Université Libre de Bruxelles Faculté de Médecine Contribution to the diagnosis and pathophysiology of Sjögren

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Université Libre de Bruxelles Faculté de Médecine

Contribution to the diagnosis and pathophysiology of Sjögren’s Syndrome

Muhammad S Soyfoo

Docteur en Médecine

Laboratoire de Chimie Biologique et de la Nutrition

Promoteur: Professeur Christine Delporte Co-Promoteur: Professeur Elie Cogan

Thèse présentée en vue de l’obtention du titre de docteur en sciences médicales

Année académique 2011-2012

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List of Abbreviations

AQP: Aquaporin

APRIL: A proliferating ligand

ATP: Adenosine triphosphate

BAFF: B cell activating factor

BCR: B cell receptor

BM: Basement membrane

CCR: Chemokine receptor

CD: Cluster of differentiation

CTLA-4: Cytotoxic T-lymphocyte antigen 4

CXCL: Chemokine ligand

CRISP 3: Cystein-rich secretory protein 3 DAMPs: Damage associated molecular patterns

DC: Dendritic cells

DHEA: Dehydroepiandrosterone

FRPL1: Formyl-receptor peptide like-1

FPR: Formyl peptide receptor

FS: Focus score

GI: Gastrointestinal

GM-CSF: Granulocyte macrophage colony stimulating factor

HLA: Human Leukocyte antigen

HMGB-1: High mobility group box -1

HSP: Heat Shock protein

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ICAM-1: Intercellular adhesion molecule-1 ICOS: Inducible costimulatory molecule

Ig: Immunoglobulin

IL: Interleukin

IFN: Interferon

IRF5: Interferon regulating factor 5 KCS: Keratoconjunctivitis sicca

KO: Knockout

LI: Labelling index

LPS: lipopolysaccharide

MHC: Major histocompatibility complex

M3R: Muscarinic M3 receptors

MyDD 88: Myeloid differentiation factor 88

NOD: Non-obese diabetic

PARP: Poly ADP ribose polymerase PBMC: Peripheral blood mononuclear cells

PKC: Protein Kinase C

PI3K: Phosphatidylinositol 3-Kinase Poly(IC): Polyinosinic:polycytidylic acid

pSS: Primary Sjögren’s syndrome

PTEN: Phosphatase and tensin homolog

PTPN 22: Protein tyrosine phosphatase nonreceptor 22

RA: Rheumatoid arthritis

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RAGE: Receptor for advanced glycation end products RbAp48: Retinoblastoma-associated protein 48

RBC: Red blood cells

SLE: Systemic lupus erythematosus

SMG: Submandibular gland

SG: Salivary gland

SS: Sjögren’s syndrome

sSS: Secondary Sjögren’s syndrome

SSDAI: Sjögren’s syndrome disease activity index

STZ: Streptozotocin

TCR: T-cell receptor

TER: Transepithelial resistance

Tg: Transgenic

Th: T helper

Tj: Tight junctions

TGF: Transforming growth factor

TLR: Toll like receptor

TNF: Tumor necrosis factor

ZO: Zonula occludens

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1. SUMMARY

Sjögren’s syndrome (SS) is a chronic autoimmune disease characterized by lymphocytic infiltration of salivary and lachrymal glands resulting in diminished production of saliva and tears. The pathophysiology of SS is very complex and its investigation is actively ongoing.

Classically it has been postulated that sicca symptoms in SS patients are a double step process whereby lymphocytic infiltration of lachrymal and salivary glands is followed by epithelial cell destruction resulting in keratoconjunctivitis sicca and xerostomia. Recent advances in the field of the pathophysiology of SS have brought in new players, such as anti muscarinic autoantibodies and aquaporin 5 (AQP5), that could explain underlying mechanistic processes and pave the way for a better understanding of this disease. In our work, we investigated potential diagnostic and prognostic markers of SS. We showed that two alarmins, HMGB-1 and S100A8/A9 are significantly increased in SS patients but could not correlate with disease activity index. Using different mouse models for SS, we showed altered distribution of AQP5 in the salivary glands of the diseased mice. Furthermore, we demonstrated that this modification of AQP5 distribution appears in the salivary glands presenting inflammatory infiltrates. Moreover, using streptozotocin-treated mice (a non-immune mouse model) characterized by significant diminished salivary flow despite the absence of inflammatory infiltrates, no modification of AQP5 distribution and expression was observed. All together, these results imply that modification of AQP5 distribution is dependent on the presence of inflammatory infiltrates in the salivary glands.

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2. INTRODUCTION

Sjögren’s syndrome (SS) is a chronic systemic autoimmune disease characterized by lymphocytic infiltration of exocrine glands and more specifically salivary and lachrymal glands (Fox, 2005). Beyond the classical sicca symptoms, systemic involvement of other organs can occur. The protean features of SS make the diagnosis of SS relatively difficult (Fox et al., 1999). Many patients either remain undiagnosed or the diagnosis is established many years after the onset of the symptoms. Early diagnosis and classification of this disease is particularly important for enabling appropriate diagnostic evaluation and optimization of therapeutic intervention (Tzioufas and Voulgarelis, 2007). The diagnosis and the classification of patients are not based on the presence of single pathognomonic signs or symptoms. The disease is usually identified by the presence of a combination of clinical and laboratory manifestations. These manifestations include the classification criteria of the disease, which usually also serve as diagnostic criteria. Ideally, the classification criteria should only be used as diagnostic criteria when their sensitivity and specificity are both close to 100%. In practice, this is rather difficult in many instances, particularly at the disease onset, when the disease manifestations are not fully overt and when the diagnosis depends largely on the ability and the expertise of the physician. However, the use of universally accepted classification criteria for disease syndromes has created a consensus between clinical researchers, facilitating the standardization of the diagnosis in patients taking part in multi- center clinical studies, thereby enabling the analysis of results in a common and unbiased fashion. In 2002, an American–European consensus new set of classification criteria for SS has been proposed (Vitali et al., 2002). Nevertheless, there is currently no diagnostic marker of SS even though some prognostic markers have been proposed (Gottenberg et al., 2007;

Gottenberg et al., 2005). In the first part of our work, we aimed to determine potential diagnostic and prognostic markers of SS.

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One of the hallmarks of Sjögren’s syndrome (SS) is the classical sicca (dry eyes and dry mouth) symptoms resulting from secretory dysfunction of the salivary and lachrymal glands (Dawson et al., 2006; Fox, 2005; Voulgarelis and Tzioufas, 2010). Although tremendous progress has been made to unearth the different inherent processes underscoring the pathophysiology of sicca symptoms in SS, the initial triggering event remains unknown despite the obvious involvement of several imbricating factors (Delaleu et al., 2008; Mariette and Gottenberg, 2010; Nikolov and Illei, 2009). As such, different mechanisms have been delineated in portraying the salivary dysfunction in SS. These mechanisms include anti muscarinic autoantibodies, altered mucin expression, salivary gland destruction, nitric oxide- mediated salivary gland dysfunction and modified aquaporin 5 (AQP5) distribution (Alliende et al., 2008; Caulfield et al., 2009; Dawson et al., 2005; Ramos-Casals and Font, 2005;

Steinfeld et al., 2001). In the second part of our work, we studied the expression and distribution of AQP5 in mouse models for SS in an attempt to further understand its possible role in secretory dysfunction in SS.

