Search

Up-regulated expression of Ro52/tripartite motif-containing protein 21 ( TRIM21), Ro60/TROVE domain family, member 2 ( TROVE2) and lupus LA protein/Sjögren's syndrome antigen B ( La/ SSB) autoantigens has been described in the salivary gland epithelial cells ( SGEC) of patients with Sjögren's syndrome ( SS). SGECs, the key regulators of autoimmune SS responses, express high levels of surface functional Toll-like receptor ( TLR)-3, whereas Ro52/ TRIM21 negatively regulates TLR-3-mediated inflammation. Herein, we investigated the effect of TLR-3-signalling on the expression of Ro52/ TRIM21, as well as Ro60/ TROVE2 and La/ SSB autoantigens, by SGECs. The effect of TLR-3 or TLR-4 stimulation on autoantigen expression was evaluated by poly I: C or lipopolysaccharide ( LPS) treatment, respectively, of SGEC lines (10 from SS patients, 12 from non- SS controls) or HeLa cells, followed by analysis of mRNA and protein expression. Poly I: C, but not LPS, resulted in a two-step induction of Ro52/ TRIM21 mRNA expression by SGECs, a 12-fold increment at 6 h followed by a 2·5-fold increment at 24-48 h, whereas it induced a late two-fold up-regulation of Ro60/ TROVE2 and La/ SSB mRNAs at 48 h. Although protein expression levels were not affected significantly, the late up-regulation of Ro52/ TRIM21 mRNA was accompanied by protein redistribution, from nucleolar-like pattern to multiple coarse dots spanning throughout the nucleus. These late phenomena were mediated significantly by interferon ( IFN)-β production, as attested by cognate secretion and specific inhibition experiments and associated with IFN regulatory factor ( IRF)3 degradation. TLR-3-signalling had similar effects on SGECs obtained from SS patients and controls, whereas it did not affect the expression of these autoantigens in HeLa cells. TLR-3 signalling regulates the expression of autoantigens by SGECs, implicating innate immunity pathways in their over-expression in inflamed tissues and possibly in their exposure to the immune system. [ABSTRACT FROM AUTHOR]

Objectives: Thymic stromal lymphopoietin (TSLP) has been implicated in primary Sjögren's syndrome (pSS) and related B-cell lymphoproliferation and lymphoma (NHL) by studies on salivary pathologic tissues and serum. The purpose of this work was to validate serum TSLP as biomarker of pSS and related lymphoproliferation by the study of two additional independent cohorts., Methods: Serum TSLP was measured by ELISA in the original published Cohort-1 from Udine, Italy, including 91 patients. Two additional cohorts were then studied for validation: Cohort-2, including 4 sub-cohorts comprising 125 patients from the Universities of Roma, L'Aquila, Pisa and Perugia, belonging to the Italian SS Study Group (GRISS), and Cohort-3, including 59 patients from the University of Athens, Greece. Overall, 159 control subjects were enrolled. Active pSS-NHL, as well as pre-lymphomatous conditions, i.e. persistent salivary gland swelling and mixed cryoglobulinaemia, were investigated in detail. In addition, serum samples from pSS-NHL in complete remission were analysed (n=27)., Results: TSLP serum levels were confirmed to be significantly higher in pSS compared to controls in both Cohort-2 and Cohort-3, in particular in patients with lymphoproliferation. Serum TSLP was much higher in pSS pre-lymphomatous conditions. Finally, active NHL showed the highest TSLP serum levels, while in NHL in remission TSLP resulted undetectable or significantly lower than in benign pSS., Conclusions: By the study of independent cohorts, it was again demonstrated that serum TSLP levels are increased in pSS, above all in more advanced B-cell lymphoproliferation and NHL. Serum TSLP can therefore represent a novel biomarker for pSS-related lymphoproliferation.

