Epithelial-immune cell interplay in primary Sjogren syndrome salivary gland pathogenesis

In primary Sjogren syndrome (pSS), the function of the salivary glands is often considerably reduced. Multiple innate immune pathways are likely dysregulated in the salivary gland epithelium in pSS, including the nuclear factor-kappa B pathway, the inflammasome and interferon signalling. The ductal cells of the salivary gland in pSS are characteristically surrounded by a CD4(+) T cell-rich and B cell-rich infiltrate, implying a degree of communication between epithelial cells and immune cells. B cell infiltrates within the ducts can initiate the development of lymphoepithelial lesions, including basal ductal cell hyperplasia. Vice versa, the epithelium provides chronic activation signals to the glandular B cell fraction. This continuous stimulation might ultimately drive the development of mucosa-associated lymphoid tissue lymphoma. This Review discusses changes in the cells of the salivary gland epithelium in pSS (including acinar, ductal and progenitor cells), and the proposed interplay of these cells with environmental stimuli and the immune system. Current therapeutic options are insufficient to address both lymphocytic infiltration and salivary gland dysfunction. Successful rescue of salivary gland function in pSS will probably demand a multimodal therapeutic approach and an appreciation of the complicity of the salivary gland epithelium in the development of pSS. Salivary gland dysfunction is an important characteristic of primary Sjogren syndrome (pSS). In this Review, the authors discuss various epithelial abnormalities in pSS and the mechanisms by which epithelial cell-immune cell interactions contribute to disease development and progression

The role of immunoactive factors of epithelial cells in the development of tissue injuries in Sjogren's syndrome

