The complexity of Sjögren's syndrome: Novel aspects on pathogenesis

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Immunology Letters 141 (2011) 1– 9

Contents lists available at ScienceDirect

Immunology Letters

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he complexity of Sjögren’s syndrome: Novel aspects on pathogenesis

oland Jonssona,b,∗, Petra Vogelsanga, Roman Volchenkova, Alexander Espinosac,arie Wahren-Herleniusc, Silke Appela

Broegelmann Research Laboratory, The Gade Institute, Laboratory Building, 5th floor, University of Bergen, N-5021 Bergen, NorwayDepartment of Rheumatology, Haukeland University Hospital, N-5021 Bergen, NorwayRheumatology Unit, Department of Medicine, Karolinska Institutet, SE-17176 Stockholm, Sweden

r t i c l e i n f o

rticle history:eceived 9 June 2011eceived in revised form 21 June 2011ccepted 22 June 2011vailable online 12 July 2011

eywords:jögren’s syndromeutoantibodiesype I interferonendritic cellsutoimmunity

a b s t r a c t

In Sjögren’s syndrome, like in most other autoimmune diseases, the enigma leading to a pathogenicattack against self has not yet been solved. By definition, the disease must be mediated by specificimmune reactions against endogenous tissues to qualify as an autoimmune disease. In Sjögren’s syn-drome the autoimmune response is directed against the exocrine glands, which, as histopathologicalhallmark of the disease, display persistent and progressive focal mononuclear cell infiltrates. Clinically,the disease in most patients is manifested by two severe symptoms: dryness of the mouth (xerosto-mia) and the eyes (keratoconjunctivitis sicca). A number of systemic features have also been describedand the presence of autoantibodies against the ubiquitously expressed ribonucleoprotein particles Ro(Sjögren’s-syndrome-related antigen A – SSA) and La (SSB) further underline the systemic nature ofSjögren’s syndrome. The original explanatory concept for the pathogenesis of Sjögren’s syndrome pro-posed a specific, self-perpetuating, immune mediated loss of acinar and ductal cells as the principalcause of salivary gland hypofunction. Although straightforward and plausible, the hypothesis, however,falls short of accommodating several Sjögren’s syndrome-related phenomena and experimental findings.Consequently, researchers considered immune-mediated salivary gland dysfunction prior to glandulardestruction and atrophy as potential molecular mechanisms underlying the symptoms of dryness in

Sjögren’s syndrome. Accordingly, apoptosis, fibrosis and atrophy of the salivary glands would representconsequences of salivary gland hypofunction. The emergence of advanced bio-analytical platforms fur-ther enabled the identification of potential biomarkers with the intent to improve Sjögren’s syndromediagnosis, promote the development of prognostic tools for Sjögren’s syndrome and the long-term goal toidentify possible processes for therapeutic treatment interventions. In addition, such approaches allowedus to glimpse at the apparent complexity of Sjögren’s syndrome.

. Introduction

Sjögren’s syndrome is a complex autoimmune rheumatic dis-ase characterized by mononuclear cell infiltration of exocrinelands and the presence of autoantibodies against the ribonucle-protein particles SSA/Ro and SSB/La. The salivary and lacrimal

lands are the principal targets of a proposed T cell mediatedhronic inflammation, with a resulting glandular atrophy andeficient function. The clinical consequences are dry eyes (kerato-

∗ Corresponding author at: Broegelmann Research Laboratory, The Gade Institute,aboratory Building, 5th floor, N-5021 Bergen, Norway. Tel.: +47 55974649;ax: +47 55975817.

E-mail addresses: [email protected] (R. Jonsson),[email protected] (P. Vogelsang), [email protected] (R.olchenkov), [email protected] (A. Espinosa), [email protected] (M.ahren-Herlenius), [email protected] (S. Appel).

