Co-culture system of human salivary gland epithelial cells and immune cells from primary Sjögren's syndrome patients: an in vitro approach to study the effects of Rituximab on the activation of the Raf-1/ERK1/2 pathway.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disorder of the exocrine glands with associated lymphocytic infiltrates in the affected glands. Dryness of the mouth and eyes results from involvement of the salivary and lacrimal glands. The efficacy of Rituximab (RTX) in pSS is still open to debate. This study delineates the signaling pathway involved in RTX-mediated down-regulation of pro-inflammatory factors in a co-culture system of pSS salivary gland epithelial cells (SGEC) with syngeneic pSS B-lymphocytes. In addition, the effects of RTX on the activation of the Raf-1/ERK1/2 pathway in pSS SGEC co-cultured with syngeneic pSS T-lymphocytes were also investigated. This study demonstrated that RTX may interfere with the ERK1/2 pathway in a syngeneic co-culture of pSS SGEC with pSS B-lymphocytes, leading to decreased cytokine production by SGEC. These novel findings reveal that syngeneic co-culture of pSS SGEC with pSS B-lymphocytes leads to a down-regulation of Raf-1 in epithelial cells that adversely regulates the activity of the ERK1/2 pathway and determines a subsequent reduction of the release of pro-inflammatory factors.

The metalloproteinase ADAM17 and the epidermal growth factor receptor (EGFR) signaling drive the inflammatory epithelial response in Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disorder that particularly compromises the function of exocrine glands. The pathogenetic mechanisms of this autoimmune exocrinopathy have not been fully elucidated. Since increasing evidence actually suggests that the epidermal growth factor receptor (EGFR) pathway has a major impact on the inflammatory/immune reactions of the epithelial cells, in the apparent effort of enhancing innate immune defense while opposing overactivation of pro-inflammatory functions, the focus of the work presented here is clarify whether the EGFR-extracellular-signal-regulated kinase (ERK) pathway plays a role in the pro-inflammatory responses mounted by pSS salivary gland epithelial cells (SGEC). Investigations revealed that the EGFR-mediated activation of the downstream effectors ERK1/2 in pSS SGEC appeared to require ADAM17-dependent release of the endogenous EGFR ligand amphiregulin and transactivation of the EGFR. Moreover, blockade of amphiregulin bioactivity using a neutralizing Ab significantly reduced EGFR transactivation and ERK1/2 phosphorylation. In addition, pSS SGEC treated with the specific ADAM17 inhibitor TAPI-1 and with the EGFR inhibitor AG1478 exhibited deactivated AREG/EGFR/ERK signaling pathway and reduced pro-inflammatory cytokines released.

ADAM17 at the interface between inflammation and autoimmunity.

The discovery of the disintegrin and metalloproteinase 17 (ADAM17), originally identified as tumor necrosis factor-a converting enzyme (TACE) for its ability as sheddase of TNF-α inspired scientists to attempt to elucidate the molecular mechanisms underlying ADAM17 implication in diseased conditions. In recent years, it has become evident that this protease can modify many non matrix substrates, such as cytokines (e.g. TNF-α), cytokine receptors (e.g. IL-6R and TNF-R), ligands of ErbB (e.g. TGF-α and amphiregulin) and adhesion proteins (e.g. Lselectin and ICAM-1). Several recent studies have described experimental model system to better understand the role of specific signaling molecules, the interplay of different signals and tissue interactions in regulating ADAM17-dependent cleavage of most relevant substrates in inflammatory diseases. The central question is whether ADAM17 can influence the outcome of inflammation and if so, how it performs this regulation in autoimmunity, since inflammatory autoimmune diseases are often characterized by deregulated metalloproteinase activities. This review will explore the latest research on the influence of ADAM17 on the progression of inflammatory processes linked to autoimmunity and its role as modulator of inflammation.

Neovascularization is prominent in the chronic inflammatory lesions of Sjögren's syndrome.

