) responsible for themodificationThis review highlights the similarities between the TGs and the PADs and their putative pathogenetic roles inautoimmune diseases Possible mechanisms of the effects of cigarette smoking and alcohol consumption onRA are discussed By reflecting the progress in CD the pathogenesis of ACPA-positive RA can be hypothesizedwhere expression and regulation of PADs play significant roles Indeed autoimmune diseases should bestudied collectively as well as individually The new insight may lead towards innovative pharmacother-apeutic principles

copy 2009 European Federation of Internal Medicine Published by Elsevier BV

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1^ Introduction

Amajority of human proteins aremodified post-translationally forinstance via glycosylation phosphorylation transamidation acyla-tion or methylation For reviews see [12] While most of theseenzyme-catalyzed reactions are essential for mammalian life there isalso a risk that an inadequate alteration might trigger the develop-ment of autoimmune diseases Based on American estimates approxi-mately 80 autoimmune conditions have been described [3] with anoverall prevalence of 5ndash8

^ [4] In addition autoimmune features have

been hypothesized in other common conditions such as atheroscle-rosis [5] and Alzheimers disease [6]

Genetic lifestyle and environmental factors may contribute to thepathogenesis but much of the basic mechanisms remain unknown formost of these autoimmune diseases During the last decade a new

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ody CD coeliac disease DTTEMA endomysial antibodiese MTX methotrexate PADome RA rheumatoid arthritisc lupus erythematosus

^

46 40 33 62 55)

ration of Internal Medicine Publish

l Peptidylarginine deiminas

insight has emerged into the pathogenesis of a T cell driven inflam-matory intestinal disorder with a special HLA-requirement namelycoeliac disease (CD) A major autoantigen in CD is the post-translationally active enzyme transglutaminase type 2 (TG2) recog-nized by anti-endomysial antibodies (EMA) [7] This landmarkobservation has lead to understanding of parts of the moleculardevelopment of CD In rheumatoid arthritis (RA) antibodies againstcitrullinated proteins (ACPA) have been detected Moreover anti-bodies against the enzyme which catalyzes the citrullination havebeen found in RA Their significance prevalence and relation to diseaseseverity indicate a possible pathogenetic role With this backgroundthe present review examines whether post-translational modifica-tions could also be involved in the pathogenesis of RA In essence wewant to highlight what can be learned from the progress made in thefield of coeliac disease

2^ Transglutaminase and the pathogenesis of coeliac disease

The calcium-dependent TGs catalyze the intermolecular cross-linking of proteins through the formation of pseudopeptide bridgesbetween γ-carbonyl groups of specific glutamine residues and ε-aminogroups of lysine residues The best-known physiologic example is the

ed by Elsevier BV

es and the pathogenesis of rheumatoid arthritis Eur J Intern Med

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cross-linking of fibrin as catalyzed by Factor XIII (FXIII) in the terminalstage of blood coagulation [8] All TG-catalyzed reactions proceed via anintermediate thioester formed between the active centre cysteine ofthe enzyme and γ-carboxamide of specific glutamine residues (Fig 1)In the traditional TG-catalyzed reaction named transamidation theprimary amino group of a lysine residue acts as a nucleophile ieis attracted to the positive charge of the polarized thioester Thus anε-(γ-glutamyl)lysine bridge is formed between the two substrates Inthe absence of a primary amino group or where pH is reduced forinstance at a site of inflammation water can serve as the secondsubstrate This results in deamidation of specific glutamine residuesyielding negatively charged glutamate Both these types of post-translational modifications are believed to constitute the molecularbasis for the initial events in the immunopathogenesis of CD (Fig 1)Basic understanding has gained light by a number of advances

bull TG2 is a major autoantigen in CDbull Calcium ions increase the affinity between TG2 and anti-TG2-antibodies from CD-patients

bull Physiological concentrations of zinc a known inhibitor of the acti-vation of TGs erase the calcium-induced augmentation of affinity

bull CD-antibodies against TG2 are present in serum but do not seem toaffect enzyme activity significantly

bull TG2 catalyzes the deamidation of specific glutamine residues ingliadins

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Fig 1^ Transglutaminase-catalyzed reactions in the initial steps of the pathogenesis of coelia

inflammation is activated by calcium Zinc counteracts the activation Dietary gluten is hydrthe gliadins function as substrates for the activated enzyme The MichaelisndashMenten intermedreduced pH such as in a site of inflammation water functions as the second substrate Thepositions In this hydrolysis the second step is rate-limiting resulting in comparatively largegliadin In the figure transamidation is illustrated by an atypical reaction when a lysine rethioester intermediate resulting in a complex between TG2 and gliadin Initially most inve

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bull In the normal TG-catalyzed reaction (transamidation) the first stepis rate-limiting

bull In the atypical reaction (deamidation) the last step is rate-limitingmeaning that the complex between TG2 and previously deamidatedglutamine residues is comparatively long-lived

bull Antibodies against specifically deamidated gliadins are presentin CD

bull Specifically deamidated gliadin fits into HLA DQ2 or DQ8

For reviews see [910] Anti-TG2 has become a specific andsensitive biomarker for CD

3^ Citrullination

During citrullination the positively charged guanidinium group ofan arginine side chain is converted into a neutral ureido group (Fig 2)This process is catalyzed by members of a family of enzymes thepeptidylarginine deiminases (PADs) Citrullination is implicated in vari-ous physiological processes such as apoptosis and terminal differenti-ation of the epidermis For a review see [11] This post-translationalmodification affects the conformation and stability of proteins In vitrostudies on the cytoskeletal proteins trichohyalin and filaggrin indicatethat citrullination of 5ndash10

^of the arginine residues results in substantial

deformation of the proteins [12] By maximal citrullination of argininesin a modified form of the hair follicle trichohyalin is transformed into a