The pathogenesis of primary Sjögren's syndrome (pSS) is complex, in part due to DNA methylation abnormalities. This study was undertaken to evaluate the importance of global DNA methylation ((5m)C) as determined in minor salivary glands (MSG) from well characterized pSS patients. Twenty-two pSS patients and ten controls were selected, and MSG were stained with anti-(5m)C, anti-(5m)C/anti-cytokeratin (KRT)19, or with anti-SSB/La antibodies (Ab). The DNA methylation status at the SSB gene promoter P1 and P1' was evaluated by methylation-sensitive restriction enzymes (MSRE) coupled with PCR. The effect of the DNA demethylating drug 5 azacytidine (5-Aza) was tested in the human salivary gland (HSG) cell line. In pSS, the reduction of global DNA methylation ((5m)C) was associated with lymphocyte infiltration, the emergence of (5m)C(low) and KRT19(high) acini, and the detection of circulating anti-SSB/La Ab, but not with disease activity (ESSDAI). Next, treating HSG cells with 5-Aza was effective in inducing SSB expression. Finally in pSS patients positive for anti-SSB/La Ab, we further observed DNA demethylation at the SSB gene promoter P1 with consequent SSB overexpression at both the transcriptional and protein levels in salivary gland epithelial cells. In conclusion, our results highlight the importance of DNA methylation in the pathophysiology of pSS and to the emergence of anti-SSB/La Ab., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Sjögren's syndrome is an autoimmune disease that targets exocrine glands, but often exhibits systemic manifestations. Infiltration of the salivary and lacrimal glands by lymphoid and myeloid cells orchestrates a perpetuating immune response leading to exocrine gland damage and dysfunction. Th1 and Th17 lymphocyte populations and their products recruit additional lymphocytes, including B cells, but also large numbers of macrophages, which accumulate with disease progression. In addition to cytokines, chemokines, chitinases, and lipid mediators, macrophages contribute to a proteolytic milieu, underlying tissue destruction, inappropriate repair, and compromised glandular functions. Among the proteases enhanced in this local environment are matrix metalloproteases (MMP) and plasmin, generated by plasminogen activation, dependent upon plasminogen activators, such as tissue plasminogen activator (tPA). Not previously associated with salivary gland pathology, our evidence implicates enhanced tPA in the context of inflamed salivary glands revolving around lymphocyte-mediated activation of macrophages. Tracking down the mechanism of macrophage plasmin activation, the cytokines IFNγ and to a lesser extent, IFNα, via Janus kinase (JAK) and signal transducer and activator of transcription (STAT) activation, were found to be pivotal for driving the plasmin cascade of proteolytic events culminating in perpetuation of the inflammation and tissue damage, and suggesting intervention strategies to blunt irreversible tissue destruction., (Published by Elsevier Ltd.)