As an introduction to our work, the anatomy, histology and secretory mechanisms of salivary glands (SG) will first be discussed. Then, the pathogenesis of SS will be reviewed and the concept of alarmins and their roles as partners in crime in inflammatory diseases will briefly be discussed.

2.1. The salivary glands 2.1.1. Generalities

Salivary glands consist of three pairs of major salivary glands (SG), the parotid, submandibular and sublingual glands, and numerous minor salivary glands distributed throughout the oral cavity within the mucosa and submucosa. On average about 0.5 litres of saliva are produced each day but the rate of saliva secretion varies throughout the day (Melvin et al., 2005a). At rest, saliva secretion rate is about 0.3 ml/min, but rises to 2.0 ml/min upon

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stimulation. The contributions from each gland to the total saliva flow are not equivalent and vary depending on the status of the glands (rest or stimulation). Indeed, at rest, the parotid produces 20%, the submandibular gland 65%, and the sublingual and minor glands 15% of the total saliva secretion, while upon stimulation, the parotid saliva secretion rises to 50%.

The nature of the saliva secretion also varies from gland to gland. Various digestive enzymes - salivary amylase - and antimicrobial agents - IgA, lysozyme, and lactoferrin - are also secreted with the saliva (Carlson and Ord, 2008).

Saliva is essential for mucosal lubrication, speech, swallowing, and initiation of the digestion process. It also performs an essential buffering role preventing demineralization of teeth occurring during carious process, as well as an antimicrobial function. When there is a marked deficiency in saliva production, xerostomia, rampant caries, and destructive periodontal disease ensue.

2.1.2. Anatomy of salivary glands (SG)

The parotid, the largest of the major salivary glands, is situated in the space between the posterior border of the mandibular ramus and the mastoid process of the temporal bone.

The submandibular gland (SMG), about half of the weight of the parotid, lies in the triangle formed by the anterior and posterior bellies of the digastric muscle, the inferior margin of the mandible, and the mylohyoid muscle. It is often referred to as the submaxillary gland because of the tendency of British anatomists to refer to the mandible as the

‘submaxilla’.

The sublingal gland, the smallest major SG, lies just deep to the floor of mouth mucosa between the mandible and genioglossus muscle. The mylohyoid muscle bound it inferiorly.

Unlike the major SG, the minor salivary glands lack a branching network of draining ducts. Instead, each salivary unit has its own single duct. The minor salivary glands are

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concentrated in the buccal, labial, palatal, and lingual regions. In addition, minor salivary glands may be found at the superior pole of the tonsils (Weber’s glands), the tonsillar pillars, the base of the tongue (von Ebner’s glands), paranasal sinuses, larynx, trachea, and bronchi.

2.1.3. Histology of the major salivary glands

SG are exocrine glands made of acinar and ductal cells. As such, acini can be serous or mucous. In the human parotid, the acini are almost entirely serous. In the submandibular gland, again, the secretory units are mostly serous. In some areas the mucinous acini have crescentic “caps” of serous cells called serous demilunes. In the sublingual gland the acini are almost entirely mucinous, although there are occasional serous acini or demilunes. The serous cells contain numerous secretory (zymogen) granules containing high levels of amylase. In addition, the acinar cells produce kallikrein, lactoferrin, and lysozyme. In mucous cells, the cytoplasm is packed with large (when HE coloration) secretory droplets (Young et al., 2006).

Myoepithelial cells are contractile cells closely related to the secretory acini and also much of the duct system. The myoepithelial cells lie between the basal lamina and the epithelial cells. Numerous cytoplasmic processes arise from them and surround the serous acini as basket cells. Those associated with the duct cells are more spindled and have fewer processes than those surrounding acini (Carlson and Ord, 2008).

There are 3 types of ducts in the SG: striated (or secretory), intercalated or interlobular, all with outer basal cells and inner luminal cells. The intercalated ducts drain into striated ducts, which coalesce into intralobular and extralobular collecting ducts. Intercalated ducts are slender ducts continuous with the terminal acini, and lined with flat, spindle-shaped cells. Secretory ducts have eosinophilic cuboïdal to columnar cells with basal striations. Both intercalated and secretory ducts are found within the parenchyma of the gland and are therefore intralobular ducts.

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2.1.4. Physiology of salivary glands 2.1.4.1. Control of saliva secretion

There is a continuous low saliva production that is stimulated by drying of the oral and pharyngeal mucosa. A rapid increase in the resting levels occurs as a reflex in response to masticatory stimuli including the mechanoreceptors and taste fibers (Proctor and Carpenter, 2007). Other sensory modalities such as smell are also involved. The afferent input is via the salivary centers, which are themselves influenced by the higher centers (figure 1). The higher centers may be facilitator or inhibitor depending on the circumstances. The efferent stimulation passes via the parasympathetic and sympathetic pathways to the SG. There are no peripheral inhibitory mechanisms. Cholinergic nerves (parasympathetic) often innervate ducts and acini. Adrenergic nerves (sympathetic) usually enter the glands along the arteries and arterioles and ramify with them.

Within the glands, the nerve fibers intermingle such that cholinergic and adrenergic axons frequently lie in adjacent invaginations of a single Schwann cell. Secretion and vasoconstriction of acini are mediated by separate sympathetic axons, whereas a single parasympathetic axon may result in vasodilatation, secretion, and constriction of myoepithelial cells. Secretory end pieces are the most densely innervated structures in the SG.

Individual acinar cells may have both cholinergic and adrenergic nerve endings. The watery part of saliva secretion, including electrolytes, results from a complex set of stimuli that are largely parasympathetic. The active secretion of proteins into the saliva depends upon the relative levels of both sympathetic and parasympathetic stimulation. Although the ducts are less densely innervated than secretory acini, they do influence the composition of the saliva.

Myoepithelial cell contraction is stimulated predominantly by adrenergic fibers, although there may be an additional role for cholinergic axons.

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Figure 1. Salivary reflex mechanisms responsible for control of salivary secretion (from Proctor and Carpenter, 2007).