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c disease TG2 in lamina propria or originating from macrophages attracted to a site ofolyzed to gliadins by peptidases Specific glutamine (Q) residues in proline rich areas ofiate formed during the reaction is a thioester In the absence of a primary amine or at acycle is repeated yielding gliadin molecules with glutamic acid (E) residues at specificconcentrations of the thioester intermediate between TG2 and previously deamidatedsidue of TG2 in the absence of a proper amine substrate nucleophilically attacks thestigators suggested that this type of complex constituted the autoantigen

es and the pathogenesis of rheumatoid arthritis Eur J Intern Med

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Fig 2^ Peptidylarginine deiminase-catalyzed citrullination

Table 1^

t11

Characteristics of peptidylarginine deiminasest12t13Isotype Chromosome

locationProposedfunctions

Number ofamino acids

Protein expression

t14PAD1 1p3613 Cornificationof epidermis

663 Epidermis uterus

t15PAD2 1p3613 Apoptosis 665 Brain spinal corduterus sweat glandssalivary glands skeletalmuscles macrophagesbone marrow pancreas

t16PAD3 1p3613 Growth of the hairfiber terminaldifferentiation

664 Hair follicles

t17PAD4 1p3613 Apoptosis 663 Blood cellst18PAD6 1p3613 Female fertility 686 Germ cells

Table 2^

t21

A comparison between transglutaminases and peptidylarginine deiminasest22t23Feature TG2 PAD24

t24Thiol-enzyme + +t25Calcium-dependent + +t26Active post-translationally + +t27Release of ammonia + +t28Zinc inhibits enzyme activity + +t29Change of substrate charge + +t210Antibodies (RACD) against the modified substrate + +t211Antibodies (RACD) against the enzyme + +t212One of several isotypes + +t213Widely distributed + +t214Peptide-bound substrate + +

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more efficient substrate for epidermal TG-catalyzed transamidation[13] Moreover post-translational citrullination of myelin basic proteinhas been implicated in multiple sclerosis For a review see [14]

Citrullination is prominently associated with immuno-pathologi-cal events The pioneering work of the groups of van Venrooij [15] andSerre [16] during the late 1990s showed that a majority of sera fromRA-patients contained antibodies against citrullinated peptides Inclinical practice ACPA is now established as a diagnostic test for RAwith a higher specificity (96ndash98

^) than the rheumatoid factor (RF)

The sensitivity is approximately 70^ Initially a synthetic Citrullinated

Cyclic Peptide (CCP) was used as antigen in the assay More recently acombination of citrullinated peptides has been utilized Moreovernew methods have been developed based on citrullinated α- and β-fibrin vimentin or collagen type II For a review see [17] In generalACPAs of the IgG class are measured but IgA-ACPA has also beenreported in RA [18] although in lower frequency Like RF ACPAs can bedetected in sera several years before the RA-symptoms occur [19] Thereason for the limited sensitivity is of great interest and may be anindicator of different subsets of RA [20]

Citrulline residues are being detected in an increasing number ofproteins such as filaggrin trichohyalin vimentin α-enolase myelinbasic protein and various formsoffibronectin keratinfibrinfibrinogenand collagen Notably several of these proteins have also been detectedin a citrullinated form in inflamed joints The appearance and amountsof citrullinated proteins in arthritic joints of experimental animalscorrelatewith the severity of the inflammation [21] This is however notthe case in human disease In collagen induced arthritis both antibodiesto citrullinated and native epitopes have shown arthrigenicityindicating that citrullination is not an obligatory requirement [22]

The conversion of arginine to citrulline in a peptide derived fromhuman vimentin dramatically increases its affinity to the RA-associated HLA DRB10401 MHC shared epitopes [23] Later studieshave confirmed an association between this haplotype and othercitrullinated candidate autoantigens [182425] A similar situation hasbeen observed in CD where a TG2-catalyzed deamidation of gliadinmolecules leads to an increase in peptide affinity and the activation ofHLA DQ2- or DQ8-restricted T cells However there is no evidence upto now showing the presence of such T-cell reactivity in RA

4^ Peptidylarginine deiminase and rheumatoid arthritis

In Japanese and Korean populations independent studies haveconfirmed the association between RA and the functional haplotype ofthe PAD4 isozyme [2627] Although PAD gene polymorphism has notturned out to be related to susceptibility to RA in Europeanpopulations [2829] it was found that PAD4 is significantly over-expressed in patients with RA Moreover a meta-analysis includingsix replication studies one from Japan and five from Europe and NorthAmerica showed a positive association between PAD4 and RA notonly in the Japanese population but also in populations of Europeandescent [30] The association is restricted to SE-carrying patients [29]

All known human PAD genes are clustered on chromosome1p36So far five isoenzymes have been identified (Table 1) PAD4 previ-

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OOFously called PAD5 is expressedmainly in granulocytes andmonocytes

while PAD2 is found in for example brain spleen pancreas andskeletal muscles For reviews see [1531] The expression of PAD2reminds of the ubiquitous TG2 distribution PAD4 and the thrombin-dependent transglutaminase FXIII have been found in monocytes andgranulocytes and PAD2 and TG2 in macrophages

In contrast to the TGs the entire family of PADs has been poorlycharacterized at the protein level However thanks to work by thegroups of Thompson [32] and Yamamoto [33] basic biochemical dataare now available for PAD4 This enzyme is a 74 kDa proteincontaining 663 amino

^acids with the proposed active site located at

Cys645 in close interaction with His471 In vitro using N-α-benzoyl-^L-

arginine ethyl ester as a substrate the deimination has a rathernarrow pH-dependence withmaximal activity at a neutral pH At a pHbelow 60 and above 85 the activity is reduced by approximately 80