165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.imlet.2011.06.007

© 2011 Elsevier B.V. All rights reserved.

conjunctivitis sicca) and dry mouth (xerostomia). Due to affectionof other organs there are a number of systemic features of Sjögren’ssyndrome.

One of the unsolved questions in this directed autoimmuneattack has been the mechanism responsible for the formation ofmononuclear cell accumulations in exocrine glands. It has beenhypothesized that primary events (e.g. infections) may occur in theglands themselves, followed in a second phase by an autoimmuneattack. Whether B cell activation is a primary cause or a secondaryeffect in Sjögren’s syndrome is not known.

The etiology and many aspects of the pathogenesis of Sjögren’ssyndrome are still elusive. Several factors such as genetic predis-position and environmental triggers influence the development ofSjögren’s syndrome, and only after irreversible organ damage has

occurred become clinical symptoms evident. Moreover, diagnosisis hampered by the heterogeneity of manifestations leading to fur-ther delay of the correct diagnosis (Fig. 1). So far, there exists no curefor this disease, and the treatment is limited to ease the symptoms.

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The most definitive test is biopsy of the minor salivary glands

Fig. 1. Proposed etiopathogenic events prior to diagnosis of Sjögren’s syndrome.dapted from Jonsson and Brun [101].

In this review we will, after giving some background informationbout Sjögren’s syndrome, highlight recent and novel aspects onhe pathogenesis.

. Epidemiology

Similarly to many of the systemic rheumatic diseases the diag-osis of Sjögren’s syndrome cannot be readily made, for instance onhe basis of a single test or symptom. Classification criteria with aist of well defined clinical and laboratory variables are an alterna-ive method of securing uniform patient populations for researchurposes. During the last three decades various national and inter-ational groups have developed multiple criteria sets for Sjögren’syndrome, giving rise to differing research results and epidemio-ogical data.

After a thorough process of validation and re-validation theriteria proposed by the European Study Group were furthereveloped and amended by European and American experts. Theesulting American–European Consensus Group criteria (AECC) forjögren’s syndrome published in 2002 [1] have gained a widecceptance (as per today the most widely cited reference for crite-ia) and they are also increasingly used as a guidance for the clinicaliagnosis of Sjögren’s syndrome. In addition to eye and/or oral siccaymptoms supplied with measurements of a decreased exocrineunction, evidence of autoimmunity is required, either as showny autoantibodies to SSA/Ro or SSB/La, or by a positive biopsy ofhe minor salivary glands of the lower lip, with a focus score of oner more.

Sjögren’s syndrome is found in all parts of the world. Regionalifferences have not been much explored. There is a large femalereponderance with a ratio of female:male of about 9:1. The disease

s found in all age groups but usually starts between the ages 40 and0 and is rarely seen in children and adolescents. In parallel withhe increase of sicca symptoms with age in the background pop-lation, the prevalence of Sjögren’s syndrome also increases withge. Often there is a several-year delay from the start of symptomso diagnosis.

As a consequence of different criteria, the prevalence estimatesave varied widely, with some studies reporting up to 3% of theopulation. Recent studies based on the American–European crite-ia show prevalences of about 0.1% with confidence intervals in theanges of <0.1–0.4% [2,3]. There are few studies on the incidence of

jögren’s syndrome but according to the most recent criteria thencidence rate is probably between 3 and 6 per 100,000 per year4].

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In addition to female gender, first grade relatives with anautoimmune disease and previous pregnancies have been identi-fied as epidemiological risk factors for the development of Sjögren’ssyndrome [5].

3. Clinical features and malignant lymphoproliferation

The hallmarks of Sjögren’s syndrome are the sicca symptoms ofthe eyes and mouth. Dryness of the eyes may be experienced asa gritty sensation, soreness or intolerance to contact lenses. Dry-ness of the mouth may give rise to difficulties in the swallowing ofdry foods without fluid, and need for frequent small sips of water,also at night. Loss of the protective and antimicrobial propertiesof saliva may increase dental caries and predispose for oral can-didiasis. In addition, patients may have other symptoms related todryness of the mucous membranes or skin, for instance nasal crustsand nose bleeds, hoarseness and speaking problems, difficulties inswallowing, halithosis, reduced sense of smell and taste, dry cough,intermittent parotid or submandibular swelling, and dyspareunia.

