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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. [ABSTRACT FROM AUTHOR]

SUMMARY CD40 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-γ) and IL-1β cytokines, but not tumour necrosis factor-alpha (TNF-α), IL-4, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IFN-α. 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... [ABSTRACT FROM AUTHOR]

ICAM.1 (CD54) is a surface protein expressed on epithelial and other nonhematopoietic cells upon activation and is known to play an important role in the stimulation of T cells by the provision of cellular adhesion and costimulatory support. Sjogren's syndrome (SS) is an autoimmune exocrinopathy, which is characterized by chronic lymphocytic infiltration of exocrine glands and aberrant activation of epithelial tissues. To address the contribution of ICAM.1 in the pathogenesis of SS, the expression of this protein was studied by immunohistochemistry and flow cytometry in minor salivary gland (SG) biopsies as well as in cultured SG epithelial cell (SGEC) lines obtained from 18 SS patients and 16 controls. In biopsies from SS patients (but not controls), strong ICAM.1 was expressed by infiltrating mononuclear cells (52%) and by a significant proportion of periacinar myoepithelial cells (18%). In addition, a patchy pattern of moderate ICAM.1 expression was detected in 31% of ductal epithelia of SS patients. These ICAM.1-expressing epithelial and myoepithelial cells were observed throughout glandular tissues and were not confined in areas proximal to lymphoid infiltrates. In support to an intrinsic activation profile of SGEC in SS, long-term cultured non-neoplastic SGEC lines derived from SS patients displayed significantly upregulated spontaneous expression of ICAM.1, compared to controls (P < 0.05). The high expression of ICAM.1 protein by the salivary epithelium of SS patients is likely suggestive of its important role in the pathogenesis of the disorder. Further, our results support a model of intrinsic activation of salivary epithelial and myoepithelial cells in SS, whereby these cells actively participate in the induction and maintenance of lymphocytic infiltrates of patients. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the immunogenicity of four synthetic peptides, representing linear B cell epitopes of the human La/SSB autoantigen: 145–164 aa (p1), 289–308 aa (p2), 301–318 aa (p3) and 349–364 aa (p4), in rabbits. New Zealand White rabbits were immunized with each of the above peptides attached in four copies on tetrameric sequential oligopeptide carriers (SOC) in duplicate. Control immunizations were also performed (one rabbit each, immunized with Freud's complete adjuvant alone or with the SOC carrier alone). Animals were bled at regular intervals and sera were analysed for anti-La/SSB activity by ELISA assays using as antigen the various synthetic peptides, as well as the whole La/SSB protein. Four months after the last immunization, the animals were killed and peripheral blood mononuclear and spleen cells were co-cultured with either the peptides, the SOC carrier, or 27 peptides, covering the entire length of the human La/SSB molecule (23 amino acids long, overlapping by eight residues to each other). A specific, IgG, anti-peptide antibody response was detected, initially directed against the priming peptide, and subsequently expanded to the other La/SSB synthetic peptides. The antibody titres remained high, even 4 months after the last immunization. Sera from rabbits immunized with either p2 or p3 reacted also with the whole La/SSB protein, as was demonstrated by ELISA and immunoblot assays. No reactivities against either Ro60 or Ro52 autoantigen were found. Rabbit spleen cell reacted not only with the epitope used for the immunization but also with other La/SSB peptides. Immunization of rabbits with the major human La/SSB B cell antigenic determinants, linked to SOC carrier, induces strong and sustained antibody and T cell responses against multiple epitopes of the human La/SSB protein. Thus, La/SSB B cell linear epitopes are probably capable also of functioning as T cell epitopes, in this experimental... [ABSTRACT FROM AUTHOR]

Counterimmunoelectrophoresis (CIE), RNA precipitation, ELISA and immunoblotting against cytoplasmic HeLa cell extract (IB-HeLa) and erythrocyte extract (IB-RBC) were applied to detect anti-Ro(SSA) antibodies in 93 sera selected from patients with various autoimmune diseases [47 were anti-Ro(SSA) positive by CIE]. The RNA precipitation assay, which demonstrated the highest sensitivity was selected as the reference method. CIE was found to be reliable with a specificity of 100% and a sensitivity of 89%. ELISA showed a comparable specificity (95%) but somewhat lower sensitivity (72%). Antibodies to 52 or 60 kDa Ro(SSA) proteins by IB-HeLa demonstrated a high specificity (95 and 97% respectively) but a low overall sensitivity (36 and 17% respectively). Anti-Ro(SSA) antibodies to 52, 54 and 60 kDa erythrocyte proteins by IB-RBC, had a variable overall specificity (95, 97 and 57%) and sensitivity (51, 13 and 34%). The anti-52 kDa antibodies detected by IB-HeLa correlated to those found by IB-RBC (<0.001) and occurred predominantly in primary Sjögren's syndrome (<0.001, sensitivity: 71 and 77%) as well as in sera with anti-Ro(SSA) and anti-La(SSB) antibodies (<0.001). These findings confirm that RNA precipitation assay has the highest sensitivity and specificity for anti-Ro(SSA) antibody detection. However, until a more sensitive ELISA is available, CIE because of its reliability appears to be the method of choice. Finally IB-RBC was found to be more sensitive than IB-HeLa for the detection of anti-Ro52 kDa antibodies. [ABSTRACT FROM PUBLISHER]