2.1.4.2. Mechanisms leading to saliva secretion

Although the saliva secreted by different SG differs in composition, the mechanism of fluid secretion remains highly conserved. Classically saliva formation involves two stages:

secretion of primary isotonic plasma-like fluid by acinar cells into the lumen of the acini (stage 1), and the modification of this NaCl-rich fluid during its transport along the ductal epithelium, where most of the NaCl is reabsorbed and K⁺ ions are usually secreted (stage 2) (Melvin et al., 2005b; Thaysen et al., 1954). Because of the poor water permeability of the ductal epithelium, the final saliva is hypotonic (Figure 2). The primary isotonic saliva, produced by the acini, results from a coordinated activation of ions transporters and water channels (Melvin et al., 2005a). The Na⁺/K⁺ ATPase, highly expressed at the basolateral membrane of acinar cells, provides an inwardly directed Na⁺ electrochemical gradient. The intracellular Cl^- concentration is increased beyond its electrochemical gradient, by the Na⁺ /K⁺/2Cl^- co-transporter (encoded by the NKCC1 gene or SLC12a2) located at the

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basolateral membrane, and the basolateral Cl^-/HCO3^- and Na⁺/H⁺ exchangers (Catalan et al., 2009a). Apical Cl^- efflux is mediated through a Ca²⁺-dependent Cl^- channel recently identified as TMEM16A(Yang et al., 2008). K⁺ channels, IK1 or SK4 (encoded by the Kcnn4 gene) and maxi K or SLO (encoded by the Kcnma1 gene), located at the basolateral membrane of acinar cells completes the Cl^- secretory process (Begenisich et al., 2004; Nehrke et al., 2003;

Romanenko et al., 2006). As a result from these ion processes, an osmotic gradient is created and favours transcellular water movement through AQP5, a water channel expressed at the apical membrane of acinar cells (Figure 2). Even if the transcellular route of water movement is predominant, it has been also established that paracellular-driven water secretion could also play a role in the secretory process (Kawedia et al., 2007). The opening of the K⁺ and Cl^- channels, located at the basolateral and apical membranes respectively, is triggered by agonist stimulation (acetylcholine) and subsequent intracellular Ca²⁺ mobilization entailing negative membrane electrical potential gradient, thereby allowing passive movement of cations across acinar cell tight junctions. Bicarbonate ion is secreted across the apical membrane via a Cl^- channel. Accumulation of secreted ions in the lumen generates a transepithelial osmotic gradient, which provides the driving force for water movement through apical AQP5 channels and paracellular pathways.

The final hypotonic secreted saliva results from the modification of the primary isotonic saliva occurring in the ductal lumen and the relative water impermeability of the ductal cells. Indeed, as the primary isotonic fluid secretion passes through the ducts, NaCl is reabsorbed through Na⁺ channels (mainly ENaC, epithelial sodium channel), Na⁺/H⁺ exchangers (Nhe2 and Nhe3 channels), Cl^– channels (CFTR (cystic fibrosis transmembrane conductance regulator) channel and Cl^-/HCO3^- exchangers), while K⁺ and HCO3^- are secreted via Cl^-/HCO3^- and K⁺/H⁺ exchangers (Catalan et al., 2009a; Melvin et al., 2005a).

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Figure 2. Mechanisms of saliva secretion; A (left figure): primary salivary secretion model. B (right figure):

Two-stage salivary gland secretion model (Catalan et al., 2009b).

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2.2. Aquaporins

2.2.1. Generalities

Aquaporins (AQPs), a family of water-permeable channels, are small transmembrane proteins of about 28 kDa, implicated in transcellular water permeability in all living organisms (Agre et al., 1993; Preston et al., 1992; Walz et al., 2009; Zardoya, 2005). It is now well agreed that transcellular water fluxes occurs through both diffusion and AQP. Water diffusion occurs at relatively low velocity and capacity, while transcellular water movement via AQP occurs at much higher velocity and capacity (Agre et al., 1993). In most tissues, AQP-mediated water flow is directed by osmotic gradients.

So far, thirteen mammalian AQPs have been identified. These AQPs can be classified into three subfamilies according to their permeability and sequences homologies (Agre et al., 2002; Rojek et al., 2008). The subfamilies include: a) the classical AQP (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6 and AQP8) only permeable to water; b) aquaglyceroporins (AQP3, AQP7, AQP9 and AQP10), permeable to water as well as to small uncharged molecules, such as glycerol and urea and c) unorthodox AQPs (AQP11 and AQP12) whose permeability still remains to be clearly established (Table 1).

2.2.2. The Structure of AQPs

AQP is made of six transmembrane helices, three extracellular loops and two intracellular loops. Both amino and carboxy terminals of the protein are intracellular. Two repeating Asn-Pro-Ala (NPA) sequences, present in the first intracellular and third extracellular loop, represent the amino acid signature sequence motifs of the AQP’s family and fold into the lipid bilayer to form the water pore according to the ‘hourglass model’ (Jung et al., 1994). However, the functionality of the AQPs requires their association in tetramers (Verbavatz et al., 1993).

Each AQP1 subunit contains six bilayer-spanning domains extracellular loops A, C, and

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E, and intracellular loops B and D. The two NPA motifs are located in loops B and E, and fold into the lipid bilayer to form the water pore as an ‘hourglass like’ structure.

Table 1. Tissue distribution of mammalian AQP

AQP Localization

Classical AQPs

AQP0 Eye lens

AQP1 RBC, kidney, eye, lung, capillary endothelia

AQP2 Kidney, epididymis

AQP4 Central nervous system, astroglia, kidney AQP5

AQP6 AQP8

Aquaglyceroporins

Salivary gland, pancreas, lung, cochlea Kidney, salivary gland

Kidney, testis, myoepithelial cells, GI tract

AQP3 Skin, kidney, eye, colon, conjunctiva

AQP7 Kidney, adipose tissue, liver, testis, heart AQP9

AQP10

Unorthodox AQPs

Liver, leucocyte, brain GI tract

AQP11 Liver, kidney, testis, brain

AQP12 Pancreas

AQP: Aquaporin; GI: gastrointestinal tract; RBC: red blood cells

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Figure 3. ’Hourglass’ structure’ of AQP1 monomer.

2.2.3. AQPs in the salivary glands

Of the known AQPs, six are expressed in the mammalian salivary glands (Table 2) (Delporte and Steinfeld, 2006). However, only AQP5 has been demonstrated to functionally contribute to saliva secretion (Krane et al., 2001; Ma et al., 1999).