Several lines of evidence indicate that salivary gland epithelial cells (SGEC) play an important role in the pathogenesis of primary Sjogren's syndrome (SS). Normal SGEC have been shown to possess functional estrogen receptors, however, the estrogenic response of SGEC in patients with SS has not been previously assessed. To address this issue, we comparatively tested cultured non-neoplastic SGEC lines from SS patients (SS-SGEC, n = 8) and from disease controls (control-SGEC, n = 12) in a standard estrogenic inhibition assay of cytokine-induced adhesion molecule expression, where the modulation of the expression of constitutive and interferon-gamma (IFNγ)-induced CD54/ICAM.1 molecules following treatment with 17β-estradiol (E2) was evaluated by flow cytometry. Similarly high ICAM.1 expression was induced by IFNγ in control-SGEC and SS-SGEC lines. E2-treatment did not modify the constitutive ICAM.1 expression in either control-SGEC or SS-SGEC lines. In line with previous results, E2-pretreatment of control-SGEC was found to impede significantly the IFNγ-induced upregulation of ICAM.1 (p = 0.003). However, such inhibition was not observed in the SS-SGEC lines (p = 0.55). Such aberrant response of SS-SGEC to estrogens did not appear to associate with altered expression of estrogen receptor (ER) proteins, as no discernible differences could be revealed by immunoblotting and immunohistochemistry in the patterns or the intensity of ERα and ERβ (ERβ1- and ERβ2-isoforms) protein expression in SGEC lines or minor salivary gland tissues between SS patients and disease controls. The deficient estrogenic responsiveness of SS-SGEC likely represents a manifestation of the intrinsic epithelial activation that characterizes SS and possibly indicates the perturbation of the immunoregulatory potential of estrogens in SS-epithelia., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: Sjögren's syndrome (SS) is a chronic autoimmune disease of unknown etiology that targets salivary and lacrimal glands and may be accompanied by multiorgan systemic manifestations. To further the understanding of immunopathology associated with SS and identify potential therapeutic targets, we undertook the present study comparing the gene expression profiles of salivary glands with severe inflammation versus those of salivary glands with mild or no disease., Methods: Using microarray profiling of salivary gland tissue from patients with SS and control subjects, we identified target genes, which were further characterized in tissue, serum, and cultured cell populations by real-time polymerase chain reaction and protein analysis., Results: Among the most highly expressed SS genes were those associated with myeloid cells, including members of the mammalian chitinase family, which had not previously been shown to be associated with exocrinopathies. Both chitinase 3-like protein 1 and chitinase 1, highly conserved chitinase-like glycoproteins (one with enzymatic activity and one lacking enzymatic activity), were evident at the transcriptome level and were detected within inflamed tissue. Chitinases were expressed during monocyte-to-macrophage differentiation and their levels augmented by stimulation with cytokines, including interferon-α (IFNα)., Conclusion: Because elevated expression of these and other macrophage-derived molecules corresponded with more severe SS, the present observations suggest that macrophages have potential immunopathologic involvement in SS and that the tissue macrophage transcription profile reflects multiple genes induced by IFNα., (Copyright © 2011 by the American College of Rheumatology.)

Salivary gland epithelial cells (SGECs) have been shown to participate in immunological responses and have been implicated in the pathogenesis of Sjögren's syndrome (SS). Experimental evidence from animal models indicates that estrogen deficiency may also participate in SS pathogenesis. However, the expression and functionality of the estrogen receptors alpha (ERalpha) and beta (ERbeta) in normal human salivary epithelium is unknown. To investigate these points, formalin-fixed, paraffin-embedded specimens and cultured non-neoplastic SGEC lines derived from nine minor salivary gland (MSG) biopsies with normal histology were studied. Immunohistochemical analyses detected the epithelial expression of ERalpha, ERbeta1, and ERbeta2 protein isoforms both in MSG tissues and in cultured SGECs. Such epithelial expression was verified by immunoblotting of various ER proteins in cellular extracts of cultured SGECs (full-length-ERalpha, ERalpha-Delta3, ERbeta1-long, ERbeta1-short, and ERbeta2-long isoforms). Estrogens did not induce growth or apoptosis in cultured SGECs. However, similarly to other cellular systems, treatment of cultured SGECs with estrogens (17beta-estradiol and the ERalpha- and ERbeta-selective agonists propylpyrazole-triol and diarylpropiolnitrile, respectively) inhibited the interferon-gamma-inducible expression of intercellular adhesion molecule-1. This finding corroborated the functionality of ER expressed by SGEC. Our results suggest that salivary epithelium expresses constitutively functional ERalpha and ERbeta proteins that apparently mediate immunomodulatory effects.