Angiogenesis is a common finding in chronic inflammatory diseases; however, its role in Sjögren's syndrome (SS) remains to be elucidated. Previous SS studies have demonstrated an increase in VEGF-A/VEGFR-2 system expression in minor salivary gland (MSG) biopsies from patients with SS, but differences in the new blood vessel formation between the different grades of disease severity have not been reported. Therefore, experiments were performed to demonstrate angiogenesis during different phases of primary SS (pSS) and to define the relationship between the microvessel density (MVD), macrophage infiltration and histiocyte distribution in SS MSG inflammatory lesions. In this series of experiments, immunohistochemistry was used to examine angiogenesis in serial sections of pSS MSG. Patients with pSS were classified accordingly with the grade of inflammatory lesions as I = low-grade (low focus score of 1 or 2), II = intermediate-grade (focus score of 3­6) and III = extensive inflammation in the MSG (high focus score of 12). Histological examination demonstrated that the MVD increased with the severity of the inflammatory lesions, and in addition, we found an increased infiltration of inflammatory and pro-angiogenic cells.These findings reveal that angiogenesis is intimately involved in the progression of pSS, may be central to the propagation of the chronic immune response observed in pSS and could represent a novel potential biomarker of pSS disease activity.

Rituximab-mediated Raf kinase inhibitor protein induction modulates NF-κB in Sjögren syndrome.

Primary Sjögren syndrome (pSS) is an autoimmune disorder characterized by an epithelial injury surrounded by dense lymphocytic infiltrates. The conditions for the long-term maintenance of human salivary gland epithelial cells from pSS patients and a co-culture system with pSS lymphocytes were used to assess the effect of Rituximab (RTX) on the inflammatory condition and progression in pSS. Quantitative real-time PCR, genes and protein array analysis, Western blot, flow cytometry, small interfering RNA transfection and nuclear factor-κB (NF-κB) DNA binding assays were used as methods. Supporting the benefits of RTX, this study demonstrates that RTX decreases NF-κB activity and interrupts the NF-κB signalling pathway through the up-regulation of the Raf-1 kinase inhibitor protein (RKIP). Over-expression of RKIP down-regulates interleukins, their receptors and the expression of genes encodes proteins that attracted lymphocytes. Silencing of the RKIP gene leads to significantly increased expression and release of pro-inflammatory mediators supporting that RKIP expression could be involved in the suppression of NF-κB activation in pSS salivary gland epithelial cells.

Chronic inflammation enhances NGF-ß/TrkA system expression via EGFR/MEK/ERK pathway activation in Sjögren's syndrome.

UNLABELLED: Primary Sjögren's syndrome (pSS) is a chronic autoimmune exocrine disease associated with variable lymphocytic infiltration of the affected organs (primarily salivary and lachrymal glands). To investigate the potential implication of nerve growth factor-ß (NGF-ß) and its high affinity receptor tyrosine kinase receptor A (TrkA) in the regulation of pSS inflammatory responses, we studied their expression in the human salivary gland epithelial cells (SGEC) cultures from pSS minor salivary glands (MSG) biopsies and their relationship with histopathological disease parameters. Here, we demonstrated an increased expression of the NGF-ß/TrkA system in pSS SGEC, correlated with the MSG inflammation grade. The results demonstrate that the pro-inflammatory cytokines TNF-α and IL-6 enhance NGF-ß production; on the contrary, NGF-ß production was reduced in the presence of both Raf-1 kinase and MEK inhibitors. Furthermore, TNF-α/IL-6 treatment increased ERK1/2 phosphorylation. Inhibition of the EGF/EGFR system also decreased NGF-ß release by pSS SGEC, indicating that the chronic inflammatory condition characteristic of pSS enhances NGF-ß production via EGFR/Raf-1/MEK/ERK pathway activation. KEY MESSAGE: NGF-ß and TrkA expression is elevated in salivary gland epithelial cells of primary Sjögren's syndrome (pSS). Overexpression of NGF-ß/TrkA system in pSS occurs via EGFR/Raf-1/MEK/ERK pathway. In pSS, NGF-ß overexpression was prevented by EGFR/Raf-1/MEK/ERK pathway inhibition.

[Paget's disease of bone: new therapeutic strategies]. / La malattia di Paget: a proposito di nuove strategie terapeutiche.

Paget's disease of bone is a chronic disorder of unknown etiology that can result in enlarged and misshapen bones. The excessive breakdown and formation of bone tissue cause affected bones to weaken, resulting in pain, misshapen bones, fractures, and arthritis in the joints. In most cases the diagnosis is achieved casually, as only 5% of patients develop burning pain at the level of affected bones. As regards therapy, the use of anti-reabsorbing drugs, such as bisphosphonates and calcitonin, appears reasonable. Given the disease pathogenesis, the administration of denosumab and tocilizumab may be a valuable alternative to inhibit RANK expression, and thus osteoclast formation, and interleukin-6 production.