AQP5 has been detected in rat and mouse parotid, submandibular and sublingual glands as well as in human parotid, submandibular, sublingual and minor labial salivary glands (King et al., 1997; Raina et al., 1995). In rat submandibular glands, AQP5 expression was observed in the apical membrane of serous acinar cells (Funaki et al., 1998; King et al., 1997; Nielsen et al., 1997). In the Sprague-Dawley rat strain, following either high or low expression of AQP5, the immunolocalization of AQP5 was different (Murdiastuti et al., 2002). In rats expressing high levels of AQP5, the AQP5 protein was localized at the apical, basal and lateral membranes of acinar cells, while in rats expressing low levels of AQP5, AQP5 was localized at apical and/or the lateral membrane of the acinar cells. Most of the studies analyzing AQP5 expression in the SG did not detect the presence of AQP5 in rat submandibular ductal cells while a few studies have reported the presence of AQP5 in the apical membranes of intercalated ducts in submandibular glands and in the intercellular ducts of parotid glands (He et al., 1997; Ishikawa et al., 2005; Nielsen et al., 1997). In human SG (parotid, submandibular, and labial glands), AQP5 expression was restricted to the apical

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membranes of acinar cells and absent from the ductal cells. Compared to wild-type mice, AQP5 knockout mice presented reduced pilocarpine-stimulated saliva secretion (~60%), hypertonic (420 mosm) and more viscous saliva, while amylase and protein secretions were not modified (Ma et al., 1999). This suggested that AQP5 played a key role in saliva fluid secretion and that high epithelial cell membrane water permeability is required for active near-isosmolar fluid transport. Further studies showed that hyposalivation was due to the significant decrease in the water permeability of salivary glands acinar cells in mice lacking AQP5 and not by changes in whole body fluid homeostasis (Krane et al., 2001). Furthermore, in the Sprague–Dawley rats, the levels of AQP5 expression observed in submandibular glands seems to be related to the variation of the saliva secretion rate observed between high and low AQP5 expressors (Murdiastuti et al., 2002). These data are in agreement with the key role of AQP5 in saliva secretion.

2.2.4. AQPs and Sjögren’s syndrome

The involvement of AQPs in the pathophysiology of the salivary dysfunction characterizing Sjögren’s syndrome (SS) (see section 2.3) has been underlined from several studies (Beroukas et al., 2002b; Delporte, 2009; Steinfeld et al., 2001; Tsubota et al., 2001).

In minor labial SG from patients suffering from SS, AQP1 distribution is significantly diminished in the myoepithelial cells while no changes are observed in endothelial cells of non-fenestrated capillaries (Beroukas et al., 2002b). However, the participation of AQP1 in the salivary dysfunction of SS is not supported from knockout AQP1 mice, in which no significant modification in the salivary flow was observed (Ma et al., 1999).

Modified AQP5 distribution has been observed in both labial SG and lachrymal glands from patients suffering from SS (Steinfeld et al., 2001; Tsubota et al., 2001). Normal labial SG and lachrymal glands essentially expressed AQP5 at the apical membrane of acinar cells, while in patients with SS the distribution of AQP5 is altered, being preferentially observed at

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the basolateral membrane and/or cytoplasmic compartment of acinar cells. However, normal distribution of AQP5 in minor SG from SS patients has also been reported (Beroukas et al., 2001). These contradictory data could be explained by the use of distinct antibodies, techniques, and/or differences existing between the investigated populations.

In an animal model for SS, the NOD mice, altered AQP5 distribution has been documented as being basolateral in acinar cells from SG (Konttinen et al., 2005).

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Table 2 AQPs expressed in salivary glands

AQP Cell type Subcellular localization Rat Human

Remarks

AQP1 Endothelial Myoepithelial

A+B A+B

AQP3 Acinar B Controversy

AQP4 Ductal B Controversy

AQP5 Acinar

Ductal

A+B A+B SG

A

AQP6 Acinar A

SG AQP8 Myoepithelial

A: Apical; B: basolateral; SG: secretory granules (Delporte, 2009)

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2.3. Sjögren’s syndrome 2.3.1. Introduction

Sjögren’s syndrome (SS) is a chronic autoimmune disease characterized by the lymphocytic infiltration of salivary and lachrymal glands leading to xerostomia and keratoconjunctivitis sicca (KCS) (Fox, 2005). The prevalence of SS is variable (depending on the geographical localization) but recent studies have estimated it to be between 0.1-0.6%

(Goransson et al., 2011). As such, SS occurs in middle-aged patients with a high female predominance of 9 to 1. SS is classified either as primary (pSS) when occurring alone or secondary (sSS) to other autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus. Besides the involvement of exocrine glands entailing the classical sicca syndrome, systemic manifestations resulting from the lymphocytic infiltration of organs can be present in up to 20% of cases. There are actually no specific diagnostic criteria for SS.

However, for clinical studies and teaching purposes, SS is classified according to the American-European classification criteria, which include subjective and objective criteria of xerostomia and KCS as well as the presence of autoimmune antibodies and histopathological salivary gland involvement (Vitali et al., 2002). Because of the lack of a “gold standard” for the diagnostic of SS, the reference standard being actually used relies on the use of American- European classification criteria by experienced clinicians. The lack of specific diagnostic tests combined with the high prevalence of sicca symptoms in the general population makes the diagnosis of SS even more complicated. This holds true especially in early disease where the symptoms and signs are usually mild and might explain the time delay for the diagnosis of SS. The importance of making the diagnosis of pSS is cardinal because of the high risk of developing lymphoma and serious systemic complications (Voulgarelis and Moutsopoulos, 2008). The morbidity of patients suffering from SS is debilitating ranging from severe fatigue to evolving arthralgia.

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2.3.2. Pathophysiology of SS

The pathophysiology of SS is very complex. Despite tremendous progress made to unearth the different mechanistic processes underlying the autoimmune abnormality, the aetiology of the disease still remains to be discovered. It is actually believed that the combination of several factors might trigger immunological abnormalities leading to overt disease (Delaleu et al., 2008).

2.3.2.1. Environmental factors 2.3.2.1.1. Viral infections

Ebstein-Barr, human T lymphotropic virus-1 and hepatitis C have been postulated to be associated with SS. However, the link between these viral infections and SS remains weak (Green et al., 1989; Haddad et al., 1992). Coxsackie virus was found to be increased in salivary glands from SS patients, but these findings have been the subject of some controversies (Gottenberg et al., 2006b; Triantafyllopoulou et al., 2004).

2.3.2.2. Sexual hormones

The high female to male predominance of SS clearly delineates the role of hormones in the pathogenesis of SS. Estrogens and androgens are thought to respectively contribute or protect to autoimmunity. Onset of SS generally occurs around menopause, when a modification of the androgen-estrogen ratio occurs. Patients with SS have been shown to possess lower systemic concentrations of dehydroepiandrosterone (DHEA) than matched aged-controls (Valtysdottir et al., 2001). Furthermore, decreased salivary DHEA levels, reduced cystein-rich secretory protein 3 (CRISP-3, a protein upregulated by DHEA) expression, alteration of CRISP-3 polarized expression in acini, altered conversion of DHEA, and decreased and abnormal expression of steroidogenesis enzymes were detected in SS patients (Laine et al., 2007; Porola et al., 2008; Spaan et al., 2009). In women, local salivary

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gland dihydrotestosterone production is totally dependent on DHEA conversion, which makes them very vulnerable to local androgen deficiency, in opposition to men who probably possess adequate systemic androgen level to satisfy local salivary gland requirement.