Sjögren’s syndrome is also a systemic disease. Many patientshave problems with fatigue and joint and muscular pain, andunspecific neurological complaints. Less common extraglandularaffections are arthritis, Raynaud’s phenomenon, skin vasculi-tis, lymphadenopathy, serositis, pulmonary fibrosis, symptomsfrom the central nervous system and renal tubular acidosis. Co-morbidity in the form of thyreoid diseases may occur in up to onethird of the patients [6–10].

Non-Hodgkin lymphoma, which may be of the mucosa asso-ciated lymphoid tissue (MALT) type, occurs at an increased ratein Sjögren’s syndrome. Previous estimates of a more than 40-foldincrease may have been too high, as a large linked registry-basedstudy showed a 16-fold increased risk [11]. Palpable purpura, lowlevels of C3 and C4, CD4+ lymphopenia, a low CD4+/CD8+ T cellratio, and parotid enlargement at first visit are presently identifiedas predictive factors for lymphoma in Sjögren’s syndrome [11,12].

With the exception of lymphoma, Theander and colleaguesfound no increased mortality or cancer incidence in Sjögren’s syn-drome in two large Swedish prospective cohort studies [11,13].

4. Diagnosis and diagnostic tests – revised EU criteria –2002

A reduced exocrine function or sicca symptoms may be causedby a variety of conditions and may be age related. Evidence ofautoimmunity as outlined in the AECC [1] helps to distinguishSjögren’s syndrome from these other conditions. The AECC isincreasingly used as a guidance or also help for the clinical diagnosisof Sjögren’s syndrome. In the AECC four out of six criteria are neededfor a diagnosis. At least one of these four must be autoantibodies ora positive labial gland biopsy.

Reduction of tear flow may be assessed by the Schirmer’s test,a measurement of the wetting during 5 min of a 30 mm filter stripplaced over the brim of the lower eye lid. Less than 5 mm of wettingis considered abnormal. Sicca symptoms of the eye may also becaused by changes of the physicochemical properties or quality ofthe tears, for which the tear film break up time is indicative. Thenumber of corneal abrasions is yet another measure of dry eyes [1].

Dryness of the mouth may be related to saliva production, whichis mostly measured by the un-stimulated salivary flow rate. Thepatient is asked to collect all of the saliva produced during 15 mininto a vial. A volume of less than 1.5 ml is considered abnormal [1].

from inside of the lower lip. If histology shows one or moremononuclear cell aggregates of 50 or more cells per 4 mm2 the testis considered positive [14].

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Autoantibodies to the SSA/Ro or SSB/La antigens are alsotrongly indicative of Sjögren’s syndrome, although they may beound in other systemic diseases like systemic lupus erythemato-us (SLE). More than 50% of Sjögren’s syndrome patients have oner more of these autoantibodies. Laboratory testing may also showhat some patients have an increased erythrocyte sedimentationate, and polyclonal hypergammaglobulinemia and rheumatoidactors may also be found [6].

The clinical examination may reveal a dry and lobulated tongue,ssures in the corners of the mouth, dry skin, and in some casesarotid or submandibular swelling, lymphadenopathia, palpableurpura especially of the lower extremities, and arthritis.

. Etiopathogenesis

.1. Genetic aspects in Sjögren’s syndrome

Genetic factors contribute to Sjögren’s syndrome, and as in manyther autoimmune diseases a strong association to specific MHClleles has been shown [15]. Association studies of other loci haveeen sparse so far [16]. Recent studies, however, revealed IRF5 andTAT4 gene variants associated with an increased risk Sjögren’syndrome [17–19]. Moreover, the largest association study per-ormed so far using combined patient material from Sweden andorway revealed genetic association also with EBF1, FAM167A-BLKnd TNFSF4 [20] as well as CHRM3 [21].