Salivary gland expression level of IκBα regulatory protein in Sjögren's syndrome.

Diagnosis and therapeutic strategies in Sjögren's syndrome (SS) might greatly benefit of the present multidisciplinary approach to studying the molecular pathogenesis of the disease. A deregulated inflammatory response has been described in the SS. The research in the last years sheds light on the importance of the NF-κB pathway regulating the pro-inflammatory cytokine production and leukocyte recruitment. These are important contributors to the inflammatory response during the development of SS. In this study we examine the expression of the NF-κB inhibitory protein termed IκBα in salivary glands epithelial cells (SGEC) comparing it with SGEC from healthy controls, to test the hypothesis that an altered expression of IκBα occurs in SGEC from SS biopsies. Real-Time PCR, western blot and immunohistochemistry demonstrated that the expression level of IκBα was significantly lower in SS with respect to healthy controls leading to an increased NF-κB activity. Our results suggest that the analysis of IκBα expression at salivary gland epithelial cell level could be a potential new hallmark of SS progression and sustain a rationale to more deeply investigate the therapeutic potential of specific NF-κB inhibitors in SS.

GRO-α/CXCR2 system and ADAM17 correlated expression in Sjögren's syndrome.

The chemokine GRO-α and its receptor CXCR2 are associated with the chronic inflammation in Sjögren's syndrome (SS). To better understand the molecular mechanisms by which the GRO-α/CXCR2 system is involved in the SS inflammatory condition, our studies were designed to clarify the role of ADAM17 activation in the modulation of the GRO-α/CXCR2 chemokine system in epithelial cells (SGEC) from SS salivary glands. The CXCR2 overexpression observed in SS SGEC was dramatically decreased by ADAM17 inhibitor TAPI-1. In addition, comparing the expression levels of ADAM17 in healthy SGEC in presence or not of GRO-α treatment, we observed that GRO-α dose-dependently influences ADAM17 activation, an effect that was inhibited by blocking the interaction of GRO-α with its CXCR2 receptor. Our data show for the first time that ADAM17 has an important role in GRO-α/CXCR2 system activity regulation, suggesting that regulating CXCR2/ADAM17 interaction could be an attractive therapeutic target in SS.

Emerging avenues linking inflammation, angiogenesis and Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune disease characterized by an inflammatory mononuclear infiltration and the destruction of epithelial cells of the lachrymal and salivary glands. The aetiology is unknown. The expression "autoimmune epithelitis" has been proposed as an alternative to SS, in view of the emerging central role of the epithelial cells in the disease pathogenesis. At the biomolecular level, the epithelial cells play an important role in triggering the autoimmune condition via antigen presentation, apoptosis, and chemokine and cytokines release. Inflammation and angiogenesis are frequently coupled in the pathological conditions associated to autoimmune diseases, and an angiogenic imbalance contributes to the pathogenesis of a number of inflammatory disorders. This work reviews the current knowledge of the molecular and cellular mechanisms underlying the pathogenesis of the inflammatory reactions that characterize SS. The literature and our data on the role of angiogenesis in the pathophysiology of the disease are discussed.

A potential role of the GRO-α/CXCR2 system in Sjögren's syndrome: regulatory effects of pro-inflammatory cytokines.

Chemokines, small pro-inflammatory cytokines, are involved in migration of inflammatory cells in inflamed tissues and recent studies established their role in angiogenesis, hematopoiesis, cancer and autoimmune conditions. Growth related oncogene-alpha (GRO-α), a member of the CXC chemokine family, and its receptor CXCR2 are involved in the inflammatory processes. Since there is no previous report that supports a possible role of GRO-α/CXCR2 receptor complex during inflammation and neovascularization existing in the autoimmune disease Sjögren's syndrome (SS), in this study, we examined CXCR2 and its ligand GRO-α expression in SS tissues. Immunohistochemistry revealed that GRO-α and its receptor CXCR2 were expressed at high levels in diseased tissues compared to healthy controls. In addition, human salivary gland epithelial cells (SGEC) cultures were submitted to a pro-inflammatory microenvironment using cytokines IL-6 and TNF-α in order to demonstrate that CXCR2 may change its initial expression pattern to another under inflammatory condition. The data show an increased expression of CXCR2 depending on the inflammatory cytokine used in culture in a time-dependent manner. Furthermore, silencing of the pro-angiogenic chemokine GRO-α is proportionally correlated with decreased expression of CXCR2 in pro-inflammatory cytokine-stimulated SGEC indicating the GRO-α/CXCR2 complex as a novel therapeutic target for the chronic inflammatory disease Sjögren's syndrome.

