**3.3 Genetic factors**

44 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

Ebstein-barr, Human T Lymphotropic Virus-1 and hepatitis C have been proposed to be associated with SS. However, the link between these viral infections and SS remains weak (Pflugfelder et al., 1993; Green et al., 1989, Haddad et al., 1992; Iwakiri et al., 2009). Coxsackie virus was found to be increased in SS salivary glands, but these findings have been the subject of some controversies (Triantafyllopoulou et al., 2004, Gottenberg et al., 2006a). Innate immunity is classically stimulated by infectious agents, which results in type I interferon production. Upon viral infection, the activation of the interferon pathway can be perpetuated by the formation of immune complexes containing viral RNA, thereby leading

Stress has been advocated as being a forerunner of SS, since pSS patients experienced a high

The high female to male predominance of SS clearly delineates the role of hormones in the pathogenesis of SS. Estrogenic action is largely imputed in the high female predominance in several autoimmune diseases, including SS (Whitacre, 2001). Estrogens and androgens are thought to respectively contribute or protect to autoimmunity. Onset of SS generally occurs around menopause, when 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 (CRISP-3, a protein upregulated by DHEA) expression, alteration of CRISP-3 polarized expression in acini, altered and decreased conversion of DHEA, and abnormal expression of steroidogenesis enzymes were detected in SS patients (Laine et al., 2007, Porola et al., 2008; Spaan et al., 2009). Women's local salivary gland dihydrotestosterone production is totally dependent on DHEA conversion, rendering them highly vulnerable to local

Estrogens play a cardinal role in targeting salivary epithelial cell and stimulating apoptosis through a Fas-mediated mechanism (Ishimaru et al., 1999). Retinoblastomaassociated protein 48 (RbAp48) induces tissue specific apoptosis in salivary glands depending on the level of estrogen deficiency (Ishimaru et al., 2008). More recently, the presence of functional estrogen receptors has been observed in salivary epithelial cells (Tsinti et al., 2009). In the latter study, estrogen was shown to block expression of ICAM-1, an adhesion molecule displaying increased expression in salivary glands of SS patients. It may therefore be speculate that estrogen deficiency might lead to increased innate

Prolactin, a pro-inflammatory hormone, stimulates estrogen activity and inhibits estrogen production, high level T cell proliferation, IL2 receptor expression, IFN- production and stimulation of antibody production (Taiym et al., 2004). Higher levels of prolactin are detected in SS patients, and may be involved with the production of autoantibodies

to plasmacytoid dendritic cell activation and production of interferon-.

degree of stress prior to the onset of disease (Karaiskos et al., 2009).

**3.1 Environmental factors 3.1.1 Viral infections** 

**3.1.2 Stress** 

**3.2 Endocrine factors 3.2.1 Sexual hormones** 

androgen deficiency.

involved in SS (Taiym et al., 2004).

immunity.

#### **3.3.1 Genetic variation**

Genetic predisposition is widely accepted as being an important etiological factor in many autoimmune diseases (Hewagama & Richardson, 2009). Several studies support the existence of predisposition to SS (Jonsson et al., 2007). Alleles within the major histocompatibility complex class II gene region, predominantly the HLA-DR and HLA-DQ (Loiseau et al., 2001), are implicated in the pathogenesis of SS. Susceptibility alleles in SS patients may also vary according to ethnic origin (Bolstad and Jonsson, 2005).

An increasing body of evidence for the implication of other gene variants outside the HLA locus association is being put forth recently. Gene polymorphisms of IRF-5 and STAT-4 genes, two transcription factors of pivotal importance in interferon pathway, have been associated with various autoimmune diseases (Martinez et al., 2008), as well as with SS (Miceli-Richard et al., 2007; Korman et al., 2008; Miceli-Richard et al., 2009; Nordmark et al., 2009, Gestermann et al., 2010). The most significantly associated single nucleotide polymorphism (SNP) of the IRF5 gene was a 4 fold repetition, instead of three, of a sequence within the promoter region (Nordmark et al., 2009). An association between this polymorphism and high levels of IRF5 mRNA was demonstrated in PBMCs and in cultured salivary epithelial cells after viral infection (Miceli-Richard et al., 2009). STAT4 polymorphism was also associated with SS (Nordmark et al., 2009; Korman et al., 2008; Gestermann et al., 2010; Palomino-Morales et al., 2010).

MECP2 and IL2-IL21 polymorphisms have also been associated with SS (Cobb et al., 2010; Maiti et al., 2010). Gene polymorphisms in IL-10, IL-6, IL-1 receptor antagonist, IL-4 receptor , TNF-, IFN- and TGF-1 have also been associated with pSS (Cobb et al., 2008).

