Postgenomic Bioinformatic Analysis of Nucleic Acid Sequences Expressed in the Salivary Gland Epithelial Cells of Primary Sjögren's Syndrome Patients in Search of Microorganisms and Endogenous Retroviruses.

Several previous studies from our laboratory have indicated that the salivary gland epithelia of primary Sjögren's syndrome (SS) patients are not only the target of autoimmune immune responses, but also key instigators of the chronic salivary gland inflammatory infiltrates of patients. In particular, the comparative analysis of salivary gland tissue specimens and of in-vitro cultured non-neoplastic salivary gland epithelial cell lines (SGEC, of ductal type) from SS-patients and non-SS disease-controls, have unequivocally highlighted the presence of intrinsic activation in the ductal epithelia of SS-patients and of aberrant expression of inflammagenic molecules thereof, that correlate with the severity of local histopathologic changes, as well as of systemic manifestations of the disease. In the same context, we have recently shown that the ductal epithelia of SS-patients manifest cell-autonomous activation of the AIM2 inflammasome owing to the presence of aberrant cytoplasmic accumulations of damaged DNA. These findings not only provide a mechanistic explanation for the intrinsic activation and inflammatory status of SS ductal epithelia, but may also point towards the putative instigating role of an exogenous or endogenous agent (i.e., a micro-organism or an endogenous retrovirus, respectively). On this basis and to further explore the nature of epithelial cell-intrinsic activation in SS, the present proposal aims to investigate the expression of endogenous retroviral and/or non-human nucleic acid sequences of microbial origin in the ductal salivary gland epithelia of SS-patients, using metagenomic analysis of high throughput DNA and RNA genome sequencing data, which will be obtained from SGEC lines derived from SS-patients and disease-controls.

Idiopathic retroperitoneal fibrosis: clinical features, treatment modalities, relapse rate in Greek patients and a review of the literature.

OBJECTIVES: Retroperitoneal fibrosis (RPF) is mostly idiopathic (iRPF); however, it can be secondary to drugs, malignancies, infections, or, as recently recognised, can be part of the IgG4-related diseases. The aim of our study was i) to describe the presenting clinical/laboratory/imaging features and treatment modalities used in patients with iRPF and ii) to evaluate factors potentially associated with disease relapse. METHODS: The medical records of patients diagnosed with iRPF and followed in four tertiary medical units in Athens, Greece from 2000 to 2018 were retrospectively evaluated. RESULTS: Sixty-seven patients with iRPF were included in the study. Seventy-three per cent were males, with a mean age at diagnosis 56.0±9.2 years. Low-back pain (63%) and constitutional symptoms (57%) were the commonest presenting symptoms. Elevated acute-phase reactants (78%), anaemia (43%) and impaired renal function (41%) were the most common laboratory findings. Serum IgG4 at diagnosis was evaluated in 36/67 patients and 36% of them had elevated levels (mean 297.7±166.3mg/dL). Diagnosis was mainly based on abdominal CT and/or MRI. Clinical/laboratory/radiological presentation did not differ between patients with elevated and normal serum IgG4 levels. Steroids were used as first-line treatment in 98%. Relapse occurred in 28.6% after a mean of 43.1±31.8 months. Relapse did not associate to initial clinical/imaging findings or to any treatment used, however patients with increased serum IgG4 had a significantly higher relapse rate (75% vs. 25%, p=0.005). CONCLUSIONS: Relapse occurred in one-fifth of patients independently of the initial clinical/radiographic presentation or treatment used. iRPF patients with baseline elevated serum IgG4 levels have a higher relapse rate.

Cell-autonomous epithelial activation of AIM2 (absent in melanoma-2) inflammasome by cytoplasmic DNA accumulations in primary Sjögren's syndrome.