.2. Immunologic findings in salivary glands, ectopic germinalenter-like structures

The pathognomonic histological finding in glandular biopsiess a progressive focal infiltration of mononuclear lymphoid cells,eplacing glandular epithelium (lymphoepithelial lesion). This cor-elates largely to the reduced salivary secretion. However, theechanisms leading to attraction and accumulation and the patho-

hysiological role of the infiltrating cells remain undefined. Thenfiltrating cells interfere with glandular function at several levels:estruction of glandular structures by cell-mediated mechanisms;ecretion of cytokines that activate pathways related to interferonsIFNs); local production of autoantibodies, etc.

The focal infiltration consists mainly of T cells, but alsoacrophages and plasma cells. Normally, lymphocytes circulate

n the blood and invade the tissue as a response to infection ornjury. This is a complex process regulated by a range of adhesion

olecules on the inflammatory cell surfaces and the endothelialells. The lymphocytes adhere to the endothelium by means ofdhesion molecules and can move from circulation to tissue. Inter-stingly, serum levels of an adhesion molecule related to epithelialells, E-cadherin, has been found to be increased in Sjögren’s syn-rome indicating the close interaction between epithelial cells and

ymphocytic organization [22].Recent studies have identified germinal center-like structures

hat could be identified in up to 1/3 of salivary gland samples andoincided with elevated titres of rheumatoid factor, other autoan-ibodies, increased IgG levels and higher focus score [23]. Theseesults indicate that formation of ectopic lymphoid microstruc-ures in non-lymphoid organs participate in the pathogenesis.nvolvement of salivary glands as a site of ectopic germinal centerormation and selection of high-affinity autoantibodies medi-ting this autoimmune state, suggest novel targets for futuremmunomodulatory therapeutic strategies.

Very recently it has been proposed in a retrospective study thathe presence of germinal center-like structures in diagnostic labiallands by light microscopy in Sjögren’s syndrome might be used as

highly predictive and easy to obtain marker for NHL development

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allowing risk stratification and possibly preventive B cell directedtherapy [24].

5.3. Dendritic cells in Sjögren’s syndrome

The immunological mechanisms behind the self-directeddestruction of salivary gland tissue and causes of glandular dys-function are still not understood despite decades of research.However, the composition of the tissue lesions seen in minorsalivary glands of patients with Sjögren’s syndrome has been exten-sively examined over the past years.

Immunohistochemical staining of patient biopsies has revealedthat B- and T cells account in large amounts for the infiltrating lym-phocytes, but also macrophages, NK cells and dendritic cells (DC)have been disclosed to constitute a minor extent of cells within theinflammatory foci [25–27]. Based on their unique ability to partic-ipate in the establishment of peripheral tolerance [28,29], DC havegained more attention regarding their potential role in autoim-mune diseases in recent years. As professional antigen presentingcells, they are able to both initiate and sustain an effective immuneresponse [30], but at the same time play a central role in themaintenance of tolerance to self-antigens. Thus, defects in DC func-tions and populations might contribute to the aberrant immuneactivation seen in patients with autoimmune disorders such asSjögren’s syndrome [31]. Auto-reactive T cells frequently escapecentral tolerance mechanisms, and in individuals with autoim-mune manifestations, the role of antigen-presenting DC controllingT cell functions in the periphery, seem to be more important thanpreviously believed (Fig. 2).