Estrogenic action is largely imputed in the high female predominance of SS, as well as other autoimmune diseases (Whitacre, 2001). Estrogens play a cardinal role in targeting salivary epithelial cell and harvesting apoptosis through a Fas-mediated mechanism (Ishimaru et al., 1999). More recently, it was demonstrated that the retinoblastoma-associated protein 48 (RbAp48) induces tissue specific apoptosis in salivary glands depending on the level of estrogen deficiency (Ishimaru et al., 2008). Transgenic mice for RbAp48 develop autoimmune exocrinopathy similar to that of SS. These recent findings uphold the fact that estrogen deficiency stimulates salivary epithelial cells through upregulation of RbAp48, to release IL-18 and IFN-, thereby leading to the expression of MHC class II molecules, CD80, CD86 and ICAM-1. This enables epithelial cells to act as antigen-presenting cells to CD4+ T cells, and promoting similar lesions to that observed in salivary glands from SS patients. More recently, the presence of functional estrogen receptors has been observed in salivary epithelial cells (Tsinti et al., 2009). In that study, estrogen was shown to block expression of ICAM-1, an adhesion molecule whose expression is increased in salivary glands of SS patients. We could speculate that estrogen deficiency might lead to increased innate immunity.

Prolactin, a pro-inflammatory hormone, stimulates estrogen activity and inhibits estrogens production at high level T cell proliferation, IL-2 receptor expression, interferon  production and stimulation of antibody production (Taiym et al., 2004). Higher levels of prolactin, detected in SS patients, may be involved with the production of autoantibodies (Taiym et al., 2004).

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2.3.2.3. Genetic factors

Several studies support the existence of genetic predisposition to SS. Alleles within the major histocompatibility complex class II gene region, predominantly the HLA-DR and HLA- DQ, are implicated in the pathogenesis of SS. Different susceptibility alleles in SS patients vary according to ethnic origin (Bolstad and Jonsson, 2005).

Gene polymorphisms of cytokines, cytokines receptors, transcription factors and PTPN22 (protein tyrosine phosphatase nonreceptor 22) have been associated with SS (Cobb et al., 2010; Cobb et al., 2008; Maiti et al., 2010).

Impaired epigenetic control has been linked to various autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis and SS (Pan and Sawalha, 2009; Richardson, 2007). In salivary glands from SS patients undergoing extracellular matrix remodelling, mechanotransduction may affect epigenetic control of gene expression (Gonzalez et al., 2011b). Global DNA methylation of salivary glands from SS patients appears to be decreased, while specific genes appear to be hypermethylated (i.e:

BP230). Over-expression of two miRNAs (miR-574 and miR-768-3p) also participates to the epigenetic control of gene expression in salivary glands from SS patients (Alevizos et al., 2011). These two micro-RNAs were associated with high degree inflammation and correlated with the histological focus score. As such, they could represent future biomarkers of inflammation in SS patients. In B6DC mice, upregulation of miRNA-150 and miRNA-146 in PBMC and target tissue was observed as compared to control mice (Lu et al., 2007).

Furthermore, miR-23A is highly expressed in salivary glands from SS patients and may regulate CUL3 expression (Gonzalez et al., 2011a).

2.3.2.4. Immune system alterations

The innate immune system is recognized as playing a fundamental role in the

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pathogenesis of SS. Following viral infection, an increase in the expression of Toll-like receptors (TLR) has been shown in the salivary glands of SS patients. The functional TLR2, TLR3 and TLR4 as well as myeloid differentiation factor 88 (MyD88) are expressed in the labial salivary glands of SS patients (Kawakami et al., 2007). Following TLR activation in salivary glands, increase in CD54 (ICAM-1) expression and IL-6 production, as well as upregulation of CD54, MHC class I and CD40 have been observed (Spachidou et al., 2007).

More recently, upregulation of TLR3 in salivary of female new Zealand/WF1 female mice, in response to poly(I:C) (Polyinosinic:polycytidylic acid), a TLR3 ligand, resulted in activation of cytokine pathways and severe loss of glandular function (Deshmukh et al., 2009). These data underline increased TLR signalling pathways in salivary glands of SS patients leading to production of proinflammatory cytokines, T cell activation and a T helper type 1 driven immune response.

Upregulated expression of human leukocyte antigen (HLA) molecules occurs in epithelial cells of salivary glands from SS patients and may be involved in antigen presentation, leading to destruction of the tissue by CD4+ T cells as well as cytokine production and stimulation of B cells proliferation and differentiation (Jonsson et al., 2002).

Several cytokines have been implicated in the pathogenesis of SS. Increased expression of IFN-regulated genes has been shown in the salivary glands from SS patients (Gottenberg et al., 2006a; Hjelmervik et al., 2005), as well as in PBMC and whole blood (Emamian et al., 2009). Increased serum levels of IFN- and IFN- have been observed in pSS patients.

Plasmacytoid dendritic cells (PDC) are the most potent producers of IFN type 1. Circulating PDC express high levels of CD40 (a marker of cellular activation), which correlated, with the expression level of several type 1 IFN-induced genes in monocytes (Wildenberg et al., 2008).

Type-1 IFN activation and secretion result in activation of immature dendritic cells, BAFF secretion, stimulation of Fas ligand expression and increased apoptosis, increased T cell

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proliferation and survival, induction of several chemokines (CXCL9, CXCL10, CXCL11) and favouring Th1 responses (Mavragani and Crow, 2010). Increased expression of TNF-, IL-1, IL-12, IL-18, and IFN- has also been shown in SS patients (Voulgarelis and Tzioufas, 2010). Recently, the role of IL-17 producing cells (Th17) has been underlined in the pathogenesis of SS (Katsifis et al., 2009; Nguyen et al., 2008; Nguyen et al., 2011; Sakai et al., 2008). The IL-23/Th17 pathway triggers autoimmune exocrinopathy and systemic autoimmunity. Mice lacking Ro 52 antigen are characterized by increased proinflammatory cytokines regulated by interferon regulated factors, tissue inflammation and systemic inflammation. Loss of IL-23 and IL-17 in Ro52 null mice, results in protection from systemic autoimmunity (Espinosa et al., 2009). Furthermore, elevated serum levels of IL-17 in patients with SS together with enhanced levels of Th17 cells and related cytokines are predominant in salivary glands and strongly correlate with histological focus score.

Once T-cell infiltration of epithelial cells is established, CD4+ T cells and PDC produce B-cell targeted cytokines and other survival factors such as B-cell-activating factor of the tumor necrosis factor family (BAFF, also known as BLYS) and APRIL (A proliferating ligand) (Lavie et al., 2004). B cell activation and proliferation occur in about 20% of patients with pSS (Fox, 2005). B cell hyperactivity has been found in SS patients (Kassan and Moutsopoulos, 2004). BAFF promotes B-cell survival and antibody secretion. BAFF- transgenic mice develop clinical features of SS, polyarthritis and lupus (Mackay and Schneider, 2009). SS patients display increased BAFF serum levels correlating with decreased BAFF-R expression on B-cells and disease activity (Sellam et al., 2007). BAFF secretion is induced by type 1-IFN in monocytes and dendritic cells, type 1 IFN in monocytes and salivary epithelial cells (Ittah et al., 2006), virus or double stranded DNA in salivary epithelial cells (Ittah et al., 2008). BAFF can be released by the epithelial salivary cells but also by B cells. As such, B cell deregulation plays a crucial role in perpetuating inflammation

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and tissue damage (Daridon et al., 2007). Decreased levels of apoptosis among BAFF- expressing cells in salivary epithelial cells result in increased levels of BAFF expression, which in turn amplifies B cell signalling and proliferation and increased production of antibody-producing plasma cells (Groom et al., 2002; Mariette et al., 2003).