Primary Sjögren's syndrome (SS) is characterized by chronic periductal inflammatory infiltrates in the salivary glands. Several previous studies have indicated that the ductal epithelia of SS patients play a pro-inflammatory role and manifest an intrinsically activated status, as demonstrated in cultured non-neoplastic ductal salivary gland epithelial cell (SGEC) lines. Herein, we investigated the activation of inflammasomes in the salivary epithelia of SS patients and non-SS controls, using salivary biopsy tissues and SGEC lines. The ductal epithelial cells of SS patients were found to display significant activation of the AIM2 (absent in melanoma-2) inflammasome. Such activation occurred in a cell-autonomous manner, as it was illustrated by the constitutively high expression of AIM2 activation-related genes, the presence of cytoplasmic ASC specks and the increased spontaneous IL-1ß production observed in patients' SGEC lines. Since AIM2 activation is known to occur in response to cytoplasmic DNA, we further searched for the presence of undegraded extranuclear DNA in the SGEC lines and SG tissues of patients and controls. This investigation revealed marked cytoplasmic accumulations of damaged genomic DNA that co-localized with AIM2 in the specimens of SS patients (but not controls). The SGEC lines and the ductal tissues of SS patients were also found to manifest impaired DNase1 expression and activity, which possibly denotes defective cytoplasmic DNA degradation in patients' cells and AIM2 triggering thereof. In corroboration, DNase1-silencing in normal SGEC was shown to lead to high AIM2-related gene expression and IL-1ß production. Our findings indicate that the cell-intrinsic activation status of ductal epithelia in SS patients owes to persistent epithelial AIM2 activation by aberrant cytoplasmic DNA build-up.

Perturbation of transcriptome in non-neoplastic salivary gland epithelial cell lines derived from patients with primary Sjögren's syndrome.

The data presented here are related to the research article titled "Impaired anti-inflammatory activity of PPARγ in the salivary epithelia of Sjögren's syndrome patients imposed by intrinsic NF-κB activation" (Vakrakou et al., Journal of Autoimmunity, in press, 2017). In the cited manuscript, using comparative analyses of salivary gland biopsy specimens and ductal salivary gland epithelial cell (SGEC) lines from SS patients and disease controls, we have demonstrated that the ductal epithelia of SS patients display constitutively reduced PPARγ expression, transcriptional activity and anti-inflammatory function that were associated with cell-autonomously activated NF-κB and IL-1ß pathways in these cells. Herein, the comparative transcriptome analysis of SGEC lines is presented. We show that the ductal epithelia of SS patients with severe lymphoepithelial lesions manifest constitutive perturbation in various inflammation- and metabolism related signaling pathways.

Systemic activation of NLRP3 inflammasome in patients with severe primary Sjögren's syndrome fueled by inflammagenic DNA accumulations.

Sjögren's syndrome (SS) patients manifest high cell-free DNA (cf-DNA) levels in serum, associated with impaired DNaseI activity. Undegraded DNA may accumulate in tissues and act as an inflammasome-activating signal. Herein, we investigated the occurrence of aberrant DNA build-up in various biologic compartments of SS patients and its correlation with the activity of NLRP3 and AIM2 inflammasomes. For this purpose, we evaluated sera, PBMC, circulating monocytes and salivary glands (SG) from different SS patient subgroups and controls. We found that SS patients at high risk for lymphoma and those with established lymphoma display high serum cf-DNA levels, substantial extranuclear DNA accumulations in PBMC and SG tissues, a unique NLRP3 inflammasome gene signature in PBMC, and significantly increased serum IL-18 and ASC levels. In these patients, the circulating monocytes manifested NLRP3 inflammasome activation and increased response to NLRP3 stimuli, whereas SG-infiltrating macrophages exhibited signs of NLRP3 activation and pyroptosis. Cell-free nucleic acids isolated from patients' sera competently primed the activation of both NLRP3 and AIM2 inflammasomes in healthy monocytes. SS patients also manifested diminished DNaseI activity in serum and DNaseII expression in PBMC, which inversely correlated with indices of inflammasome activation. DNaseII gene-silencing in healthy monocytes led to cytoplasmic DNA deposition and activation of inflammasome-related genes and of caspase1. Our data reveal the occurrence of systemic NLRP3 inflammasome activation in severe SS, which is associated with widespread extranuclear accumulations of inflammagenic DNA and impaired DNA degradation. These findings can provide novel biomarkers and new therapeutic targets for the management of SS patients with adverse outcomes.