DC are relatively rare populations of cells in human peripheralblood and to date, two main subsets of DC have been character-ized: myeloid DC (mDC) and plasmacytoid DC (pDC). According toexpression of distinct surface markers, mDC can be further sub-divided into the BDCA-1 (CD1c) expressing mDC1 subset and theBDCA-3 (CD141) expressing mDC2 subset [32]. Due to the lowcell numbers in blood, little is known about the functions of thesemDC subsets so far, but it recently became evident that mDC2 andnot mDC1 are able to cross-present necrotic cell antigens [33–36].Plasmacytoid DC (pDC) are characterized by expression of BDCA-2(CD303) and BDCA-4 (CD304) and are known as the main producersof IFN-� [37].

Monocytes are classically known to constitute precursors formacrophages, but since Sallusto and Lanzavecchia demonstratedthe ability to generate DC from monocytes in vitro in 1994,monocyte-derived DC became the most commonly used DC modelin human research [38]. In 1998, Randolph and colleagues provedthe differentiation of human monocytes into DC using an elegantmodel of transendothelial trafficking [39]. It is now widely acceptedin the field that monocytes differentiate into monocyte-derived DCunder inflammatory conditions also in vivo [40]. These so-calledtipDC are characterized by production of tumor necrosis factor-�(TNF-�) and inducible nitric oxide synthase (iNOS). So far, tipDChave only been reported and investigated in mice, thus their rolein human autoimmune processes still needs to be elucidated. Nev-ertheless, it was shown that monocyte-derived DC from patientswith SLE overexpress CD86, and might therefore be overactive instimulating T cells [41].

IFN-� is a cytokine, which plays an important role in manyimmune regulatory processes. Thus, pDC as a putative source ofIFN-� might influence the pathogenesis of Sjögren’s syndromeby activating several other immune cells through inappropriatesecretion of IFN-� (Fig. 3). Initially, the presence of IFN-� con-

taining cells in minor salivary gland biopsies from patients withprimary Sjögren’s syndrome provided the first evidence that pDCindeed participate in formation of inflammatory foci [42]. More-over, gene expression profiling of minor salivary glands revealed

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Fig. 2. Hypothesis of how dysfunctional dendritic cells might initiate an autoimmune disease such as Sjögren’s syndrome. An initial viral infection could stimulate plas-macytoid dendritic cells (pDC) to secrete IFN-�. This increased IFN-� level could then wrongly stimulate autoantigen-presenting myeloid DC (mDC). In a non-predisposedindividual, tolerance would be sustained by recruitment of regulatory T cells (Treg), induction of T cell anergy and apoptosis of the antigen-presenting cell. In a predisposedi ctive TM

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ndividual, however, this malfunctioning of DC could lead to stimulation of autoreaodified from Vogelsang et al. [31].

n up-regulation of IFN regulated genes (IFN signature) in patientsith Sjögren’s syndrome [43]. The locally increased IFN signatureas later been confirmed along with the presence of pDC in thealivary glands by direct staining with CD123 and BDCA-2 pDCarkers [44]. Besides the occurrence of pDC in the inflammatory

oci, reduced levels of pDC were found in peripheral blood fromatients with Sjögren’s syndrome [45,46], suggesting the recruit-ent of pDC to the target organ, but a direct connection still needs

o be established. The increased IFN signature in patients withjögren’s syndrome seems not to be only locally restricted to thealivary glands, as a recent study identified an increased expressionf IFN inducible genes also in monocytes from Sjögren’s syndromeatients [45].

Apart from pDC, other DC types have been analyzed in bothalivary glands and peripheral blood from patients with Sjögren’syndrome. CD1a+ and CD83+ cells have been detected in minoralivary gland biopsies from patients with Sjögren’s syndromeut not controls [47]. CD83 is expressed only on activated DC,hereas CD1a is predominately known as a marker for Langer-ans cells, a certain type of dermal DC, as well as monocyte-derived

C. Decreased levels of the CD141+ mDC2 subset were found ineripheral blood from patients with Sjögren’s syndrome [46]. Nev-rtheless, it still needs to be proven whether mDC2 are actuallyresent in the salivary glands of these patients.

cells and proliferation of autoantibody producing B cells.