Toll-like receptors (TLR) constitute essential receptors of the immune system and pay significant contribution to the defense of the organism. Up to now, in humans, there have been described at least 11 TLR molecules that are expressed differentially in almost every cell type. The TLRs have the ability to recognize highly conserved components of bacteria, fungi and viruses. Upon triggering, TLRs activate intracellular signaling pathways that result in the initiation of inflammatory responses. More specifically, TLR triggering leads to the induction of pro-inflammatory cytokines and chemokines, costimulatory and adhesion molecules. Moreover, triggering of TLRs, has been found to induce apoptotic cell death in several types of cells, probably aiming towards the elimination of infected cells along with the infectious particle. However, beside the pathogenic constituents, TLRs have the ability to recognize and respond to endogenous ligands expressed in tissues, a fact that supports the hypothesis that TLR signaling can be associated with the pathogenesis of autoimmune diseases. This hypothesis is further supported by data displaying differential expression of the TLRs in tissues of autoimmune patients and induction or perpetuation of organ-specific autoimmune lesions in experimental animal models upon TLR triggering. Primary Sjogren’s syndrome (pSS) or autoimmune epithelitis is a relative common autoimmune exocrinopathy, that is characterized by chronic dysfunction and destruction of salivary and lacrimal glands associated with chronic lymphocytic infiltrations. The clinical, histological and laboratory characteristics of pSS render it an excellent model of studying not only autoimmunity but also the role of epithelial tissue in the development of autoimmune responses. The establishment of long-termed culture of non-neoplastic salivary gland epithelial cell (SGEC) lines derived from SS patients or from patients with non-specific sialoadenitis, constitutes a valuable mean for studying the physiology of salivary glands and their pathophysiological role in the syndrome. Using this approach, it has been shown that the salivary gland epithelial cells are immunologically active, since they express constitutively several immunoactive molecules. Moreover the cells derived from SS-patients were found to be intrinsically activated since they express these molecules in higher levels than the control-patients. In this study, given the importance of epithelial cells in the pathogenesis of inflammatory reactions of pSS patients and the crucial role of TLR signalling in the development of inflammatory responses, we aimed to assess in parallel the expression and function of several TLR molecules in cultured non-neoplastic SGEC lines obtained from pSS patients and disease controls. Thus, we triggered culture non-neoplastic SGEC with TLR-2, -3 and -4 ligands. The quantitative expression of TLR, was assessed in mRNA and protein level, and their function was determined by the induction of the expression of several immunoactive molecules. In confirmation of their central role in tissue immunity, salivary gland epithelial cells displayed high constitutive expression of TLR-1, -2, -3 and -4 molecules. These receptors were found to be functional as their triggering results in the up-regulation of the expression of the adhesion molecule CD54/ICAM.1, of the costimulatory molecule CD40 and of the antigen-presenting molecule MHC-I. In addition, SGEC derived from pSS patients were found to display significantly higher constitutive mRNA expression of TLR-1, -2, -3 and -4 molecules compared to disease controls, a fact, which supports the intrinsic activation that characterises the epithelial cells in pSS patients.Οι υποδοχείς Toll-like (TLR) αποτελούν ουσιώδεις υποδοχείς του ανοσολογικού συστήματος και συμβάλλουν σημαντικά στην άμυνα του οργανισμού αφού συντελούν στην ενεργοποίηση τόσο της φυσικής όσο και της επίκτητης ανοσίας. Στον άνθρωπο έχουν περιγραφεί μέχρι στιγμής τουλάχιστον 11 υποδοχείς που εκφράζονται σχεδόν σε όλους τους κυτταρικούς τύπους, παρουσιάζοντας βέβαια αποκλίσεις στα επίπεδα έκφρασής τους στους διάφορους ιστούς. Οι TLR έχουν την ικανότητα να αναγνωρίζουν υψηλά συντηρημένα συστατικά βακτηρίων, μυκήτων ή ιών. Ύστερα από τη διέγερσή τους, ενεργοποιούν ενδοκυττάριες οδούς μεταγωγής σήματος που καταλήγουν στην έναρξη φλεγμονωδών αποκρίσεων. Πιο συγκεκριμένα η διέγερση των TLR οδηγεί στην επαγωγή προ-φλεγμονωδών κυτταροκινών και χημειοκινών, συνδιεγερτικών και προσκολλητικών μορίων. Παράλληλα, όμως, η διέγερση των υποδοχέων TLR έχει καταδειχθεί ότι επάγει και αποπτωτικό θάνατο σε διάφορους κυτταρικούς τύπους, με πιθανό στόχο την απαλοιφή των προσβεβλημένων κυττάρων και συνεπακόλουθα και του μολυσματικού παράγοντα. Ωστόσο, εκτός από τα στοιχεία των παθογόνων μικροοργανισμών, οι TLR έχουν την ικανότητα να αναγνωρίζουν και να αντιδρούν σε ενδογενή μόρια των ιστών, γεγονός που συνέβαλε στην ενίσχυση της υπόθεσης ότι η ενεργοποίηση των υποδοχέων συμμετέχει στην παθογένεια αυτοάνοσων νοσημάτων. Η τελευταία υπόθεση βασίζεται επίσης και σε αναφορές που καταδεικνύουν τη διαφορική έκφραση των υποδοχέων στους ιστούς ασθενών με αυτοάνοσα νοσήματα, καθώς και την επαγωγή ή την επέκταση των οργανοειδικών αυτοάνοσων βλαβών σε διάφορα πειραματικά ζωικά μοντέλα ύστερα από διέγερση των υποδοχέων TLR. Το πρωτοπαθές σύνδρομο Sjogren ή αυτοάνοση επιθηλιίτιδα είναι μία σχετικά κοινή αυτοάνοση εξωκρινοπάθεια, που χαρακτηρίζεται από χρόνια δυσλειτουργία και καταστροφή των σιελογόνων και δακρυϊκών αδένων, σχετιζόμενες με χρόνιες λεμφοκυτταρικές διηθήσεις. Τα κλινικά, εργαστηριακά και ιστολογικά χαρακτηριστικά του συνδρόμου Sjogren το καθιστούν άριστο πρότυπο μελέτης, όχι μόνο για την αυτοανοσία, αλλά και για την έρευνα του ρόλου του επιθηλιακού ιστού στην εκδήλωση της αυτοάνοσης απόκρισης. Η σύσταση μακρόχρονων καλλιεργειών μη-νεοπλασματικών επιθηλιακών κυτταρικών σειρών (ΕΚΣΑ), που προέρχονται από σιελογόνους αδένες ασθενών με σύνδρομο Sjogren και ασθενών με μη-ειδική σιελαδενίτιδα, αποτέλεσε ένα πολύτιμο μέσο για τη μελέτη της φυσιολογίας των σιελογόνων αδένων, καθώς και του παθοφυσιολογικού τους ρόλου στο σύνδρομο. Χρησιμοποιώντας αυτή την προσέγγιση έχει αποδειχθεί τόσο η ανοσολογική λειτουργία των επιθηλιακών κυττάρων, όπως καταδεικνύεται από την ικανότητά τους να εκφράζουν αυθόρμητα διάφορα ανοσοδραστικά μόρια, όσο και η ενδογενής ενεργοποίηση των κυττάρων που προέρχονται από τους ασθενείς με σύνδρομο Sjogren, αφού τα τελευταία εκφράζουν, αυθόρμητα, σε υψηλότερα επίπεδα τα εν λόγω μόρια. Δεδομένης της συμμετοχής των επιθηλιακών κυττάρων στην παθογένεια των φλεγμονωδών αντιδράσεων που εμφανίζονται στους ασθενείς με πρωτοπαθές σύνδρομο Sjogren, και του σημαντικού ρόλου της σηματοδότησης μέσω των υποδοχέων TLR στην ανάπτυξη των φλεγμονωδών αποκρίσεων, στην παρούσα μελέτη επιδιώξαμε να καθορίσουμε παράλληλα την έκφραση και τη λειτουργία ορισμένων υποδοχέων TLR στα καλλιεργημένα μη-νεοπλασματικά ΕΚΣΑ, που προέρχονται από ασθενείς με πρωτοπαθές σύνδρομο Sjogren και από ασθενείς μάρτυρες (ασθενείς με μη-ειδική σιελαδενίτιδα)