^

5^ Common features between transglutaminases and

peptidylarginine deiminases

TGs and PADs share many biological and biochemical features(Table 2) [3435] The expression of the subclasses of the two enzymefamilies in tissues seems to be similar They are all calcium-dependent

^thiol-enzymes of similar molecular weights and act in the first step ofthe reaction by a nucleophilic attack on a polarized carbon atom of thesubstrate In both cases the reaction products are ammonia and aprotein with altered electrical charges Their substrates are specific

^peptide-bound glutamine and arginine residues respectively Thesubstrate properties are affected by the presence of adjacent prolineresidues Physiologically they are implicated in the terminal differen-tiation of cytoskeletons and in apoptosis [1134]

6^ Antibodies against peptidylarginine deiminase in RA

In CD antibodies can be detected against deamidated gliadin andagainst TG2 the enzyme that catalyzes the hydrolysis of specificglutamine residues to glutamic acids In RA the interest first focused

es and the pathogenesis of rheumatoid arthritis Eur J Intern Med

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on IgG-antibodies against citrullinated peptides Later stimulated bythe progress made in the field of CD six groups so far have reportedantibodies against PAD as well (Table 3) Takizawa et al [33] and Zhaoet al [36] used a hexa-histidine-tagged recombinant human PAD4as the antigen while Halvorsen et al [37] used an antigen preparedfrom a recombinant fused complex between glutathione S-transferaseand human PAD4 Nissinen et al [38] and Roth et al [35] preferred acommercial PAD purified from rabbit skeletal muscle and with re-tained enzyme activity This enzyme is a counterpart of human PAD2and is highly homologous to human PAD4 Harris et al [24] usedimmunoprecipitation with in vitro-translated radio-labelled humanPAD4

As illustrated in Table 3 other methodological conditions varied aswell From the experiences with CD we have learnt that laboratoryconditionsmight have a dramatic and sometimes overlooked effect onthe ELISA results For instance if the TG2 is in a native state theinclusion of calcium during the coating procedure will increase theaffinity between the enzyme and CD-antibodies several fold [39] Thisis true also in low-titre CD-samples collected from infants less thanone year of age [40] However if the zymogen is denatured and lackspotential enzyme activity an effect caused by the addition of calciumis unlikely Another pitfall affecting for example radio-bound assaysmight be the contamination of native enzymes originating from thecell components such as a lysate of rabbit reticulocytes utilized toexpress recombinant human radio-labelled enzymes [40]

Furthermore since^thiol-enzymes such as PADs and TGs easily

undergo oxidation addition of a reducing agent may be considered Inthe ELISAs with PAD Nissinen et al [38] added calcium dithiothreitol(DTT) and a small amount of EDTA during the coating procedureLearning from the field of CD and in order to avoid any interactionswithpotential inhibiting ions such as zinc Roth et al [35] used calcium butnot DTT or EDTA Since RA-patients are mostly ACPA-positive a recentsuggestion [31] was that PAD might be self-deiminating meaning thatthe proposed antibodies against PADwould simply reflect the presenceof citrulline residues in theenzymemolecule Since allmammalianPADsare strictly calcium-dependent Zhao et al [36] addressed this issue bynot including calciumduring the expression of rh-PAD4 thus excludingself-deimination Yet they were able to show antibodies in RA-samplesagainst PAD4 in a calcium-free environment

Considering the divergent conditions used by the six groups it iseven more impressive that they all reported a significantly increasedprevalence of IgG anti-PAD in RA In other words the situation in CDwith antibodies against the modified substrate (deamidated gliadin)as well as against the enzyme (TG2) that catalyzes the modification ismirrored in RAwith antibodies against both citrullinated peptides andagainst PAD

The specificity of anti-PAD remains to be fully explored but mostdata support high specificity for RA [41] So far only Nissinen et al[38] have reported a significantly enhanced prevalence of anti-PAD insera from patients with SLE (44

^) and primary Sjoumlgren syndrome

(pSS) (84^) The experimental conditions such as a high pH (80)

UNC

Table 3^

Antibodies against peptidylarginine deiminases Conditions and results are presented from

Antigen Ca2+ EDTA DTT pH RA

Rabbit muscle PAD + minus minus 74 40113^a (35

^

1771^b (24

^)

Rabbit muscle PAD + + + 80 5057 (88^)

His-tagged rhPAD4 minus minus minus ns 2142 (50^)

rhPAD4 + minus minus 75 96414 (23^)

His-tagged rhPAD4 minus minus minus ns 49109 (46^)

rhPAD4 minus + minus 74 1638 (46^)

rhPAD4 minus + minus 74 46129 (36^)

ns = not specified nd = not determined

^^

a Not treated with methotrexate (MTX) or other disease-modifying agent

^^

b Treated with MTX

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and presence of DTT and EDTA may be an explanation for theseunconfirmed results Furthermore the 19 pSS-patients were diag-nosed according to older non-stringent pSS criteria

Four of the six research teams also studied the association betweenanti-PAD and ACPA and they all found a positive correlation [2435ndash37]Moreover Zhao et al [36] reported a positive correlation between anti-PAD and disease activitymeasured as DAS 28 Similarly Halvorsen et al[37] reported a positive association between anti-PAD and increasedphysical disability radiographic damage scores and signs of arthritisHarris et al [24] found that anti-PAD4 antibodies were associatedwith more severe joint destruction in RA However in most analysesmedication is not reported which makes the studies difficult to judgeInterestingly Roth et al [35] showed that patients treated withmethotrexate (MTX) displayed lower levels of anti-PAD than untreatedcases while MTX did not seem to affect the levels of ACPA It is notknown if anti-PAD is present already in the pre-clinical phase of RA