2.3.2.5. Autoantibodies

Autoantibodies against Ro (SSA) and La (SSB) are found in the serum of pSS and sSS patients (Garcia-Carrasco et al., 2002). These autoantibodies are linked to the onset, severity, duration and extraglandular manifestations of SS (Skopouli et al., 2000; Ter Borg et al., 2011). It is still undetermined if these antibodies play a direct pathogenic role in the glandular damage. Nonetheless, there is evidence supporting a clear cut role of anti Ro and anti La antibodies in the local autoimmune response. Indeed, autoantibodies to Ro and La have been found in saliva and infiltrating cells of salivary glands from SS patients Furthermore, increased mRNA production of La in acinar epithelial cells and translocation of La protein, resulting in membrane localization, in the conjunctiva epithelial cells have been observed in SS patients (Halse et al., 1999; Hammi et al., 2005; Routsias and Tzioufas, 2010; Tzioufas et al., 1999; Yannopoulos et al., 1992).

Autoantibodies against -fodrin, a major constituent of the cytoskeleton of eukaryotic cells, have also been detected in sera from patients with SS (Haneji et al., 1997). Abnormal location of -fodrin has been detected on the surface of apoptotic-induced cells, thereby relating the role of -fodrin in SS through apoptotic pathways (Maruyama et al., 2004;

McArthur et al., 2002; Wang et al., 2006). Several studies have suggested that aberrant proteolysis of -fodrin results in its expression at the external surface of apoptotic epithelial cells entailing the autoimmune process. The prevalence of -fodrin antibodies ranges from 20 to 38% (Locht et al., 2008), with a specificity of 30% for SS (Willeke et al., 2007).

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Autoantibodies to muscarinic M3 receptors (M3R), found in the serum of SS patients, induce the inhibition of the synapse between the efferent nerves and the salivary glands, leading to decreased saliva production (Fox and Stern, 2002; Sumida et al., 2010). More recently, antibodies against carbonic anhydrase II, VI and XIII have been described in relation to renal manifestations of SS (Pertovaara et al., 2011).

2.3.2.6. Autonomic system

In addition to autoimmune damage, autonomic dysfunction is also considered as a central feature in the pathogenesis of SS. (Dawson et al., 2000; Dawson et al., 2006; Nikolov and Illei, 2009). Involvement of the parasympathetic nervous system, that mainly controls exocrine function, might be responsible for glandular dysfunction and diminished salivary and lachrymal production (Waterman et al., 2000). There is growing body of evidence supporting the role of autonomic dysfunction in SS pathogenesis as shown by the presence of antimuscarinic M3 receptors (M3R) autoantibodies in 50 % of SS patients (Naito et al., 2005;

Nakamura et al., 2008) .In the salivary glands of SS patients, there is an upregulation of M3R, which might be the corollary of antagonistic M3R autoantibodies or impaired release of acetylcholine (Beroukas et al., 2002a; Zoukhri and Kublin, 2001). Furthermore, in a murine model for autoimmune sialoadenitis using M3R^-/- →Rag1^-/- mice (recombination activating gene deficient mice), whereby M3R^-/-were immunized with murine M3R peptides and the splenocytes of the former were then transferred in Rag1^-/- mice, it was clearly shown that M3R T cells play a fundamental role in autoimmune sialoadenitis (Iizuka et al., 2010). An additional mechanism that could contribute to autonomic dysfunction is elevated levels of acetylcholinesterase in the salivary glands of patients with SS (Dawson et al., 2000).

Decreased levels of acetylcholine due to increased cholinesterase levels result in glandular dysfunction and diminished production of saliva (Dawson et al., 2001).

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2.3.2.7. Epithelial cells activation

Impaired function and/or architectural destruction of epithelial cells occur in salivary glands from SS patients. Epithelial cells are now considered as playing active roles in immune defences (Manoussakis and Kapsogeorgou, 2010). Epithelial salivary gland cells possess all features to act as antigen-presenting cells, (Manoussakis et al., 1999; Matsumura et al., 2001).

In fact, epithelial cells might act as non-professional antigen-presenting cells, thereby participating actively in autoimmune responses leading to the development of SS (Tsunawaki et al., 2002). This is illustrated by increased expressions of CD40 and adhesion molecules (such as ICAM-1), B7 molecules which induce costimulation signals in CD4+ T cells, HLA class II, as well as the local production of lymphoid chemokines, cytokines and B-cell activating factor by the epithelial cells (Dimitriou et al., 2002; Kapsogeorgou et al., 2001;

Lavie et al., 2004; Xanthou et al., 2001). These immunoactive molecules mediate lymphoid cell homing and antigen presentation, and amplify the interactions between the epithelial and immune cells (Manoussakis and Kapsogeorgou, 2010) (Figure 4). Proinflammatory cytokines and other factors are capable to induce the activation of surrounding epithelial cells (Abu- Helu et al., 2001). Furthermore, as a result of apoptosis and formation of exosomes, epithelial cells present intracellular autoantigens such as the Ro and La autoantigens, further contributing to autoimmune process. Besides, following type 1 IFN stimulation and viral infection of epithelial cells, the latter release BAFF thereby activating B cells (Ittah et al., 2009; Kapsogeorgou et al., 2005; Mitsias et al., 2006).

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Figure 4: The concept of autoimmune epithelitis: role of epithelial cells in the pathogenesis of SS (Mitsias et al., 2006)

2.3.2.8. Apoptosis

A pivotal role for apoptosis as a pathogenic mechanism in SS-related glandular damage has been demonstrated in several studies (Manganelli and Fietta, 2003). Increased apoptosis of the glandular ductal and acinar epithelia occurs in pSS patients. Upregulation of the expression of several apoptotic-related molecules has been described in lymphocytes and epithelial cells from salivary glands of patients with SS. Epithelial cell apoptosis contributes to the glandular destructive lesions through the upregulation of apoptotic-related molecules leading to the proteolysis of exocrine autoantigens and ensuing glandular damage. It has been postulated that apoptotic disequilibrium between pro-apoptotic signals and anti-apoptotic mechanisms might act as the basis of epithelial cell destruction of exocrine glands in pSS.

