Both type I and II interferons (IFNs) have been implicated in the pathogenesis of Sjogren's syndrome (SS). We aimed to explore the contribution of type I and II IFN signatures in the generation of distinct SS clinical phenotypes including lymphoma development. Peripheral blood (PB) from SS patients (n=31), SS patients complicated by lymphoma (n=13) and healthy controls (HC, n=30) were subjected to real-time PCR for 3 interferon inducible genes (IFIGs) preferentially induced by type I IFN, 2 IFIGs preferentially induced by IFNγ as well as for IFNα and IFNγ genes. The same analysis was performed in minor salivary gland tissues (MSG) derived from 31 SS patients, 10 SS-lymphoma patients and 17 sicca controls (SC). In PB and MSG tissues, overexpression of both type I and type II IFIGs was observed in SS patients versus HC and SC, respectively, with a predominance of type I IFN signature in PB and a type II IFN signature in MSG tissues. In SS-lymphoma MSG tissues, lower IFNα, but higher IFNγ and type II IFIG transcripts compared to both SS and SC were observed. In receiver operating characteristic curve analysis, IFNγ/IFNα mRNA ratio in MSG tissues showed the best discrimination for lymphoma development. Discrete expression patterns of type I and II IFN signatures might be related to distinct SS clinical phenotypes. Additionally, IFNγ/IFNα mRNA ratio in diagnostic salivary gland biopsies is proposed as a novel histopathological biomarker for the prediction of in situ lymphoma development in the setting of SS.
Objective. Exosomes are membrane vesicles of endosomal origin that are distinct from apoptotic bodies and are thought to represent an acellular mechanism for antigen transfer to classic antigen-presenting cells, as well as for direct antigen presentation with the capacity to induce immune response or tolerance. Nevertheless, it is not known whether exosomes are involved in the induction or regulation of immune responses against intracellular autoantigens that characterize autoimmune diseases. Exosomes have been shown to be secreted by several types of cells, whereas studies of non-neoplastic epithelial cells are lacking. This study was undertaken to investigate the capacity of nonneoplastic salivary gland epithelial cells (SGECs) to release exosomes, and to determine whether epithelial exosomes contain RNPs, which are major autoantigens in systemic rheumatic diseases.Methods. Exosomes were isolated by ultracentrifugation from culture supernatants of 26 non-neoplastic SGEC lines established from patients with various rheumatic disorders. They were analyzed by electron microscopy, immunoblotting, or immunoprecipitation.Results. All SGEC lines were found to release comparable and significant amounts of exosomes. Similar to other cell systems, exosome secretion was constitutive and was unrelated to activation or apoptotic processes. SGEC-derived exosomes were found to contain the autoantigenic Ro/SSA, La/SSB, and Sm RNPs, as well as epithelial-specific cytokeratins.Conclusion. SGECs constitutively secrete exosomes that contain the major autoantigens Ro/SSA, La/SSB, and Sm. This mechanism may represent a pathway whereby intracellular autoantigens are presented to the immune system with an immunogenic or tolerogenic outcome.Autoimmune diseases are characterized by the development of humoral responses against self antigens, most of which are intracellular proteins. Although thought to involve typical antigen-driven responses, the precise mechanism(s) involved in the presentation of such intracellular autoantigens to the immune system is unclear (1). Apoptosis and the resulting release of autoantigen-containing apoptotic vesicles are considered potential pathways for the generation of autoimmune responses (2).During the last decade, a novel cell-free mechanism of antigen presentation has been recognized (3) that involves small (30-100 nm) membrane vesicles, which are termed exosomes and are distinct from apoptotic bodies. These are different from the exosome protein complex, which is implicated in RNA processing and degradation processes. Exosomal vesicles are secreted following the fusion of multivesicular late endosome/lysosomes with the plasma membrane. Exosome production has been observed in a variety of cell types, including reticulocytes, platelets, cytotoxic T lymphocytes and B lymphocytes, dendritic cells, and neoplastic intestinal epithelial cells, whereas studies of non-neoplastic epithelial cells are lacking. Several physiologic roles have been assigned to exosomes, including the expulsion of obsolete membran...