7^ Antibodies against transglutaminases in RA

The prevalence of anti-TG2 in RA-sera has been studied by severalgroups Riente et al [42] did not find elevated levels of antibodies in RAwhen using either bovine or human recombinant TG2 as antigens Incontrast Picarelli et al [43] using native erythrocyte or recombinanthumanTG2 as antigens andRoth et al [35] using guinea pig TG2 foundsignificantly increased prevalence of anti-TG2 in RA All patients wereEMA-negative and the levels of anti-TG2 were low compared withthose observed in CD-patients Patients treated with MTX displayedlower titres of anti-TG2 which could reflect a disease-suppressingeffect of the drug [35] It is unclear whether anti-TG2 plays a patho-genetic role or is an epiphenomenon secondary to the disease processThis can also be said regarding the low titres of anti-TG2 recentlyreported in patients with cardiovascular diseases [44]

8^ Regulation of the activities of TGs and PADs

If post-translational modifications participate in the pathogenesisof autoimmune diseases factors affecting the regulation of the activityof enzymes involved are of interest Regulation of the expression ofthe enzymes and their activating agents such as calcium are the twopossible modifying factors RA and CD are more prevalent in womenHowever the gender difference in the incidence of RA is not seen inthe elderly [45] which could implicate sex hormones as a factor insusceptibility to disease or pathogenesis Experimentally the PAD2gene can be induced by 17β-oestradiol which gives rise to cyclicchanges in the levels of enzyme in the uterus and pituitary gland [46]Pregnancy is associated with a substantial reduction in onset of RAwhile the risk is increased in the post-partum period [45] Moreovermost pregnant RA-patients experience a remission [47] These obser-vations lend indirect support to possible involvement of PAD in thepathogenesis of RA Similarly the expression of TG2 is upregulated inthe subepithelial lamina propria of patients with CD

the various ELISA- and RBA-measurements

pSS SLE MS Controls Ref

) 278 nd 185 1100 Roth et al [35]

1619 1943 020 590 Nissinen et al [38]04 219 05 140 Takizawa et al [33]nd 1284 nd 9148 Halvorsen et al [37]248 667 441 5106 Zhao et al [36]nd nd nd 032 Harris et al [24]nd nd nd 032 Harris et al [24]

es and the pathogenesis of rheumatoid arthritis Eur J Intern Med

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Expression of TGs undergoes changes in the course of cell matu-ration Human monocytes contain FXIII exclusively but during thedifferentiation of the cell the expression of FXIII is down-regulated andreplaced by TG2 in the mature macrophage [48] Accordingly humanmacrophages collected at bronchial alveolar lavage only display TG2-activity Inanearly studybasedonagarosegel electrophoresis combinedwith activity staining TG-activity was detected in synovial fluids fromRA-patients sometimes originating fromFXIII and sometimes fromTG2in what appeared to be an irreproducible pattern [49] In retrospect theswitch of expression from FXIII to TG2 in the monocytemacrophagesystem is a likely explanation Similarly onewould expect the presenceof both PAD4 and PAD2 originating from monocytes and macrophagesrespectively in the synovial fluid of an inflamed joint [50] The messageis that depending on cell maturity one member of an enzyme familycan replace a specific function of another member

The metal ion dependence of the enzymes allows in vivo modu-lation of the activities of TGs and PADs PAD4 seems to be specificallydependent on calcium [32] while TGs can also be activated bystrontium and magnesium However the levels of calcium (1ndash2 mM)required for full activation of these enzymes in vitro are one orderof magnitude higher than the concentrations present intracellularly(1ndash100 μM) For decades TG2 was considered to be a strictly intra-cellular enzyme and sometimes even called cellular transglutami-nase It is now known that TG2 is also present extracellularly wherecalcium concentrations may reach activating levels Although nosimilar evidence has emerged for the PADs it is conceivable that theymay be activated when released during apoptosis Moreover in vitrothe presence of phospholipids reduces the calcium dependence ofPAD2 by almost twofold [51]

A novel approach to modulate TG2 and PAD activity could be theemployment of a physiological agent affecting the calcium-inducedactivationWe propose that zinc could be such an agent By competingwith calcium for the binding to the active site zinc is a very efficientinhibitor of the calcium-induced activation of TG2 and FXIII in vitro[52] Low concentrations of zinc also abolish the calcium-inducedincrease in affinity between CD-antibodies and TG2 in the ELISA tests[39] Zinc is also a potent inhibitor of PAD4 [32] Calcium and zinc maycontribute to the pathogenesis of yet another autoimmune diseasetype-1 diabetes Recently a zinc-transporting protein ZnT8 wasshown to be a major autoantigen in type-1 diabetes [53] Moreover acalcium-dependent thiol enzyme glutamic acid decarboxylase(GAD65) is investigated as a putative autoantigen in this disease[54] and might play an important role in the pathogenesis of type-1diabetes The high concentration of zinc in the insulin-producingpancreatic beta cells could also participate in GAD65 inhibition Thezinccalcium balance may thus have pathogenetic roles in autoim-mune diseases and could form the basis for therapeutic intervention

9^ RA smoking and alcohol

The site of the disease-triggering immunization leading to ACPA-positive RA is unknown Compelling evidence has identified smokingas a risk factor to develop RA An Australian study [55] found smokingto be associated with an increased prevalence of autoantibodies inparticular ANA Several later studies have amply confirmed this earlyreport [2056] Recent data from Klareskog et al [57] have shown thatthe association is dose-dependent but restricted to the ACPA- and SE-positive subset of RA Moreover as a consequence of upregulation ofPAD2 expression broncho-alveolar cells mainly macrophages fromsmokers but not from non-smokers stain positive for citrullinatedproteins [58] Long time smoking may lead to an increased expressionof PADs while inhibition of the enzyme activity would be anticipatedduring the instant exposure of thiol enzymes to cigarette smoke Thismay be explained theoretically by a reaction between electrophiliccomponents of cigarette smoke and the nucleophilic active site cys-teine of PADs or TGs Enzyme activity of TG2 in murine bone marrow-