EPITHELIUM

Endocrine

Stress

EXOSOMES

DC

Ag- Release

T T T T T

T T T T Ag-Presentation

B B

EPITHELIUM Persistent Virus Genetic Make-up

CD4 0

APOPTOSI S

Fas FasL

B7

T

B

Cytokines/

Chemokines I CAM.1

CK receptor

EPITHELIUM

La/ SSB La/ SSB

MHC- I I

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Apoptotic cell death might also function in a specific fashion favouring abnormal exposure of nuclear and cytoplasmic autoantigens thereby providing a way for presentation with autoreactive T cells. Furthermore, anti-Ro and anti-La autoantibodies can activate caspase-3 and cleave PARP (Poly ADP ribose polymerase) and trigger apoptosis. Furthermore, these autoantibodies can also activate extrinsic apoptotic pathways by transcriptional upregulation and activation of caspase-8. As such, anti-Ro and anti-La autoantibodies could trigger apoptosis resulting into tissue destruction (Ramos-Casals and Font, 2005).

2.3.2.9. Tight junction alterations

Alterations of tight junction (Tj) proteins such as zonula occludens-1 (ZO-1), claudins and occludin are involved in the pathogenesis in SS. Furthermore, cytokines may affect their expression and induce morphological changes.

In SG from SS patients, ZO-1 and occludin were strongly downregulated, while claudin-1 and claudin-4 were overexpressed (Ewert et al., 2010). In SS patients, the expression of ZO-1 and occludin was decreased at the apical membrane, while claudin-3 and claudin-4 were redistributed to the basolateral plasma membrane. In vitro exposure of isolated normal acini to TNF-α (Tumor necrosis alpha) and IFN-γ (interferon gamma) reproduced these alterations. Ultrastructural analysis associated tight junction disorganization with the presence of endocytic vesicles containing electron-dense material that may represent tight junction components. Local cytokine production, such as TNF-α and IFN-γ, in large salivary glands from SS patients may contribute to the secretory gland dysfunction by altering tight junction integrity of epithelial cells, thereby decreasing the quality and quantity of saliva (Baker et al., 2008). Chronic exposure of polarized rat parotid gland (Par-C10) epithelial cell monolayer to TNF-α and IFN-γ decreases transepithelial resistance (TER) and anion secretion. Treatment of Par-C10 cell monolayer with TNF-α and IFN-γ increased paracellular permeability to normally impermeable proteins, altered cell and TJ morphology, and

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downregulated the expression of the TJ protein, claudin-1, but not other TJ proteins expressed in Par-C10 cells. Therefore, cytokine production is an important contributor to secretory dysfunction in SS by disrupting TJ integrity of salivary epithelia (acinar + ductal cells).

2.3.2.10. Basement membrane alterations

The basement membrane (BM) may be submitted to similar functional and morphologic changes as the tight junctions. The BM supports the tissue architecture, and allows a flow of information that is transmitted by cell surface receptors to modulate cell differentiation and migration. Early changes of the BM components have been involved in the pathogenesis of inflammation (McArthur et al., 1993). In SS, the BM is disorganized, or locally even totally lacking due to the increased gelatinolytic activity of metalloproteases degrading type IV collagen (Goicovich et al., 2003; Konttinen et al., 1998). Modifications of the BM components precede the immunological events in salivary glands. The high state of disorganization of the basal lamina of acini and ducts could then allow invasion by cytotoxic T lymphocytes in SS (Molina et al., 2006).

An increase in laminin or a laminin-like substance in salivary ductal epithelia of SS patients suggests a potential role for laminin in the pathogenic mechanism and the use of increased laminin expression as a marker for SS (Defilippi et al., 1992).

2.4. Diagnosis of SS

2.4.1. Current tools for the diagnosis of SS

There are actually no diagnostic criteria for pSS. The task of physicians is fraught with difficulties by the absence of diagnostic markers for SS.

A more detailed review of the diagnosis, clinical and pathophysiological features of SS can be found in the manuscripts n°1 and n°2, annexed hereafter (see 2.4.3. and 2.4.4.).

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2.4.2. Possible future tools for the diagnosis of SS 2.4.2.1. Generalities on the alarmins

Alarmins have been ushered as potential markers in several inflammatory diseases (Bianchi, 2007; Foell et al., 2007b). It remains however currently unknown if alarmins could represent potential tools for the diagnosis of SS.

The alarmins (also called intracellular DAMPs, damage associated molecular patterns) are endogenous molecules that activate the innate immune system in response to pathogens and cell death (Foell et al., 2007b; Kono and Rock, 2008). As such the alarmins possess several intrinsic characteristics: 1) they are released essentially following non-programmed cell death; 2) they can be produced by cells of immune system; 3) they can recruit and activate receptor-expressing cells of the innate immune system and can therefore indirectly promote adaptive immunity responses; 4) they can restore homeostasis by favouring tissue reconstruction. The receptors detecting the alarmins are less fully understood. Some responses to alarmins are mediated through toll-like receptors (TLRs); for example TLR4 is activated not only by LPS, but also by S100A8/A9 (Vogl et al., 2007). A table presenting the putative alarmins, their receptors and their pro-inflammatory activities is shown below (Table 3).

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Table 3. Alarmins and their pro-inflammatory activity

Alarmins Pro-inflammatory activity Receptors

HMGB1 Inflammation in response to liver

Injury blocked by neutralizing antibody;

neutrophil recruitment; cytokine induction;

chemotaxis

RAGE, TLR2, 4

S100 Neutrophil recruitment and cytokine induction;

chemotaxis

RAGE, TLR4

IL-33 Neutrophil recruitment ST2

IL-1 Induction of cytokine production, neutrophil recruitment

IL-1aR

HSPs Cytokine induction TLR2,4;CD14,CD9

1,CD40, Scavenger receptors

Defensins Chemotaxis CCR6, TLR4

Cathedelicins Chemotaxis FPRL1

Galectins Monocyte recruitment and chemotaxis CD2 and others containing - galactose Uric acid Gout induced by purified molecule,neutrophil

recruitment, cytokine production N-formylated

peptides

Chemotaxis, neutrophil recruitment FPR and FPRL1 Chromatin,

nucleosomes and DNA

Neutrophil recruitment, cytokine induction, B-cell activation

TLR9 (with BCR or Fc receptor)

Thioredoxin Chemotaxis

Adenosine and ATP Chemotaxis and exacerbation of nephritis by purified molecule

P1, P2X and P2Y,A1,A2A,A2B and A3 receptors

BCR: B-cell receptor; CCR: chemokine receptor; FPR: formyl peptide receptor; FRPL1: formyl-receptor peptide like-1; HMGB1: high mobility group box 1 protein; HSP: heat-shock protein; RAGE: receptor for advanced glycation end-products; TLR: toll-like receptor. (adapted from Kono and Rock, 2008).

2.4.2.2. HMGB-1

HMGB1 is an intracellular DNA-binding protein stabilizing nucleosomes and binding DNA to regulate transcription (Sims et al., 2010). For HMGB1 to act as a cytokine, it must be released into the extracellular space. Two major pathways of HMGB1 release occur during invasion or injury, one classified as active and the other passive based on molecular mechanisms, release kinetics and downstream signalling responses (Figure 5) (Andersson and Tracey, 2011).