Elucidating the molecular pathways active in pathologic tissues has important implications for defining disease subsets, selecting therapy, and monitoring disease activity. The development of therapeutics directed at IFN-α or IFN-γ makes the discovery of probes that report precisely on the activity of different IFN pathways a high priority. We show that, although type I and II IFNs induce the expression of a largely overlapping group of molecules, precise probes of IFN-γ activity can be defined. Used in combination, these probes show prominent IFN-γ effects in Sjögren syndrome (SS) tissues. In contrast, dermatomyositis muscle shows a dominant type I IFN pattern. Interestingly, heterogeneity of IFN signatures exists in patients with SS, with some patients demonstrating a predominant type I pattern. The biochemical patterns largely distinguish the target tissues in patients with SS from those with dermatomyositis and provide a relative weighting of the effects of distinct IFN pathways in specific biopsies. In SS, type I and II IFN effects are localized to the same epithelial cells, surrounded by inflammatory cells expressing IFN-γ–induced proteins, suggesting reinforcing interactions. Precise probes of the different IFN pathways active in tissues of complex rheumatic diseases will be critical to classify disease, elucidate pathogenesis, and select therapy.
Objective Increased type I interferon (IFN-I) and a broad signature of IFN-I-induced gene transcripts are observed in patients with SLE and other systemic autoimmune diseases. To identify disease-relevant triggers of the IFN-I pathway we investigated whether endogenous virus-like genomic repeat elements, normally silent, might be expressed in patients with systemic autoimmune disease, activate an innate immune response and induce IFN-I. Methods Expression of IFN-I and long interspersed nuclear element-1 (LINE-1; L1) was studied in kidney tissue from lupus patients and minor salivary gland (MSG) tissue from patients with primary Sjogren’s syndrome (SS) by PCR, western blot and immunohistochemistry. Induction of IFN-I by L1 was investigated by transfection of plasmacytoid dendritic cells (pDCs) or monocytes with an L1-encoding plasmid or L1 RNA. Involvement of innate immune pathways and altered L1 methylation were assessed. Results L1 mRNA transcripts were increased in lupus nephritis kidneys and in MSG from SS patients and correlated with IFN-I expression and L1 DNA demethylation. L1 open reading frame 1/p40 protein and IFNβ were expressed in MSG ductal epithelial cells and in lupus kidneys, and IFNα was detected in infiltrating pDCs. Transfection of pDCs or monocytes with L1-encoding DNA or RNA induced IFN-I. Inhibition of TLR7/8 reduced L1 induction of IFNα in pDCs and an inhibitor of IKKε/TBK1 abrogated induction of IFN-I by L1 RNA in monocytes. Conclusion L1 genomic repeat elements represent endogenous nucleic acid triggers of the IFN-I pathway in SLE and SS and may contribute to initiation or amplification of autoimmune disease.
Objective. To investigate the expression of B7 costimulatory molecules in the lymphoepithelial lesions of salivary gland (SG) biopsy tissues and in SG epithe-lial 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-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 epithe-lial 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.
SUMMARYCD40 has been identified in an expanding list of haematopoietic and non-haematopoietic cells and has received an increased interest based on its role in a variety of cell-mediated responses and its potential to participate in the pathogenesis of chronic inflammatory disorders. Sjögren's syndrome (SS) is an autoimmune exocrinopathy, which is characterized by chronic lymphocytic infiltration of exocrine glands and aberrant activation of epithelial tissues. We studied the expression of CD40 protein in cultured non-neoplastic salivary gland epithelial cell (SGEC) lines as well as in minor SG biopsies obtained from 17 SS patients and 12 controls. Immunocytochemical and flow cytometric analyses had revealed the occurrence of constitutively expressed CD40 molecules on the surface of long-term cultured SGEC lines, which could be further induced by interferon-gamma (IFN-g) and IL-1b cytokines, but not tumour necrosis factor-alpha (TNF-a), IL-4, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IFN-a. Triggering of SGEC through CD40 enhanced the surface expression of the adhesion molecule intercellular adhesion molecule-1 (ICAM-1)/CD54, but not MHC class I and class II (HLA-DR) molecules. Spontaneous CD40 expression was significantly higher in SGEC lines derived from SS patients, compared with controls (P < 0·001), which is suggestive of their intrinsically activated status. In SG biopsies, CD40 was constitutively expressed by lymphocytes, ductal epithelial cells and endothelial cells but not by other glandular cell types, such as acinar cells, myoepithelial cells and fibroblasts. In addition, CD40L staining was also detected in 30-50% of the infiltrating lymphocytes in the biopsies of SS patients. Our findings indicate the immunoregulatory potential of SGEC and lend further support to a model of intrinsic activation in salivary epithelia in SS, whereby these cells actively participate in the induction and maintenance of lymphocytic infiltrates of patients.
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