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derived macrophages is significantly inhibited by aqueous extracts ofgas-phase cigarette smoke while the FXIII zymogen that exposes noreactive cysteine residues before being activated by thrombin andcalcium is not affected [59] However after activation FXIII is alsoinhibited [60] Inhibition of TGs in the broncho-alveolar tract causedby cigarette smoking is an attractive hypothesis in order to explainimpaired phagocytosis among smokers In RA interplay betweenaugmented PAD and dysfunctional TG2 could contribute to impairedremoval of the smoke-induced increase in PAD-catalyzed citrullinatedmaterial due to inhibited TG2-catalyzed phagocytosis or apoptosis

A case^ndashcontrol study on women from a population based cohort

of SLE in southern Sweden investigating environmental risk factorsrevealed unexpectedly that alcohol consumption appeared to be pro-tective in a dose-dependentway [61] This observationwas followed upin the collagen-IImodel of arthritis inmiceand showedprotectionwhenthe mice were fed 10

^ ethanol rather than water [62] More recently

two large Scandinavian casendashcontrol studies in RA reported dose-related protection [63] Moreover alcohol consumption in Koreanpatients with RA was protective against extra-articular manifestations[64] The mechanisms involved in these apparently salutary effects ofalcohol in connection with rheumatoid diseases remain unknown It isplausible that acetaldehyde the major metabolite of ethanol couldreact with nucleophiles such as the active site cysteine of PADs thusinhibiting enzyme activity Such effects have in fact been reported forTGs [65]

10^ The pathogenesis of ACPA-positive RA

^mdash^a hypothesis of

the initial steps

ACPA positivity seems to define a subset of RA constituting 60ndash70^

of all patients The subset is distinct in a number of ways as discussedabove regarding presence of SE relationship to smoking and erosive-ness The molecular pathogenesis probably differs from other subsetsof RA Therefore this discussion concerns ACPA-positive RA and theputative roles of PADs and TGs

Based on the intriguing interplay between endogenous TG2 anddietary gluten in CD the similarities between TGs and PADs (Table 2)and the emerging knowledge of ACPA and PAD immunity in RA wepropose the following hypothesis for the pathogenesis of ACPA-positive RA

Primary immunization

bull A local inflammation eg of the lung caused by an infection inha-lation of cigarette smoke or organic solvents or by other injuriousfactors [66] activates the innate immune system and increases thecitrullinating capacity by attracting macrophages to the site ofinflammation Genetic host factorsmay be operative in these events

bull Activation of PADs is induced by calcium or perhaps facilitated byreduced zinc availability caused by the inflammation

bull Specific arginine residues of endogenous proteins in the broncho-alveolar tract are consequently deiminated

bull The altered conformations caused by the citrullination render theproteins immunogenic by allowing attachment into the fourthanchoring groove of the SEs activating effector T cells During theenzymatic reaction PAD forms a complex with already partlydeiminated proteins and fragments of PAD are also presented toT cells

bull The evoked immune response results in expression of antibodiesagainst both citrulline-containing proteins and against PAD

Start of the symptomatic stage of RA (may occur several years afterthe primary immunization)

bull Changes in the balance of sexual hormones induce an increasedexpression of potential PAD

^activity in the joints

bull Fibrinogen fibronectin collagens and other proteins in the joint arecitrullinated by PADs (PAD2 and PAD4)

es and the pathogenesis of rheumatoid arthritis Eur J Intern Med

C

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525526527528529530531532533534535Q2536537Q3538539Q4540541542543544545546547Q5548549Q6550551552Q7553554555556557558Q8559560Q9561562563564565566Q10567568

6 P Stenberg et al European Journal of Internal Medicine xxx (2009) xxxndashxxx

ARTICLE IN PRESS

UNCO

RRE

bull The citrullinated proteins activate the immune system in theindividual previously immunized in the lung

bull More PAD-containing monocytes and macrophages are attracted tothe inflamed joint and a vicious circle is induced

bull Local acidosis in the inflamed joint affects the kinetics of the PAD-catalyzed reaction and contributes to increased stability of the com-plex between PAD and previously deiminated substrates leading tochronicity

bull A contra-lateral increase in vascular function and afferent neuralactivity leads to an inflammation with symmetric joint involvement[67]

11^ PADs and pharmacological interventions

In order to prevent irreversible destruction of the joints thepresent strategy is to treat RA with disease-modifying drugs in thevery first stage of the disease A novel theoretically attractiveapproach of an early intervention would be inhibition of the PADswhich are involved in the pathogenesis To this end a number of newsubstances with potential PAD4-inhibiting capacity have beensynthesized and are at present evaluated in vitro [68] A similarapproach has been taken towards development of inhibitors againstTGs In order to achieve specificity a backbone which resembles asubstrate localizes the inhibitor to the active site of the calcium-activated enzyme Then the warhead a carbon atom activated by anadjacent halogen alkylates the thiolate of the active site cysteine ofthe enzyme with the halo atom serving as the leaving group [32] Theresult will be an irreversible inhibition of PAD4 with the main part ofthe inhibitor covalently attached to the active site Toxic and immu-nological effects of this approach remain to be studied Moreover oneshould be aware of possible redundancy with an increased expressionof other PAD isoenzymes Similarly TG2- or PAD2-knock-out micemight function normally simply because other isoenzymes can takeover their missions