PTPN22 (protein tyrosine phosphatase nonreceptor 22), primarily expressed in lymphoid tissues, has been suggested to have prominent roles in T-cell signaling. The 1858 T allele of PTPN22 has been shown to be a risk factor for SS in one Columbian study whilst other studies did not find any significant association with SS (Gomez et al., 2005; Ittah et al., 2005). PTEN, a tumour suppressor gene, displayed a rare mutations shown to be associated concomitantly with SS and Cowden disease (Raizis et al., 1998) and may be associated, in diseases, with the latter occurrence of non-Hodgkin lymphoma.

Very recently, a large Swedish-Norwegian study has associated potentially muscarinic receptor-3 gene variant with SS (Appel et al., 2011). In this study, focus scores, abnormal Schimer's test and autoantibody presence were associated with muscarinic receptor-3 SNPs.

Recent data have suggested an increased association between immune system genes and the pathogenesis of primary SS. Indeed, an increase in the copy number of 2 genes linked to immune regulation-FCGR3B and CCL3L1-that can confer susceptibility to SS (Mamtani et al., 2010). A similar study revealed, besides confirming association of STAT4 and IRF5/TNPO3, three new loci as being associated as well with SS. However, the SNPs studied were not associated with the presence of anti-SSA/anti-SSB antibodies; though they are all involved in B-cell differentiation and activation (Nordmark et al., 2011).

Finally, in contrast one polymorphic variant, 168His of the minor histocompatibilty antigen HA-1, has been described as protective, lowering the risk of pSS (Harangi et al., 2005).

#### **3.3.2 Epigenetic control**

Epigenetic mechanisms are currently and increasingly being associated in disease processes, including autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus,

Primary Sjögren's Syndrome: Current Pathophysiological, Diagnostic and Therapeutic Advances 47

(Mavragani and Crow., 2010). Increased expression of TNF-, IL-1, IL-12, IL-18, and IFN has also been shown in SS patients (Vougarelis and Tzioufas, 2010, Becker et al., 2010). Recently, the role of IL-17 producing cells (Th17) has been underlined in the pathogenesis of SS (Nguyen et al., 2008; Espinosa et al., 2009). The IL-23/Th17 pathway triggers autoimmune exocrinopathy and systemic autoimmunity. Mice lacking Ro 52 antigen are characterized by increased proinflammatory cytokines, 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). Elevated serum levels of IL-17 and Th17 cells in patients with SS and related cytokines are predominant in salivary glands and strongly

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 (Lavie et al., 2004). Ectopic germinal centre-like structures are a hallmark of B cell activation and proliferation and occur in about 20% of patients with pSS (Garcia-Carrasco et al., 2002). 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 and B cells. B cell dysregulation plays a crucial role in perpetuating inflammation and tissue damage (Pers 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 signaling and proliferation and increased production of antibody-producing

Autonomic system dysfunction is also considered as a central feature in the pathogenesis of SS (Fox and Stern, 2002; Dawson et al., 2001). It might be responsible for glandular dysfunction and diminished salivary and lachrymal production (Waterman et al., 2000; Humphreys-Beher et al., 1999). The role of autonomic dysfunction in SS pathogenesis is supported by the presence of anti muscarinic M3 receptors (M3R) autoantibodies in 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) or yet modified epigenetic control on M3R (Appel et al., 2010). Furthermore, M3R Tcells 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

Autoantibodies against Ro (SSA) and La (SSB) are found in the serum of pSS and sSS patients (Garcia-Carrascos et al., 2002). They are linked to the onset, severity, duration and

correlate with histological focus score (Katsifis et al., 2009).

plasma cells (Mariette et al., 2003; Groom et al., 2002; Jonsson et al., 2005).

**3.5 Autonomic system** 

of saliva (Dawson et al., 2001).

**3.6 Autoantibodies** 

systemic sclerosis and SS (Pan and Sawalha., 2009; Richardson, 2007; Hewagama & Richardson, 2009; Brooks et al., 2010). In salivary glands from SS patients undergoing extracellular matrix remodelling, mechanotransduction may convey organ structural modifications that will inturn affect epigenetic control of gene expression (Gonzalez et al., 2011a). Indeed, the global DNA methylation of salivary glands from SS patients appears to be decreased, while specific genes appear to be hypermethylated (Gonzalez et al., 2011b).