Impaired anti-inflammatory activity of PPARγ in the salivary epithelia of Sjögren's syndrome patients imposed by intrinsic NF-κB activation.

Sjögren's syndrome (SS) patients manifest inflammation in the salivary glands (SG) and evidence of persistent intrinsic activation of ductal SG epithelial cells (SGEC), demonstrable in non-neoplastic SGEC lines derived from patients (SS-SGEC). The peroxisome-proliferator-activated receptor-γ (PPARγ) mediates important anti-inflammatory activities in epithelial cells. Herein, the comparative analysis of SG biopsies and SGEC lines obtained from SS patients and controls had revealed constitutively reduced PPARγ expression, transcriptional activity and anti-inflammatory function in the ductal epithelia of SS patients that were associated with cell-autonomously activated NF-κB and IL-1ß pathways. Transcriptome profiling analysis revealed several differentially expressed proinflammatory and metabolism-related gene sets in SS-SGEC lines. These aberrations largely correlated with the severity of histopathologic lesions, the disease activity and the occurrence of adverse manifestations in SS patients studied, a fact which corroborates the key role of the persistently-activated epithelia in the pathogenesis of both local and systemic features of this disease. The treatment of control SGEC lines with PPARγ agonists was found to diminish the NF-κB activation and apoptosis induced by proinflammatory agents. In addition, the in-vitro application of PPARγ agonists and pharmacologic inhibitors of IL-1ß and NF-κB had significant beneficial effects on SS-SGEC lines, such as the restoration of PPARγ functions and the reduction of their intrinsic activation, a fact which may advocate the future clinical study of the above agents as therapeutic modalities for SS.

Decreased serum DNase1-activity in patients with autoimmune liver diseases.

Deoxyribonuclease1 (DNase1) is involved in chromatin degradation of apoptotic cells. Its deficiency results in accumulation of self-DNA, which in turn may induce inflammation and autoimmunity. We assessed for the first time serum DNase1-activity in a large consecutive cohort of treatment-naïve patients with autoimmune liver diseases (ALD). DNase1-activity was determined by single radial enzyme-diffusion (SRED) at diagnosis of 224 patients with autoimmune hepatitis (AIH), 249 with primary biliary cirrhosis (PBC) and 36 with primary sclerosing cholangitis (PSC). Sera from 146 patients with chronic hepatitis B or C, 140 with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) and 114 healthy individuals served as disease and healthy controls. Available serum samples during remission from 50 AIH and 39 PBC patients were also investigated by paired analyzes. DNase1-activity was significantly lower in AIH, PBC and PSC compared to viral hepatitis (p < 0.02, p < 0.001, p = 0.03), NAFLD/NASH (p < 0.001) and healthy (p < 0.001). No significant difference was found in between each specific ALD. In AIH, DNAse1-activity was positively correlated with aspartate aminotransferase (AST) (p < 0.02), bilirubin (p < 0.01) and increased IgG (>1400 mg/dl; p < 0.05); in PBC, with AST (p < 0.01), alanine aminotransferase (ALT) (p < 0.03) and anti-mitochondrial antibodies (AMA) (p = 0.008). In PSC, DNase1-activity was inversely associated with alkaline phosphatase (ALP) (p < 0.05). In AIH, complete responders were characterized by increased baseline DNase1-activity compared to partial responders, relapsers and non-responders (p < 0.02), whereas it was significantly increased after achievement of remission (p < 0.001). Serum DNase1-activity is significantly decreased in ALD patients, indicating its potential implication in their pathogenesis. Furthermore, DNase1-activity could be used as a new surrogate biomarker for predicting response to AIH treatment.

Biologic treatment for rheumatic disease: real-world big data analysis from the Greek country-wide prescription database.