Screening of older pharmaceuticals for PAD-inhibitory capacitymay be an efficient and safe way to avoid unexpected adverse effects[69] A number of well-known drugs seem to have affinity for theactive site of PAD4 Especially streptomycin and a number oftetracyclines such as chlortetracycline turned out to be reversiblelow-potent inhibitors of PAD4 in vitro

If the autoantigen can be specifically identified an induction ofimmune tolerance (tolerization) may be a way forward This approachis entering phase III in type-1 diabetes [54] in order to inducetolerance against a known autoantigen (GAD65) However recentlyOak et al [70] reported that anti-GAD65 antibodies are present innormal individuals in complex with anti-idiotypic antibodies Type-1diabetic patients showed a lack of such anti-idiotypic antibodies Thusthe situation is more complex than expected In CD with TG2 as theautoantigen the situation may be more straightforward However toour knowledge attempts to induce immune tolerance in CD byinjecting TG2 have not been performed perhaps because gluten-freediet is an efficient treatment In RA so far the autoantigens have notbeen clearly defined Induction of tolerance against type II collagen isunder investigation [71] However attempts to suppress establishedRA remain unsuccessful In fact it remains to be proven that inductionof immune tolerance is a successful way in any type of autoimmunedisease

12^ Conclusions

Better understanding of initiating basicmechanismswould have thepotential to device preventing strategies against autoimmune diseasesProgress in the field of CDmay point to new therapeutic possibilities Inthe initial steps of CD a post-translational TG2-catalyzeddeamidation ofspecific glutamine residues in dietary gluten results in the formation ofantibodies against bothmodified gliadin and against TG2 In this review

Please cite this article as Stenberg P et al Peptidylarginine deiminas(2009) doi101016jejim200908007

TEDPR

OOF

we have highlighted the similarities between TGs and PADs and alsoindicated some similar immunological features of CD and RA In a subsetconstituting 60ndash70

^ of all

^RA-patients ACPAs constitute a specific

biomarkerMoreover auto^antibodies against PADcan bedetected in RA

Thus in two major T cell-dependent autoimmune diseases autoanti-bodies canbedetected against a post-translationallymodifiedprotein aswell as against the calcium-dependent thiol enzyme responsible for thesubstrate modification

By reflecting the progress in CD the pathogenesis of RA can behypothesized where expression and regulation of PADs play signifi-cant roles It may be helpful to apply comparative as well as individualapproaches to the study of autoimmune diseases Moreover the hopeis that new insights into the pathological mechanisms may lead tonovel pharmacotherapeutic principles

Learning points

bull Post-translational modifications of proteins are associated withautoimmune diseases

bull TG2 is a major autoantigen in CDbull Antibodies against citrullinated peptides (ACPA) are a specificmarker for a distinct subset of RA

bull Antibodies against the citrullinating enzyme PAD are present in RAbull Presence of anti-PAD indicates a more severe form of RAbull Expression of PAD and regulation of zymogen activation may playsignificant roles in the pathogenesis of RA

bull Cigarette smoking and alcohol consumption affect the risk of RA indifferent ways

bull PAD is a putative therapeutic target in RA

References

[1] Doyle HA Mamula MJ Post-translational protein modifications in antigenrecognition and autoimmunity Trends Immunol 200122(8)443ndash9

[2] Doyle HA Mamula MJ Posttranslational modifications of self-antigens Ann N YAcad Sci 200510501ndash9

[3] Fox PC Autoimmune diseases and Sjogrens syndrome an autoimmuneexocrinopathy Ann N Y Acad Sci 2007109815ndash21

[4] Committee TADC Progress in autoimmune disease research a report tocongress US Department of Health and Human Service National Institutes ofHealth National Institute of Allergy and Infectious Diseases (Access at wwwniaidnihgovpublicationspdfADCCFinalpdf) 2005

[5] Milioti N et al Antigen-induced immunomodulation in the pathogenesis ofatherosclerosis Clin Dev Immunol 20082008723539

[6] Jellinger KA et al Biomarkers for early diagnosis of Alzheimer disease lsquoALZheimerASsociated genersquomdasha new blood biomarker J Cell Mol Med 200812(4)1094ndash117

[7] Dieterich W et al Identification of tissue transglutaminase as the autoantigen ofceliac disease Nat Med 19973(7)797ndash801

[8] Lorand L Factor XIII and the clotting of fibrinogen frombasic research tomedicineJ Thromb Haemost 20053(7)1337ndash48

[9] Dieterich W Esslinger B Schuppan D Pathomechanisms in celiac disease Int ArchAllergy Immunol 2003132(2)98ndash108

[10] Stenberg P Roth EB Sjoberg K Transglutaminase and the pathogenesis of coeliacdisease Eur J Intern Med 200819(2)83ndash91

[11] Gyorgy B et al Citrullination a posttranslational modification in health anddisease Int J Biochem Cell Biol 200638(10)1662ndash77

[12] Tarcsa E et al Protein unfolding by peptidylarginine deiminase Substratespecificity and structural relationships of the natural substrates trichohyalin andfilaggrin J Biol Chem 1996271(48)30709ndash16

[13] Tarcsa E et al The fate of trichohyalin Sequential post-translational modificationsby peptidyl-arginine deiminase and transglutaminases J Biol Chem 1997272(44)27893ndash901

[14] Harauz G Musse AA A tale of two citrullinesmdashstructural and functional aspects ofmyelin basic protein deimination in health and disease NeurochemRes 200732(2)137ndash58

[15] Vossenaar ER et al PAD a growing family of citrullinating enzymes genesfeatures and involvement in disease Bioessays 200325(11)1106ndash18

[16] Girbal-Neuhauser E et al The epitopes targeted by the rheumatoid arthritis-associated antifilaggrin autoantibodies are posttranslationally generated onvarious sites of (pro)filaggrin by deimination of arginine residues J Immunol1999162(1)585ndash94