In general, low cell amounts and constricted numbers of dis-tinctive markers hamper the field of DC research in humans. Thus,detailed knowledge on human DC subsets and their functions hasto be acquired in order to link the aberrant DC frequencies with thepathogenesis of autoimmune manifestations.

In conclusion, the recruitment of DC to the salivary glands hasnow repeatedly been demonstrated and increased expression ofIFN induced genes are found locally and systemically in patientswith Sjögren’s syndrome. However, the pathological relevance ofthese findings needs to be confirmed.

5.4. Th17 and regulatory T cells

The immune system carefully balances the different T cellsubsets. They cross-regulate each other by secretion of variouscytokines, and a certain disease phenotype will be dependent onthe predominance of a specific T cell subset [48].

Th17 cells comprise a population of CD4+ T cells mainly definedby secretion of IL-17, but they also express other cytokines suchas IL-22. IL-17, a pro-inflammatory cytokine, has been shown to be

involved in several inflammatory and autoimmune diseases [49].In patients with Sjögren’s syndrome, both presence of Th17 cellsin salivary glands [50–52] as well as systemically elevated levelsof IL-17 [51,53] have been reported by several groups, indicating

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Fig. 3. Possible involvement of dendritic cells in the pathogenesis of Sjögren’s syndrome. In response to a viral infection or self-RNA containing immune complexes,plasmacytoid DC (pDC) are recruited from the blood to salivary glands where they produce IFN-�. Inflammation further recruits myeloid DC (mDC), monocytes, T and B cells.IFN-� might then lead to maturation of autoantigen-presenting mDC in predisposed individuals. The local production of IFN-� might lead to upregulation of IFN-stimulatedg entingB s coulM

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enes, explaining the IFN-signature seen in a subgroup of patients. Autoantigen-pres cells to produce autoantibodies. In this inflammatory setting, recruited monocyteodified from Vogelsang 2010 [102].

n involvement of this cell population in this autoimmune disease.hese observations are further substantiated by the essential rolef Th17 cells demonstrated in animal models of Sjögren’s syndromend SLE [54].

Interestingly, polymorphisms in the Ro52/Trim21 gene haveeen associated with both Sjögren’s syndrome and SLE [55–57],nd mice with genetically modified Ro52 have been reported toevelop a lupus-like condition with high levels of IL-17 and Th17ells [58]. Although the role of IL-17 and the Th17 pathway in SLEnd Sjögren’s syndrome is only beginning to be understood, sev-ral studies implicate a role for Th17 effector cells also in theseutoimmune conditions [50–52,59,60].

Regulatory T cells (Treg) play a central role in controllingutoimmunity in animal models [61–63]. However, Gottenberg ando-workers could recently show that Sjögren’s syndrome patientsave functionally normal regulatory T cells [64], making a defect ofhese cells in the pathology of Sjögren’s syndrome rather improb-ble. The presence of Treg in salivary glands has been associatedith severity of inflammation [65,66].

.5. Autoantibodies and breakdown of tolerance

One of the characteristic features of Sjögren’s syndrome is theresence of autoantibodies against SSA/Ro (Ro52 and Ro60) andSB/La. These autoantibodies are non-organ specific and their role

mDC could stimulate autoreactive T cells, which in turn could activate autoreactived develop into tip-DC producing even more inflammatory cytokines.

in the pathogenesis of Sjögren’s syndrome is not clearly understood.A better understanding of the function of the targeted proteins mayhelp to understand why these specific molecules become targets ofthe autoimmune response in Sjögren’s syndrome.