Saponins from Tribulus terrestris L. protect human keratinocytes from UVB-induced damage.

Chronic exposure to solar UVB radiation damages skin, increasing the risk to develop cancer. Hence the identification of compounds with a photoprotective efficacy is essential. This study examined the role of saponins derived from Tribulus terrestris L. (TT) on the modulation of apoptosis in normal human keratinocytes (NHEK) exposed to physiological doses of UVB and to evaluate their antitumoral properties. In NHEK, TT saponins attenuate UVB-induced programmed cell death through inhibition of intrinsic apoptotic pathway. In squamous cell carcinomas (SCC) TT saponins do not make the malignant keratinocytes more resistant to UVB and determine an enhanced apoptotic response. The photoprotective effect of TT saponins is tightly correlated to the enhancement of NER genes expression and the block of UVB-mediated NF-κB activation. Collectively, our study shows experimental evidence that TT has a preventive efficacy against UVB-induced carcinogenesis and the molecular knowledge on the mechanisms through which TT saponins regulate cell death suggests great potential for TT to be developed into a new medicine for cancer patients.

Altered IκBα expression promotes NF-κB activation in monocytes from primary Sjögren's syndrome patients.

AIMS: To study the importance of IκBα in NF-κB signal transduction, we analysed the IκBα expression in monocytes from Sjögren's syndrome (SS) patients versus healthy controls. METHODS: Monocytes were obtained from the peripheral blood of 30 SS patients and 23 healthy subjects. IκBα expression was studied by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), real-time PCR, immunoblotting, flow cytometry and enzyme linked immunosorbent assay (ELISA). RESULTS: Analysis of the gene and protein expression profiles of SS monocytes revealed a down-regulation of IκBα, and in all the Sjögren's syndrome cases examined, serum IκBα levels were significantly decreased in comparison with controls. CONCLUSIONS: Our findings clearly demonstrate changes in the levels of IκBα in SS monocytes, suggesting that the attenuated expression of IκBα could contribute to the deregulation of NF-κB pathways in the SS pathogenesis. Decreased expression of IκBα may specifically amplify cytokines production and inflammatory response linked to Sjögren's syndrome.

Neuropilin-1 is upregulated in Sjögren's syndrome and contributes to pathological neovascularization.

Neuropilin-1 (NRP1) is a transmembrane co-receptor for members of the vascular endothelial growth factor family. Recent studies revealed an important role of NRP1 in angiogenesis and progression of many diseases. The role of NRP1 in the development of Sjögren's syndrome (SS), one of the most common rheumatic diseases, has not yet been investigated. Molecular studies and protein expression techniques were performed to elucidate the gene and protein expression profile of NRP1 in human salivary gland epithelial cells (SGEC) from primary SS. We used human microarrays and transient transfection with a mutant form of the negative inhibitory κBα proteins (IκBαDN) to investigate whether selective inhibition of nuclear Factor-κB (NF-κB) improves NRP1-mediated pro-angiogenic factors release from SS SGEC. The selective NRP1 function inhibition with an antibody to human NRP1, was employed to evaluate the therapeutic potential of targeting NRP1. We demonstrate that NRP1 is expressed in SGEC of both human healthy biopsies and in SS samples, and increased NRP1 expression in SS SGEC is significantly associated with pro-angiogenic factors release. Neutralizing anti-NRP1 antibody decreased pro-angiogenic factor production from SS SGEC and blocking NF-κB activation could be a way to inhibit NRP1-mediated angiogenesis in Sjögren's syndrome.

Sjögren's syndrome autoantibodies provoke changes in gene expression profiles of inflammatory cytokines triggering a pathway involving TACE/NF-κB.