MicroRNAs (miRNA) are an emerging field of epigenetic gene expression control, with potential involvement in autoimmune diseases (Dai & Ahmed, 2011; Alevizos & Illei, 2010a). miRNAs are small (20-22 nucleotides RNAs), resulting from a complex cellular processing, leading to RNA sequestration or destruction. miRNAs 17 & 92 have been shown to be associated with SS (Alevizoz et al., 2011a). Another study showed that over-expression of 2 miRNAs (miR-574 and miR-768-3p) participates to the epigenetic control of gene expression in salivary glands from SS patients (Alevizos et al., 2011b). These two micro-RNAs were associated with a high degree of inflammation and correlated with the histological focus score. As such, they could represent future biomarkers of inflammation in SS patients (Alevizos & Illei, 2010b, 2011). In animal model of SS, upregulation of miRNA 150 and 146 in PBMC and target tissue was observed (Lu et al., 2010). Furthermore, miR23A is highly expressed in salivary glands from SS patients and may regulate CUL3 expression (Gonzalez et al., 2011a).

#### **3.4 Immune system alterations**

The innate immune system plays a fundamental role in the 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 TLR 2, 3, 4 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, an 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). Upregulation of TLR3 in salivary glands of female new Zealand/WF1 female mice, in response to 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 signaling 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 salivary glands from SS patients (Gottenberg et al., 2006; Hjelmervik et al., 2005), as well as in PBMC and whole blood (Emamian et al., 2009). Increased serum levels of IFN- and have been observed in pSS patients. Circulating PDC express high levels of CD40 (a marker of cellular activation), which correlate 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 proliferation and survival, induction of several chemokines and favoring 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 (Vougarelis and Tzioufas, 2010, Becker et al., 2010). Recently, the role of IL-17 producing cells (Th17) has been underlined in the pathogenesis of SS (Nguyen et al., 2008; Espinosa et al., 2009). The IL-23/Th17 pathway triggers autoimmune exocrinopathy and systemic autoimmunity. Mice lacking Ro 52 antigen are characterized by increased proinflammatory cytokines, 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). Elevated serum levels of IL-17 and Th17 cells in patients with SS and related cytokines are predominant in salivary glands and strongly correlate with histological focus score (Katsifis et al., 2009).

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 (Lavie et al., 2004). Ectopic germinal centre-like structures are a hallmark of B cell activation and proliferation and occur in about 20% of patients with pSS (Garcia-Carrasco et al., 2002). 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 and B cells. B cell dysregulation plays a crucial role in perpetuating inflammation and tissue damage (Pers 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 signaling and proliferation and increased production of antibody-producing plasma cells (Mariette et al., 2003; Groom et al., 2002; Jonsson et al., 2005).

#### **3.5 Autonomic system**

46 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

systemic sclerosis and SS (Pan and Sawalha., 2009; Richardson, 2007; Hewagama & Richardson, 2009; Brooks et al., 2010). In salivary glands from SS patients undergoing extracellular matrix remodelling, mechanotransduction may convey organ structural modifications that will inturn affect epigenetic control of gene expression (Gonzalez et al., 2011a). Indeed, the global DNA methylation of salivary glands from SS patients appears to be decreased, while specific genes appear to be hypermethylated (Gonzalez et al., 2011b). MicroRNAs (miRNA) are an emerging field of epigenetic gene expression control, with potential involvement in autoimmune diseases (Dai & Ahmed, 2011; Alevizos & Illei, 2010a). miRNAs are small (20-22 nucleotides RNAs), resulting from a complex cellular processing, leading to RNA sequestration or destruction. miRNAs 17 & 92 have been shown to be associated with SS (Alevizoz et al., 2011a). Another study showed that over-expression of 2 miRNAs (miR-574 and miR-768-3p) participates to the epigenetic control of gene expression in salivary glands from SS patients (Alevizos et al., 2011b). These two micro-RNAs were associated with a high degree of inflammation and correlated with the histological focus score. As such, they could represent future biomarkers of inflammation in SS patients (Alevizos & Illei, 2010b, 2011). In animal model of SS, upregulation of miRNA 150 and 146 in PBMC and target tissue was observed (Lu et al., 2010). Furthermore, miR23A is highly expressed in salivary glands from SS patients and may regulate CUL3 expression (Gonzalez

The innate immune system plays a fundamental role in the 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 TLR 2, 3, 4 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, an 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). Upregulation of TLR3 in salivary glands of female new Zealand/WF1 female mice, in response to 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 signaling pathways in salivary glands of SS patients leading to production of proinflammatory cytokines, T cell

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

Several cytokines have been implicated in the pathogenesis of SS. Increased expression of IFN-regulated genes has been shown in salivary glands from SS patients (Gottenberg et al., 2006; Hjelmervik et al., 2005), as well as in PBMC and whole blood (Emamian et al., 2009). Increased serum levels of IFN- and have been observed in pSS patients. Circulating PDC express high levels of CD40 (a marker of cellular activation), which correlate 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 proliferation and survival, induction of several chemokines and favoring Th1 responses

stimulation of B cells proliferation and differentiation (Jonsson et al., 2002).

et al., 2011a).