Ro52 is an interferon-inducible protein [67,68] belonging to thetripartite motif (TRIM) protein family [69]. The Ro52/Trim21 geneis located on chromosome 11 in humans, and chromosome 7 inmouse. It consists of seven (human) or eight (mouse) exons witha conserved exon/intron structure spanning approximately 8 kb. Inboth the human and mouse genomes, the Trim21 gene is part ofa large Trim gene cluster containing Trim2, 6, 22 and 34 in addi-tion to Trim21. The Ro52 protein comprises 475 amino acids inhumans and 470 in mouse, with the TRIM motif constituting abouthalf of the protein at the N-terminal end [70–72]. The C-terminaldomain of Ro52 is denoted B30.2 and contains the subdomainssp1A and RyR (SPRY) and associated with SPRY (PRY), togetherreferred to as the PRYSPRY domain. An alternatively spliced Ro52mRNA transcript, where exon 4 is deleted, has been described anddetected in a variety of tissues, including fetal heart and salivaryglands [73,74]. The significance of this alternative transcript is how-ever unclear, as the existence of the corresponding Ro52 isoform,

denoted Ro52-�, has never been demonstrated at the protein levelin vivo.

The TRIM motif contains a RING domain, one or two B-box motifsand a coiled-coil region. As several other members of the TRIM

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Fig. 4. Ro52 is a negative feed back regulator of proinflammatory cytokine production. (A) Through TLRs, IRFs are activated to induce proinflammatory cytokine productionduring infection. Produced interferons induce expression of Ro52, which acts as a negative regulator of the activity of several IRFs. (B) In cells deficient of Ro52 this negativef cytokd

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eedback loop is abrogated, resulting in aberrant production of proinflammatory

ifferentiation, tissue inflammation and systemic autoimmunity.

rotein family, Ro52 has E3 ligase activity and functions in the ubiq-itination of proteins [75,76]. During the ubiquitination process,he 76 amino acid polypeptide ubiquitin is first bound and acti-ated by an E1 enzyme and transferred to an ubiquitin conjugatingnzyme (E2 or Ubc). E3 ligases mediate the transfer of the activatedbiquitin from an E2/Ubc to the substrate protein to be ubiquiti-ated. Depending on whether the substrate is modified by poly-r monoubiquitination it is degraded by the 26S proteaosome, tar-eted for lysosomal degradation or functionally modified [77,78].he E3 ligase recruits the substrate protein and is thus essential inegulating cellular levels and activity of specific proteins.

Several proteins have been suggested as substrates for Ro52-ediated ubiquitination, including p27 [79], IgG [80], IKK-� [81]

nd several members of the interferon regulatory factor (IRF)ranscription factor family [58,82–84]. Observations from Ro52-eficient mice support a biologic relevance of Ro52-mediatedbiquitination of the IRF transcription factors as mice lacking func-ional Ro52 aberrantly express type I interferons and cytokines IL-6,L-12/IL-23p40 as well as TNF-� [58,85]. These pro-inflammatoryytokines are all regulated by IRF transcription factors, and these

bservations indicate a central role for Ro52 as negative regu-ator of IRFs and pro-inflammatory cytokines. Further, the miceubsequently developed tissue inflammation and systemic autoim-unity with high levels of IL-17 and Th17 cells [58]. This phenotype

ines including IL-12/IL-23p40, TNF and IL-6 with a net effect of promoting Th17

was abolished after crossing the mice to IL-23p19−/− mice, demon-strating that the systemic autoimmunity developing in the absenceof Ro52 is dependent on the IL-23/Th17 pathway (Fig. 4).

In unstimulated cells, Ro52 resides in the cytoplasm. Upon inter-feron stimulation, both upregulation and translocation into thenucleus is induced [68]. Ro52 can act as an E3 ligase in both com-partments, as it interacts with the cytoplasmic E2/Ubc H5 as well asthe nuclear E2/Ubc H6 [86]. This upregulation and nuclear translo-cation of Ro52 might thus be a functional part of the negativefeedback loop suppressing interferon mediated immune activation.Further, it relates Ro52 deficiency to dysregulation of interferonpathways [87,88].