Salivary epithelial cells from Sjogren's syndrome patients are highly sensitive to anoikis induced by TLR-3 ligation

In certain types of cells, Toll-like receptor-3 (TLR-3) ligation by viral dsRNA induces apoptotic death, likely engaged into the elimination of virus-infected cells. We have previously shown that TLR-3 ligation on cultured non-neoplastic salivary gland epithelial cells (SGEC) with polyI:C (a synthetic analogue of viral dsRNA) results in the induction of surface immunoactive molecules, however, the pro-apoptotic effect of such signaling has not been addressed. In this study, polyI:C-treated SGEC were found to suffer severe detachment from substratum and subsequent apoptosis, a phenomenon suggestive of anoikis or anoikia (detachment-induced apoptosis). PolyI:C-induced anoikis in SGEC was associated with the upregulation of the pro-apoptotic Bmf, BimEL and Bax and the down-regulation of the pro-survival Bcl-2 (real-time PCR analyses). Finally, the comparative analysis of SGEC lines derived from primary Sjogren’s syndrome (SS) patients (SS-SGEC) and non-SS controls had revealed that SS-SGEC are particularly susceptible to TLR-3-induced anoikis, as it was triggered by suboptimally low concentrations of polyI:C. This finding correlated with significantly higher constitutive surface TLR-3 expression by SS-SGEC, a feature indicative of their intrinsic activation status. In conclusion, TLR-3 signaling pathway in the salivary epithelium appears to extend beyond the induction of innate immune responses and to involve the activation of programmed-cell death via anoikis. In the same context, the increased vulnerability of SS-SGEC to the injurious effect of TLR-3 ligation is likely associated with the intrinsic activation processes that apparently operate in the epithelia of SS patients, and a feature of key pathogenetic importance for the disorder. (C) 2010 Elsevier Ltd. All rights reserved

RNA interference of interferon regulatory factor-1 gene expression in THP-1 cell line leads to toll-like receptor-4 overexpression/activation as well as up-modulation of annexin-II