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Figure 5. Release and biological effects of HMGB1 (from Andersson and Tracey, 2011).

The role of HMGB1 as a pro-inflammatory molecule has been supported by the facts that it mediates cytokine release, inflammation, maturation of dendritic cells, epithelial cell barrier and endothelial activation (Andersson and Tracey, 2011; Muller et al., 2004; Scaffidi et al., 2002). Furthermore, the inflammatory activities are exacerbated in the presence of exogenous TLR agonists and other pro-inflammatory cytokines.

HMGB1 interacts with different receptors that have been previously identified for their abilities to transduce activation signals from exogenous (TLR2, TLR4 and TLR9) and endogenous (RAGE) ligands (Andersson and Tracey, 2011). HMGB1 interacts with RAGE, but the interaction with TLR4 is critical for HMGB1 activation of cytokine release in macrophages (Sims et al., 2010; Yang and Tracey, 2010) (Figure 6).

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Figure 6. HMGB1 receptors and their intracellular signalling cascade (from Andersson and Tracey, 2011)

2.4.2.3. S100 proteins

The S100s proteins consist in a family of more than 20 calcium-binding proteins exclusively expressed in vertebrates (Heizmann, 2002). These small, acidic proteins (10- 12kDa) are characterized by two calcium EF-hand motifs (helix-loop-helix structural domains): a C-terminal canonical EF and an N-terminal pseudo EF-hand connected by a central hinge (Donato, 2001). In the presence of increased intracellular calcium concentrations, calcium binding to the EF-hand motif triggers conformational changes that enable interactions with target proteins. The structural homology of the S100s permits the formation of active heterodimers, tetramers and higher-order multimeric structures that alter binding and physiological properties (Roth et al., 2003). S100s proteins have a wide spectrum

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of physiological roles including modulation of calcium homeostasis, cytoskeletal organization, cell cycle progression, and cell growth and differentiation. (Foell and Roth, 2004). Besides, they can be used as markers of disease activities in inflammatory diseases (Foell and Roth, 2004).

Three S100 proteins (S100A8, S100A9, S100A12) are specifically related to innate immune functions by their expression in cells of myeloid origin. S100A12 is expressed mainly by granulocytes and under distinct conditions in subsets of activated monocytes and macrophages and has been described as an endogenous ligand of RAGE (Foell et al., 2007b).

S100A8 and S100A9 exist mainly as S100A8/S100A9 heterodimers and are expressed by granulocytes, monocytes and early differentiation states of macrophages.

S100A12 is overexpressed at sites of local inflammation and serum concentrations correlate with individual disease activity in patients with inflammatory disorders (Foell and Roth, 2004). S100A12 is released either in the inflamed tissue or in the main blood stream by activated granulocytes. Secreted S100A12 binds to RAGE on the endothelial cells, which leads to increased expression of ICAM-1. Besides, S100A12 possesses chemotactic properties promoting extravasations of granulocytes and monocytes to the sites of inflammation.

S100A8/S100A9 has been shown to be not only a useful marker in several inflammatory conditions (especially rheumatoid arthritis and inflammatory bowel disease) but also a key player in the pathogenesis of inflammatory disorders (Ehrchen et al., 2009) . S100A8/S100A9 is secreted by activated phagocytes at the sites of inflammation. The interaction between endothelium and phagocytes is an important stimulus for S100A8/A9 secretion. The extracellular effects of S100A8/S100A9 during the inflammatory process are depicted in figure 7.

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Figure 7. Extracellular effects of S100A8–S100A9 and S100A12 during inflammatory processes (Roth et al., 2003). CD: cluster of differentiation; NFKB: Nuclear factor Kappa light chain enhancer of activated B cells;

PKC: protein kinase C; RAGE: receptor for advanced glycation end products.

More recently, S100A8/S100A9 has been identified to bind to TLR4 on the surface of phagocytes and promote lethal, endotoxin-induced shock (Vogl et al., 2007). Importantly, S100A8/S100A9 are not only involved in promoting the inflammatory response in infections, but was also identified as a potent amplifier of inflammation in autoimmunity. This pro- inflammatory action of S100A8/S100A9 involves autocrine and paracrine mechanisms in phagocytes, endothelium and other cells. The net outcome is the extravasations of leucocytes into inflamed tissues and their subsequent activation is also increased (Ehrchen et al., 2009).

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2.4.3. Manuscript n° 1: Primary Sjögren’s syndrome: current pathophysiological, diagnostic and therapeutic advances.

Delporte C, Perret J and Soyfoo MS. Huang F.P. (eds). Autoimmune disorders, Intech Publishers Inc., Rijeka, Croatia, ISBN: 978-953-307-653-9, pp41-66, 2011

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2.4.4. Manuscript n° 2: Diagnostic and prognostic features of Sjögren’s syndrome.

Soyfoo MS and Cogan E. Rheumatic diseases, 2011, Intech publishers.

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2.5. Mouse Models for SS

To understand the complex pathogenesis of SS, different mouse models for SS have been developed. They represent unique tools to study the different mechanistic processes underlying SS. The general characteristics of the different mouse models for SS and the detailed characteristics of the mouse models for SS used in the thesis work (NOD, IQI/JIC and R1ΔT/R2n mice) are given hereafter.

2.5.1. Manuscript n°3: Usefulness of Mouse models to study the pathogenesis of Sjögren’s syndrome.

Soyfoo MS, Steinfeld SD and Delporte C. Oral Diseases 2007 ;13 :366-75.

A detailed review of the different mouse models for SS is presented in Manuscript n°3. The general characteristics of the different mouse models for SS are represented in tables 4 and 5.

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Table 4. Characteristics of spontaneous mouse models for SS

NOD NFS/sld MRL/lpr NZB/W IQI/jic aly/aly

Occurance F>M F>M F=M F>M F>M F=M

Loss of secretory function

Yes At 4 mo

Yes*

At 18 mo

No

Yes At 6 mo

ND ND

Inflammatory Infiltrates

CD4+>CD8+

At 1 mo

CD4+>CD8+

At 1mo

CD4+>CD8+

At 2 mo

CD4+>CD8+

At 4 mo

CD4+ in small foci B cells in large foci

At 2 mo

CD4+>CD8+

At 3 mo

Serological

AntiSSA/SSB

-fodrin AAB

ASDA

-fodrin AAB

ANA ANA ANA NO

Chronicity Yes Yes Yes Yes Yes Yes

Involvement Other organs

Yes Yes Yes Yes Yes Yes

ND: Not Determined; AAB: autoantibodies; ANA: antinuclear antibodies; ASDA: Anti salivary duct antibodies; F: female M:male; mo:months. Loss of salivary function in the nfs/sld mice is thought to be an age-related process rather than due to autoimmune disease.

Université Libre de Bruxelles Faculté de Médecine Contribution to the diagnosis and pathophysiology of Sjögren