The phosphoprotein La (SSB) is a member of the RNA recognitionmotif protein family and is a part of the Ro/La ribonucleoprotein(RNP) complex that associates with small cytoplasmic RNAs andalso viral RNAs. Recently, Bitko and colleagues discovered that Lashields viral leader RNA (leRNA) of respiratory syncytial virus (RSV)from retinoid acid-inducible gene I (RIG-I), a receptor involved inrecognition of viral RNA [89]. As a result, IFN activation is preventedand viral growth continues. Amino acids 104–235 of La were identi-

fied as the approximate region responsible for RSV leRNA-binding,and several epitopes targeted by La autoantibodies of patients withSjögren’s syndrome are located in the same area [90,91]. Anotherstudy revealed that Epstein–Barr virus (EBV) encoded small RNA is

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eleased from cells in complexes with La and that such complexesnduce TLR3-related secretion of type I IFN and TNF [92].

It might therefore be speculated that at the time of viral infec-ion of salivary gland epithelial cells, Ro52 is overexpressed as aefensive mechanism both to suppress viral replication through

nhibition of NF-�B signaling and to protect the host from pro-onged activation of the type I IFN system. At the same time, theathogen may utilize La protein from the host to escape from the

mmune response, thereby also leading to its overexpression. Nev-rtheless, as proposed by Bitko and co-workers, small amounts ofeRNA might escape La and that amount could be sufficient for thectivation of IFN [89]. IFN would drive inflammation and during cellysis and apoptosis overexpressed La and Ro could be mistargetedy antigen-presenting cells [reviewed in [93]]. Another option ishat Ro and La can undergo oxidative damage in the same man-er as shown in SLE [94]. Such modified proteins might lead tohe formation of neoepitopes and epitope spreading, productionf autoantibodies and breakdown of self-tolerance. Later, autoan-ibodies against Ro and La in combination with apoptotic materialrom cells could induce and sustain the production of IFN-� by DC42] and stimulate the production of pro-inflammatory cytokinesNF-� IL-6 and IL-8 by salivary gland epithelial cells [95] thus lead-ng to the continuous tissue inflammation.

It has been shown that for the breakdown of self-tolerance thectivation of dendritic cells alone is not enough in the absence ofufficient amount of specific CD4+ T helper cells [96]. Such specific

helper cells might appear as a result of molecular mimicry as haseen shown in studies with Ro60, another component of Ro/La RNPhat is involved in quality control of RNA. Early reports indicatedequence homology of Ro60 major linear B-cell epitope recognizedy serum of Sjögren’s syndrome patients, and Coxsackie virus 2Brotein [97]. Moreover, in SLE, molecular mimicry between Ro60nd Epstein–Barr virus nuclear antigen-1 (EBNA-1) was suggestednd Ro60 was proposed as an initiating lupus antigen responsibleor epitope spreading [98]. This was further supported by data fromtudies of SLE patients in whom Ro60 autoantibodies appear manyonths prior to disease onset and before the appearance of anti-

o52 and anti-La autoantibodies [99]. Unfortunately, such studiesave not been performed in Sjögren’s syndrome. However, in anxperimental model of Sjögren’s syndrome it was shown that thepitope spreading and the development of sicca symptoms can berevented by oral feeding of Ro60 peptide or whole Ro60 [100].

. Conclusions

The etiology and many aspects of the pathogenesis of Sjögren’syndrome are still elusive. There is no cure, and today’s therapiesely merely on relieving the symptoms as good as possible. There iso single symptom for diagnosis of Sjögren’s syndrome, and theymptoms occur long after the onset of the disease. It is there-ore not unusual that several years pass by before the patients areiagnosed correctly. The biggest challenge for us remains there-ore to improve therapy and diagnosis of Sjögren’s syndrome. Morefforts are needed in order to understand the true complexity of theathogenic events of this disease.

cknowledgements

We are indebted to all the patients with Sjögren’s syndrome whoave participated in our studies.

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