Interferon regulatory factor-1 (IRF-1) is a candidate transcription factor for the regulation of the Toll-like receptor-4 (TLR-4) gene. Using a small interfering RNA-based (siRNA) process to silence IRF-1 gene expression in the leukemic monocytic cell line THP-1, we investigated whether such a modulation would alter TLR-4 expression and activation status in these cells. The siIRF-1 cells expressed elevated levels of TLR-4 mRNA and protein compared to controls by 90% and 77%, respectively. ICAM.1 protein expression and apoptosis levels were increased by 8.35- and 4.25-fold, respectively. The siIRF-1 cells overexpressed Bax mRNA compared to controls. Proteomic analysis revealed up-modulation of the Annexin-II protein in siIRF-1 THP-1 cells. Myelodysplastic syndrome (MDS) patients with an absence of full-length IRF-1 mRNA also overexpressed Annexin-II. It is plausible that this overexpression may lead to the activation of TLR-4 contributing to the increased apoptosis characterizing MDS. Copyright © 2007 Neoplasia Press, Inc. All rights reserved

RNA Interference of Interferon Regulatory Factor-1 Gene Expression in THP-1 Cell Line Leads to Toll-Like Receptor-4 Overexpression/Activation As Well As Up-modulation of Annexin-II1

Interferon regulatory factor-1 (IRF-1) is a candidate transcription factor for the regulation of the Toll-like receptor-4 (TLR-4) gene. Using a small interfering RNA-based (siRNA) process to silence IRF-1 gene expression in the leukemic monocytic cell line THP-1, we investigated whether such a modulation would alter TLR-4 expression and activation status in these cells. The siIRF-1 cells expressed elevated levels of TLR-4 mRNA and protein compared to controls by 90% and 77%, respectively. ICAM.1 protein expression and apoptosis levels were increased by 8.35- and 4.25-fold, respectively. The siIRF-1 cells overexpressed Bax mRNA compared to controls. Proteomic analysis revealed upmodulation of the Annexin-II protein in siIRF-1 THP-1 cells. Myelodysplastic syndrome (MDS) patients with an absence of full-length IRF-1 mRNA also overexpressed Annexin-II. It is plausible that this overexpression may lead to the activation of TLR-4 contributing to the increased apoptosis characterizing MDS

Expression of functional Toll-like receptors by salivary gland epithelial cells: Increased mRNA expression in cells derived from patients with primary Sjögren's syndrome

Toll-like receptors (TLR) play an essential role in the activation of both innate and adaptive immune responses. Salivary gland epithelial cells (SGEC) may participate in the development of glandular inflammatory reactions that characterize primary Sjögren's syndrome (pSS). In this study we sought to assess the expression and function of several TLR molecules in cultured non-neoplastic SGEC obtained from pSS patients and disease controls. Long-term cultured non-neoplastic SGEC derived from pSS patients (SS-SGEC) and disease controls (control-SGEC), as well as the monocytic cell line THP-1 (positive control cell line), were examined by reverse transcription-polymerase chain reaction (RT-PCR) analysis and quantitative real-time PCR for mRNA expression of TLR1, -2, -3 and -4 molecules. TLR function was assessed by the induction of the expression (flow cytometry) of the immunoregulatory molecules CD54/intercellular adhesion molecule-1 (ICAM-1), CD40, CD86/B7·2, major histocompatibility complex (MHC) class I and MHC class II following treatment with the TLR ligands: Staphylococcus aureus peptidoglycan (TLR2), the synthetic dsRNA analogue polyinosinic:cytidylic acid (TLR3) and Escherichia coli lipopolysaccharide (TLR4). SGEC were found to express functional TLR2, -3 and -4 molecules, as attested by dose-dependent up-regulation of surface ICAM-1, CD40 and MHC-I expression (as well as of reciprocal TLR mRNA) following treatment with the respective TLR-ligands. SS-SGEC lines displayed significantly higher constitutive expression of TLR1 (P = 0·0027), TLR2 (P = 0·01) and TLR4 (P = 0·03) mRNA compared to control-SGEC. This study demonstrates that cultured SGEC express functional TLR molecules; the high constitutive TLR expression by SS-SGEC is probably suggestive of the intrinsic activation of epithelial cells in pSS and further supports the role of this type of tissue in pathogenesis of the disorder. © 2007 British Society for Immunology