**3.4 Immune system alterations** 

activation and a T helper type 1 driven immune response.

Autonomic system dysfunction is also considered as a central feature in the pathogenesis of SS (Fox and Stern, 2002; Dawson et al., 2001). It might be responsible for glandular dysfunction and diminished salivary and lachrymal production (Waterman et al., 2000; Humphreys-Beher et al., 1999). The role of autonomic dysfunction in SS pathogenesis is supported by the presence of anti muscarinic M3 receptors (M3R) autoantibodies in 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) or yet modified epigenetic control on M3R (Appel et al., 2010). Furthermore, M3R Tcells 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).

#### **3.6 Autoantibodies**

Autoantibodies against Ro (SSA) and La (SSB) are found in the serum of pSS and sSS patients (Garcia-Carrascos et al., 2002). They are linked to the onset, severity, duration and

Primary Sjögren's Syndrome: Current Pathophysiological, Diagnostic and Therapeutic Advances 49

caspase-3 and cleave PARP and trigger apoptosis. Furthermore, these autoantibodies have been shown to also activate extrinsic apoptotic pathways by transcriptional upregulation and activation of caspase-8. Anti-Ro and anti-La autoantibodies could trigger apoptosis

Aquaporins (AQP) are small transmembrane proteins involved in water flow across cell membranes. Several AQPs are expressed in salivary glands and lachrymal glands (Delporte, 2009). AQP5, expressed on salivary acinar cells, contributes to salivary flow (Ma et al., 1999). A modified distribution of AQP5 expression has been documented in both human salivary and lachrymal glands from pSS patients (Steinfeld et al., 2001; Tsubota et al., 2001) and in SS animal models (Konttinen et al., 2005; Soyfoo et al., 2007, Sasaki et al., 2007; Ohashi et al., 2008). Treatment by rituximab restored apical localization of AQP5 (Ring et al., 2006). A decreased expression of AQP1 in salivary glands of SS patients was observed (Beroukas et al., 2001; Beroukas et al., 2002). Both AQP5 and AQP1 could participate to the pathogenesis

An active remodelling of the basal lamina is taking place in acinar and ductal cells from SS patients. In normal salivary glands, laminin 5 bridges the basal lamina with epithelial cells by forming adhesion complexes through specific integrin 64 (Velozo et al., 2009). Modified expression of basal laminins, laminins 1 and 5, occurs during different stages of SS (Kwon et al., 2006; Laine et al., 2004). In salivary acinar cells from SS patients, altered distribution of 64 integrin results in the destruction epithelial cell complexes with laminins (Velozo et al., 2009). Tight junction protein levels and distribution of ZO-1, occluding, claudin-1 and claudin-4 were modified in patients with SS (Ewert et al., 2010). Therefore, maintenance of equilibrium between cell-cell and cell-basal lamina attachment is

Xerophtalmia is often less prominent than xerostomia. It is therefore necessary to follow a detailed anamnestic investigation to detect symptoms of ocular dryness. The main complaint of xeropthalmia is foreign-body sensation, but other symptoms such as grittiness, thick rope like secretions at the inner canthus, photosensitivity, burns, and sensation of having a veil before the eyes, absence of tears after irritation or emotion are all frequent features of xerophtalmia. Xerophtalmia is due to the lymphocytic infiltration of lachrymal glands leading to reduced lachrymal flow and tear composition, thereby altering corneal and conjunctival epithelia, characterizing the known condition of keratoconjunctivitis sicca (KCS). In severe disease, functional disability with visual

More than 90% of patients with SS complain of symptoms resulting from functional alteration of salivary glands. The symptoms range from dry mouth and lips, the need to drink more water when eating, to difficulties in the mastication process. In the early phase

of SS, although they can not account for salivary and lachrymal secretory defects.

resulting into tissue destruction (Ramos-Casals and Font, 2005).

**3.9 Alterations of proteins involved in glandular function** 

necessary to ensure gland cell survival.

**4. Clinical manifestations** 

impairment may occur (Fox, 2005).