[17] van Venrooij WJ van Beers JJ Pruijn GJ Anti-CCP antibody a marker for the earlydetection of rheumatoid arthritis Ann N Y Acad Sci 20081143268ndash85

[18] Snir O et al Multiple antibody reactivities to citrullinated antigens in sera frompatients with rheumatoid arthritis association with HLA-DRB1 alleles AnnRheum Dis 200968(5)736ndash43

es and the pathogenesis of rheumatoid arthritis Eur J Intern Med

569Q11570571572Q12573574575Q13576577578Q14579580Q15581582583Q16584585586Q17587588589Q18590591Q19592593594Q20595596Q21597598599Q22600601Q23602603Q24604605606Q25607608609610611Q26612613614Q27615616Q28617618619Q29620621622623624625Q30626627628Q31629630631632Q32633634Q33635636Q34637638

639640641Q35642643644Q36645646Q37647648649650651Q38652653654Q39655656657658659Q40660661662Q41663664Q42665666667668669Q43670671Q44672673674Q45675676677678679Q46680681682Q47683684Q48685686687Q49688689690691692693694695696697Q50698699700701Q51702703704Q52705706

707

7P Stenberg et al European Journal of Internal Medicine xxx (2009) xxxndashxxx

ARTICLE IN PRESS

CORR

EC

[19] Rantapaa-Dahlqvist S et al Antibodies against cyclic citrullinated peptide and IgArheumatoid factor predict the development of rheumatoid arthritis ArthritisRheum 200348(10)2741ndash9

[20] Klareskog L et al A new model for an etiology of rheumatoid arthritis smokingmay trigger HLA-DR (shared epitope)-restricted immune reactions to autoanti-gens modified by citrullination Arthritis Rheum 200654(1)38ndash46

[21] Lundberg K et al Citrullinated proteins have increased immunogenicity andarthritogenicity and their presence in arthritic joints correlates with diseaseseverity Arthritis Res Ther 20057(3)R458ndash67

[22] Uysal H et al Structure and pathogenicity of antibodies specific for citrullinatedcollagen type II in experimental arthritis J Exp Med 2009206(2)449ndash62

[23] Hill JA et al Cutting edge the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB10401 MHC class II molecule J Immunol 2003171(2)538ndash41

[24] Harris ML et al Association of autoimmunity to peptidyl arginine deiminase type4 with genotype and disease severity in rheumatoid arthritis Arthritis Rheum200858(7)1958ndash67

[25] van Gaalen FA et al Association between HLA class II genes and autoantibodies tocyclic citrullinated peptides (CCPs) influences the severity of rheumatoid arthritisArthritis Rheum 200450(7)2113ndash21

[26] Kang CP et al A functional haplotype of the PADI4 gene associated with increasedrheumatoid arthritis susceptibility in Koreans Arthritis Rheum 200654(1)90ndash6

[27] Suzuki A et al Functional haplotypes of PADI4 encoding citrullinating enzymepeptidylarginine deiminase 4 are associated with rheumatoid arthritis Nat Genet200334(4)395ndash402

[28] Burr ML et al PADI4 genotype is not associated with rheumatoid arthritis in alarge UK Caucasian population Ann Rheum Dis 2009

[29] Poor G et al Genetic background of anticyclic citrullinated peptide autoantibodyproduction in Hungarian patients with rheumatoid arthritis Ann N Y Acad Sci2007111023ndash32

[30] Iwamoto T et al Association between PADI4 and rheumatoid arthritis a meta-analysis Rheumatology (Oxford) 200645(7)804ndash7

[31] Mechin MC et al Update on peptidylarginine deiminases and deimination in skinphysiology and severe human diseases Int J Cosmet Sci 200729(3)147ndash68

[32] Kearney PL et al Kinetic characterization of protein arginine deiminase 4 atranscriptional corepressor implicated in the onset and progression of rheumatoidarthritis Biochemistry 200544(31)10570ndash82

[33] Takizawa Y et al Peptidylarginine deiminase 4 (PADI4) identified as aconformation-dependent autoantigen in rheumatoid arthritis Scand J Rheumatol200534(3)212ndash5

[34] Roth B Transglutaminase and peptidylarginine deiminase in the pathogenesis ofautoimmune diseases Doctorial Thesis Lund University 2008

[35] Roth EB et al Antibodies against transglutaminases peptidylarginine deiminaseand citrulline in rheumatoid arthritis

^mdashnew pathways to epitope spreading Clin

Exp Rheumatol 200624(1)12ndash8[36] Zhao J et al Prevalence and significance of anti-peptidylarginine deiminase 4

antibodies in rheumatoid arthritis J Rheumatol 200835(6)969ndash74[37] Halvorsen EH et al Serum IgG antibodies to peptidylarginine deiminase 4 in

rheumatoid arthritis and associations with disease severity Ann Rheum Dis200867(3)414ndash7

[38] Nissinen R et al Peptidylarginine deiminase the arginine to citrulline convertingenzyme is frequently recognized by sera of patients with rheumatoid arthritissystemic lupus erythematosus and primary Sjogren syndrome Scand J Rheumatol200332(6)337ndash42

[39] Roth EB Sjoberg K Stenberg P Biochemical and immuno-pathological aspects oftissue transglutaminase in coeliac disease Autoimmunity 200336(4)221ndash6

[40] Agardh D et al Calcium activation of tissue transglutaminase in radioligandbinding and enzyme-linked autoantibody immunoassays in childhood celiacdisease Clin Chim Acta 2005358(1ndash2)95ndash103

[41] Roth E et al Pathogenesis of autoimmune diseases antibodies againsttransglutaminase peptidylarginine deiminase and protein-bound citrulline inprimary Sjogrens syndrome multiple sclerosis and Alzheimers disease Scand JImmunol 200867(6)626ndash31