Sjogren's syndrome (SS) is an exocrinopathy characterized by T cell infiltrates, salivary gland epithelial cell (SGEC) apoptosis and high Fas and FasL expression. To address the participation of T cell-derived cytokines and of Fas apoptotic pathway in SS glandular lesions, we utilized non-neoplastic SGEC lines established from SS patients and controls. Possibly attesting to their intrinsic activation, cell lines derived from SS patients displayed significantly higher constitutive Fas and FasL than controls. Surface co-expression of Fas and FasL was not associated with spontaneous fratricide apoptosis. SGEC were resistant to anti-Fas-mediated apoptosis (possibly owing to the constitutive expression of anti-apoptotic proteins cFLIP and Bcl-2), but became sensitive after protein or RNA synthesis inhibition. IFN-gamma and TNF-alpha were able to upregulate surface Fas and FasL, whereas IL-1beta downregulated surface FasL. IFN-gamma (but not several other cytokines) reduced the survival of SGEC in a dose- and time-dependent manner and induced Fas/FasL-mediated apoptosis, directly and via anoikia. Dexamethasone inhibited the upregulation of Fas and FasL by IFN-gamma and the induction of SGEC apoptosis and detachment by anti-Fas mAb or IFN-gamma. Our findings indicate the injurious role of IFN-gamma for the salivary epithelia of SS patients through the induction of Fas-mediated apoptosis and anoikia., (Copyright 2001 Academic Press.)

B7 molecules expressed on classic APC play a critical role in the regulation of immune responses by providing activation or inhibitory signals to T cells, through the ligation with CD28 or CTLA4 receptors, respectively. We have recently described the expression of B7 molecules by the salivary gland epithelial cells (SGEC) of patients with Sjögren's syndrome (also termed autoimmune epithelitis). The role of such expression needs to be clarified. Thus, in the present study, we sought to address the existence and function of B7.2 proteins on cultured nonneoplastic SGEC lines derived from Sjögren's syndrome patients. The occurrence of B7.2 proteins on SGEC was verified by flow cytometry, immunocytochemistry, immunoprecipitation, and immunoblotting. The assessment of several cell lines in costimulation assays had revealed that the constitutive expression of B7.2 molecules is sufficient to provide costimulatory signals to anti-CD3-stimulated T cells. SGEC-derived costimulation induced IL-2-dependent proliferation of CD4(+) T cells, which was associated with low production of IL-2, but probably also with the secretion of yet undefined autocrine T cell growth factor(s). B7.2 proteins expressed by SGEC were found to display distinctive binding properties denoted by the functional interaction with CD28 receptor and reduced binding to CTLA4. Finally, the detection of a functional soluble form of B7.2 protein in cell-free culture supernatants of both SGEC and EBV-transformed B cell lines is demonstrated. These findings imply a critical role for epithelial cells in the regulation of local immune responses in the salivary glands.

Objective: To investigate the expression of B7 costimulatory molecules in the lymphoepithelial lesions of salivary gland (SG) biopsy tissues and in SG epithelial cell lines derived from patients with Sjögren's syndrome (SS)., Methods: B7.1 and B7.2 protein expression was studied by immunohistochemistry in minor SGs obtained from 11 patients with SS and 10 disease control patients with nonspecific sialadenitis and in cultured SG epithelial cell lines obtained from minor SGs from 15 SS patients and 15 control patients. B7.1 and B7.2 messenger RNA (mRNA) expression by SG epithelial cell lines was examined by reverse transcription-polymerase chain reaction (RT-PCR)., Results: In biopsy tissues from SS patients, but not control patients, ductal and acinar epithelial cells showed increased expression of both B7.1 and B7.2. Intense spontaneous B7.1 protein expression (as well as HLA-ABC, but not B7.2 or HLA-DR) was also found in 73% of SG epithelial cell lines from SS patients versus 13% of those from control patients (P < 0.01). Interferon-y treatment induced, or up-regulated, B7.1, B7.2, and HLA-DR expression in all SG epithelial cell lines tested. B7.1 and B7.2 expression by SG epithelial cell lines was also verified at the mRNA level by RT-PCR., Conclusion: Human SG epithelia are intrinsically capable of expressing B7 proteins upon activation. In SS patients, the expression of B7 molecules by SG epithelial tissues and by SG epithelial cell lines indicates the activated status of SG epithelial cells in this disorder and, possibly, their capacity for presenting antigens to T cells.