**4.1 Xeropthalmia** 

**4.2 Xerostomia** 

extraglandular manifestations of SS (Jonsson et al., 2002; Skopouli et al., 2005). It is still undetermined if these antibodies play a direct pathogenic role in the glandular damage. Nonetheless, there is evidence supporting a 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 in patients with SS. Increased mRNA production of La in acinar epithelial cells and translocation of La protein, resulting in membrane localization, in the conjunctival epithelial cells have been observed in SS patients (Hammi et al., 2005; Tzioufas et al., 1999).

Autoantibodies against fodrin, a major constituent of the cytoskeleton, have also been detected in sera from patients with SS. Abnormal location of -fodrin on the surface of apoptotic-induced cells suggests the role of -fodrin in SS through apoptotic pathways. Aberrant proteolysis of -fodrin results in its expression at the surface of apoptotic epithelial cells entailing the autoimmune process (Locht et al., 2008; Willeke et al., 2007).

Autoantibodies to muscarinic M3 receptors, 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).

#### **3.7 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 defenses (Manoussakis and Kapsogeorgou, 2010). Epithelial salivary glands cells, even if not proven to act as antigen-presenting cells possess all the features to do so (Manoussakis et al., 1999; Matsumura et al., 2001). Epithelial cells might act as nonprofessional antigen-presenting cells, and thereby participate in autoimmune responses leading to the development of SS (Tsunawaki et al., 2002; Xanthou et al., 2001; Dimitriou et al., 2002; Lavie et al., 2004). Proinflammatory cytokines and other factors can induce 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 the autoimmune process. Besides, following type 1 IFN stimulation and viral infection of epithelial cells, the latter releases BAFF thereby activating B cells (Kapsogeorgou et al., 2005; Ittah et al., 2009).

#### **3.8 Apoptosis**

A pivotal role for apoptosis as a pathogenic mechanism in SS-related glandular damage has been demonstrated. Increased apoptosis of the 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 molecules leading to the proteolysis of exocrine autoantigens and ensuing glandular damage. Disequilibrium between pro-apoptotic signals and anti-apoptotic mechanisms might act as the basis of epithelial cell destruction of exocrine glands in pSS.

Apoptotic cell death might also function in a specific fashion favoring abnormal exposure of nuclear and cytoplasmic autoantigens thereby providing mechanism of antigen presentation to autoreactive T cells. Furthermore, anti-Ro and anti-La autoantibodies can activate caspase-3 and cleave PARP and trigger apoptosis. Furthermore, these autoantibodies have been shown to also activate extrinsic apoptotic pathways by transcriptional upregulation and activation of caspase-8. Anti-Ro and anti-La autoantibodies could trigger apoptosis resulting into tissue destruction (Ramos-Casals and Font, 2005).

#### **3.9 Alterations of proteins involved in glandular function**

Aquaporins (AQP) are small transmembrane proteins involved in water flow across cell membranes. Several AQPs are expressed in salivary glands and lachrymal glands (Delporte, 2009). AQP5, expressed on salivary acinar cells, contributes to salivary flow (Ma et al., 1999). A modified distribution of AQP5 expression has been documented in both human salivary and lachrymal glands from pSS patients (Steinfeld et al., 2001; Tsubota et al., 2001) and in SS animal models (Konttinen et al., 2005; Soyfoo et al., 2007, Sasaki et al., 2007; Ohashi et al., 2008). Treatment by rituximab restored apical localization of AQP5 (Ring et al., 2006). A decreased expression of AQP1 in salivary glands of SS patients was observed (Beroukas et al., 2001; Beroukas et al., 2002). Both AQP5 and AQP1 could participate to the pathogenesis of SS, although they can not account for salivary and lachrymal secretory defects.

An active remodelling of the basal lamina is taking place in acinar and ductal cells from SS patients. In normal salivary glands, laminin 5 bridges the basal lamina with epithelial cells by forming adhesion complexes through specific integrin 64 (Velozo et al., 2009). Modified expression of basal laminins, laminins 1 and 5, occurs during different stages of SS (Kwon et al., 2006; Laine et al., 2004). In salivary acinar cells from SS patients, altered distribution of 64 integrin results in the destruction epithelial cell complexes with laminins (Velozo et al., 2009). Tight junction protein levels and distribution of ZO-1, occluding, claudin-1 and claudin-4 were modified in patients with SS (Ewert et al., 2010). Therefore, maintenance of equilibrium between cell-cell and cell-basal lamina attachment is necessary to ensure gland cell survival.