[42] Riente L et al Antibodies to tissue transglutaminase and Saccharomyces cerevisiaein ankylosing spondylitis and psoriatic arthritis J Rheumatol 200431(5)920ndash4

[43] Picarelli A et al Anti-tissue transglutaminase antibodies in arthritic patients adisease-specific finding Clin Chem 200349(12)2091ndash4

[44] Di Tola M et al Antitissue transglutaminase antibodies in acute coronarysyndrome an alert signal of myocardial tissue lesion J Intern Med 2008263(1)43ndash51

UN

Please cite this article as Stenberg P et al Peptidylarginine deiminas(2009) doi101016jejim200908007

TEDPR

OOF

[45] Silman AJ Pearson JE Epidemiology and genetics of rheumatoid arthritis ArthritisRes 20024(Suppl 3)S265ndash72

[46] Senshu T et al Peptidylarginine deiminase in rat pituitary sex difference estrouscycle-related changes and estrogen dependence Endocrinology 1989124(6)2666ndash70

[47] Gerosa M et al Rheumatoid arthritis a female challenge Womens Health(Lond Engl) 20084(2)195ndash201

[48] Seiving B et al Transglutaminase differentiation during maturation of humanblood monocytes to macrophages Eur J Haematol 199146(5)263ndash71

[49] Wollheim FA Stenberg P Marsal L Characterisation of transglutaminases inplasma and synovial fluid from patients with arthritis 15th International Congressof Rheumatology 1981 Paris

[50] Vossenaar ER et al Expression and activity of citrullinating peptidylargininedeiminase enzymes in monocytes and macrophages Ann Rheum Dis 200463(4)373ndash81

[51] Musse AA et al Kinetics of human peptidylarginine deiminase 2 (hPAD2)mdashreduction of Ca2+ dependence by phospholipids and assessment of proposedinhibition by paclitaxel side chains Biochem Cell Biol 200886(5)437ndash47

[52] Credo RB Stenberg P Tong YS Lorand L Inhibition of fibrinoligase andtransglutaminase by zinc ions Fed Proc 1976 San Fransisco

[53] Wenzlau JM et al The cation efflux transporter ZnT8 (Slc30A8) is a majorautoantigen in human type 1 diabetes Proc Natl Acad Sci U S A 2007104(43)17040ndash5

[54] Agardh CD et al Clinical evidence for the safety of GAD65 immunomodulation inadult-onset autoimmune diabetes J Diabetes Complications 200519(4)238ndash46

[55] Mathews JD et al Association of autoantibodies with smoking cardiovascularmorbidity and death in the Busselton population Lancet 19732(7832)754ndash8

[56] Silman AJ Newman J MacGregor AJ Cigarette smoking increases the risk ofrheumatoid arthritis Results from a nationwide study of disease-discordant twinsArthritis Rheum 199639(5)732ndash5

[57] Klareskog L et al Immunity to citrullinated proteins in rheumatoid arthritis AnnuRev Immunol 200826651ndash75

[58] Makrygiannakis D et al Smoking increases peptidylarginine deiminase 2 enzymeexpression in human lungs and increases citrullination in BAL cells Ann RheumDis 2008

[59] Roth WJ et al Characterization of two distinct transglutaminases of murine bonemarrow-derived macrophages effects of exposure of viable cells to cigarettesmoke on enzyme activity J Leukoc Biol 198742(1)9ndash20

[60] Galanakis DK Laurent P Janoff A Cigarette smoke contains anticoagulants againstfibrin aggregation and factor XIIIa in plasma Science 1982217(4560)642ndash5

[61] Bengtsson AA et al Risk factors for developing systemic lupus erythematosusa casendashcontrol study in southern Sweden Rheumatology (Oxford) 200241(5)563ndash71

[62] Jonsson IM et al Ethanol prevents development of destructive arthritis Proc NatlAcad Sci U S A 2007104(1)258ndash63

[63] Kallberg H et al Alcohol consumption is associated with decreased risk ofrheumatoid arthritis results from two Scandinavian casendashcontrol studies AnnRheum Dis 200968(2)222ndash7

[64] Kim SK et al Anti-cyclic citrullinated peptide antibody smoking alcohol con-sumption and disease duration as risk factors for extraarticular manifestations inKorean patients with rheumatoid arthritis J Rheumatol 200835(6)995ndash1001

[65] Suchocki EA Brecher AS The effect of acetaldehyde on human plasma factor XIIIfunction Dig Dis Sci 200752(12)3488ndash92

[66] Matzinger P Tolerance danger and the extended family Annu Rev Immunol199412991ndash1045

[67] Kelly S Dunham JP Donaldson LF Sensory nerves have altered functioncontralateral to a monoarthritis and may contribute to the symmetrical spreadof inflammation Eur J Neurosci 200726(4)935ndash42

[68] Luo Y et al Inhibitors and inactivators of protein arginine deiminase 4 functionaland structural characterization Biochemistry 200645(39)11727ndash36

[69] Knuckley B Luo Y Thompson PR Profiling protein arginine deiminase 4 (PAD4) anovel screen to identify PAD4 inhibitors Bioorg Med Chem 200816(2)739ndash45

[70] Oak S et al The lack of anti-idiotypic antibodies not the presence of thecorresponding autoantibodies to glutamate decarboxylase defines type 1diabetes Proc Natl Acad Sci U S A 2008105(14)5471ndash6

[71] Ju JH et al Oral administration of type-II collagen suppresses IL-17-associatedRANKL expression of CD4+ T cells in collagen-induced arthritis Immunol Lett2008117(1)16ndash25

es and the pathogenesis of rheumatoid arthritis Eur J Intern Med