We explore the association of the inflammatory gene expression profile observed in the chronic inflammatory autoimmune disorder Sjögren's syndrome (SS) with changes in TNF-α converting enzyme (TACE), tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB levels showing that pathways that include TNF-α signaling converge on NF-κB contributing to exacerbate the diseases. The treatment of human salivary gland epithelial cells (SGECs) with SS anti-Ro/SSA autoantibodies (Abs) result in a progressive increase in NF-κB-DNA binding, that includes a marked enhancement in NF-κB subunit p65 protein-DNA binding. A human cytokine multi-analyte array demonstrated that the NF-κB proinflammatory target genes, increased by anti-Ro/SSA Abs treatment, includes CXC chemokines (CXCL1, CXCL6 and CXCL9), CC chemokines (CCL2, CCL13 and CCL20), interleukins (IL-1α, IL-1ß, IL-1F8, IL-6, IL-8, IL-9, IL-13, IL-17 and IL-22) and their receptors (IL-1RN, IL-10Rα, IL-13Rα, CCR1, CCR2, CCR3, CCR4 and CXCR1). Blockade of TACE through the use of the specific inhibitor TAPI-1 regulates proinflammatory cytokines production in SGEC treated with anti-Ro/SSA Abs inhibiting NF-κB nuclear translocation and activation. To further investigate the role of NF-κB on anti-Ro/SSA Abs-determined proinflammatory gene expression, we used the inhibitory protein IκB-α dominant negative super-repressor as inhibitor of NF-κB-DNA binding, demonstrating that transfection with dominant-negative IκB-α in anti-Ro/SSA-treated SGEC determined a marked reduction of proinflammatory cytokines gene expression. Although further studies are needed to clarify the mechanisms underlying SS, our results demonstrate that SS Abs exert their pathogenic effects via triggering the TACE/TNF-α/NF-κB axis.

A failure of TNFAIP3 negative regulation maintains sustained NF-κB activation in Sjögren's syndrome.

Sjögren's syndrome (SS) is characterized by the features of systemic autoimmunity and exocrine gland dysfunction and inflammation. Deregulated cytokine production is known to contribute to the etiology of SS but the underlying molecular mechanism is still remains to be unclear. TNF-α-induced protein 3 or TNFAIP3 is involved in the negative feedback regulation of nuclear factor-κB (NF-κB) signaling in response to specific pro-inflammatory stimuli in different cell types. To define the contribution of TNFAIP3 to SS, the levels of TNFAIP3 expression in human salivary gland epithelial cells (SGEC) derived from active primary SS patients were analyzed. Histological analysis was performed on paraffin-embedded human Sjögren's samples and healthy tissues. In separate experiments, immunofluorescence staining, western blot analysis and quantitative real-time PCR for TNFAIP3 was conducted in SGEC from SS and healthy subjects. Our findings clearly demonstrate changes in levels of the protein and gene expression between healthy controls and SS patients, depicting a very weak positivity for TNFAIP3 in SS samples. TNFAIP3 was found down-regulated in SGECs derived from SS patients in comparison with controls, and the cells with down-regulated TNFAIP3 expression exhibited enhanced NF-κB activities. In addition, to investigate the role of TNFAIP3 in the activation of NF-κB, we depleted TNFAIP3 expression by siRNA in healthy SGEC after treatment with or without TNF-α. Intriguingly, the silencing of TNFAIP3 by its siRNA in healthy SGEC increased NF-κB activation that could explain the deregulated cytokines production observed in SS.

Advances in the understanding of the Fc gamma receptors-mediated autoantibodies uptake.

Receptors for the Fc fragment of immunoglobulin G (FcγRs) are important molecules not only to mediate and control the effectors' functions of IgG antibodies, but they also control the autoimmunity-tolerance balance in the periphery. In humans, three different types of FcγRs, belonging to the Ig gene superfamily, have been identified; FcγRI (cluster of differentiation (CD64), FcγRII (CD32) and FcγRIII (CD16). A wide range of inflammatory and autoimmune diseases, such as vasculitis, glomerulonephritis, and autoimmune hemolytic anemia, seems to be mediated, in part, by FcγRs. Recent findings supposed that, under certain conditions, FcγRs are involved in the penetration of antibodies into cells and FcγRs constitute one of the main effector mechanisms through which autoantibodies exert their action. In this review, we concentrate on the role of human FcγRs in autoantibodies penetration and summarize the current knowledge on the structure, ligand binding capacity and their role in autoimmunity and pathogenic effect of autoantibodies.

Expression of pro-inflammatory TACE-TNF-α-amphiregulin axis in Sjögren's syndrome salivary glands.

The tumor-necrosis-factor-converting-enzyme (TACE)-TNF-α-Amphiregulin (AREG) axis plays an important pathogenic role in inflammatory and autoimmune disorders. However, the pathological roles of these proteins in the chronic autoimmune disease Sjögren's syndrome (SS) remain to be elucidated. It is known that the TACE-AREG axis is clearly part of a larger cascade of signals that starts with the activation of Furin, responsible for maturation of TACE that, in turn, determines the production of active TNF-α, directly involved in the up-regulation of AREG expression. This study showed that Furin, TACE, TNF-α, and AREG proteins, detected in acinar and ductal cells of human salivary glands from SS patients, increased remarkably in comparison with biopsies of labial salivary glands from healthy controls. The changes in Furin, TACE, TNF- α, and AREG proteins' level detected in salivary glands biopsies of SS patients could be responsible for pro-inflammatory cytokines overexpression characterizing Sjögren's syndrome.

RETRACTED: Blockade of TNF-α signaling suppresses the AREG-mediated IL-6 and IL-8 cytokines secretion induced by anti-Ro/SSA autoantibodies.

The aim of this study was to analyze the Furin-TNF-α-converting enzyme (TACE)-amphiregulin (AREG)-IL-6/IL-8 secretion pathway in non-neoplastic human salivary gland epithelial cells (SGECs) stimulated with anti-Ro/SSA autoantibodies (Abs). We examined whether anti-Ro/SSA Abs-mediated TACE activation is responsible for AREG activation. As recent studies have demonstrated that AREG could induce proinflammatory cytokines secretion in epithelial cells, we discuss how TACE-mediated AREG shedding, caused by anti-Ro/SSA Abs treatment, could have a critical role in TNF-α-induced IL-6 and IL-8 secretion by SGEC. Furthermore, the effects of TNF-α blockade on AREG expression and TNF-α-AREG-mediated IL-6 and IL-8 secretion were evaluated. We have discovered that the upregulation of AREG occurs through TNF-α produced after anti-Ro/SSA Abs uptake via Fcγ receptors. Biological drug adalimumab and the gene silencing technique were used to study the AREG-IL-6/IL-8 secretion pathway, demonstrating that (i) adalimumab-mediated TNF-α blocking and TNF-α gene silencing provoke a significant decrease of proinflammatory cytokines production and AREG expression in anti-Ro/SSA Abs-treated SGEC; (ii) AREG gene silencing has a potent inhibitory effect on TNF-α-induced IL-6 and IL-8 secretion in SGEC treated with anti-Ro/SSA Abs; (iii) an inspection of the kinetics of cytokine production after exogeni TNF-α and AREG addition, and the use of cycloheximide in the presence of exogenous TNF-α as stimulant, clarified that TNF-α induces IL-6 and IL-8 secretion through AREG.Laboratory Investigation advance online publication, 20 September 2010; doi:10.1038/labinvest.2010.168.

Pro-inflammatory role of Anti-Ro/SSA autoantibodies through the activation of Furin-TACE-amphiregulin axis.

Prolonged inflammation can be detrimental because it may cause host toxicity and tissue damage. Indeed, excessive production of inflammatory cytokines is often associated with many autoimmune diseases. In this study we demonstrate that the anti-Ro/SSA autoantibodies (Abs) stimulate the production of pro-inflammatory cytokines IL-6 and IL-8 by human healthy salivary gland epithelial cells (healthy SGEC). The secretion of these cytokines is due to amphiregulin (AREG) that is overexpressed in healthy SGEC treated with anti-Ro/SSA Abs and in Sjögren's syndrome. We have discovered that the up-regulation of AREG occurs through TNF-alpha produced following anti-Ro/SSA Abs treatment. The gene silencing technique was used to study the AREG-TNF-alpha-IL-6/IL-8 secretion pathway, demonstrating that: (i) TNF-alpha gene silencing provokes a significant decrease of proinflammatory cytokines production and AREG expression in anti-Ro/SSA Abs-treated healthy SGEC; (ii) AREG gene silencing has a potent inhibitory effect on TNF-alpha-induced IL-6 and IL-8 secretion in healthy SGEC treated with anti-Ro/SSA Abs. These findings indicate that TACE-mediated AREG shedding plays a critical role in TNF-alpha-induced IL-6 and IL-8 secretion by the human healthy salivary gland epithelial cells, suggesting that this may be one of the possible intracellular mechanisms involved in the salivary glands inflammatory response in Sjögren's syndrome.