OBJECTIVES: To directly assess the prevalence of inflammatory rheumatic disease under treatment with biologic disease modifying anti-rheumatic drugs (b-DMARDs) and compare treatment patterns between rheumatoid arthritis (RA) and spondyloarthropathy (SpA), including psoriatic arthritis. METHODS: The obligatory country-wide prescription electronic database covering 10.223.000 Greek citizens (95.1% of the population, 99.5% Caucasian), all of whom with fully reinbursed access to b-DMARDs, was used to retrospectively capture all patients under b-DMARDs for RA/SpA between June 2014-May 2015. Age, gender and medications for RA/SpA and co-morbid classical cardiovascular risk factors (hypertension, dyslipidaemia, diabetes) were retrieved and analysed. RESULTS: A total of 9.824 RA (61.2±14.0 years, 79% women) and 9.279 SpA patients (51.4±13.1 years, 41% women) using pharmacy-dispensed prescriptions for b-DMARDs were identified (overall prevalence 0.19%). Tumour necrosis factor inhibitors were used in 73% and 99% of RA and SpA patients, respectively. B-DMARD monotherapy (RA: 18.71%, SpA: 52.11%), b-DMARD switching during 12 months (RA: 7.73%, SpA: 6.26%), and use of methotrexate (RA: 50.25%, SpA: 27.35%) and corticosteroids (RA: 55.8%, SpA: 23.63%) differed between the two patient subgroups. In both subgroups, women received more often than men methotrexate, leflunomide, hydroxychloroquine and corticosteroids, and less often b-DMARD monotherapy. After adjustments for age, gender and concomitant drugs, the probability for anti-hypertensive and lipid-lowering drug prescription was higher in SpA than RA [OR=1.41 (95%CI: 1.29-1.54) and 1.24 (1.14-1.36), respectively, p<0.001], whereas for anti-diabetics it was similar. CONCLUSIONS: In the first country-wide study that examines the characteristics of rheumatic disease patients under b-DMARD we show that their exact prevalence is 0.19%, with RA patients being older by 10 years, only slightly more numerous, and less likely to receive treatment for hypertension and dyslipidaemia than their demographically matched SpA counterparts. Longitudinal studies should assess the implications of these novel findings on the differential financial burden of rheumatic diseases, as well as on cardiovascular morbidity and mortality of these patients.

Treat-to-target biologic therapy in patients with rheumatoid arthritis is more efficacious and safe compared to delayed initiation of biologics: a real-world study.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic, devastating disease. Treat-to-target strategy (T2T) more than the usual care, reduces disease activity by using aggressively all therapeutic options. The aim of the study was to evaluate our hypothesis that T2T strategy using biologic disease-modifying anti-rheumatic drugs (bDMARDs), when needed, is also safer than the usual care characterised by delayed initiation of bDMARDs. METHODS: Disease activity was regularly measured by DAS-28 until the end of treatment with the first bDMARD. All adverse events (AEs) and their severity were recorded. Cox proportional-hazards models were performed examining the association of treatment groups, with the risk of first AE. RESULTS: There were 113 patients in T2T and 250 patients in usual care group. The likelihood (adjusted hazard ratio, HR) of achieving remission or LDA was 71% higher in the T2T group than in the usual care group, as it has been already shown by others. The novel finding of our work was that AEs, including cancers, were less frequent in the T2T group with the corresponding HRs being less than 0.50 for serious AEs, infections and serious infections (significant or marginally non-significant results). There were 15 new cancer cases in usual care and 1 in T2T group (IR 1.99 vs. 0.4, p=0.027). CONCLUSIONS: Treat-to-target treatment with bDMARDs offers a safer, rapid and better long-term outcome to patients with RA.

Impaired clearance of early apoptotic cells mediated by inhibitory IgG antibodies in patients with primary Sjögren's syndrome.

OBJECTIVES: Deficient efferocytosis (i.e. phagocytic clearance of apoptotic cells) has been frequently reported in systemic lupus erythematosus (SLE). Todate, patients with primary Sjögren's syndrome (SS) have not been assessed for phagocytosis of apoptotic cells (ApoCell-phagocytosis) and of particulate targets (microbeads, MB-phagocytosis). DESIGN: ApoCell-phagocytosis and MB-phagocytosis were comparatively assessed by flow cytometry in peripheral blood specimens and monocyte-derived macrophage (MDM) preparations from healthy blood donors (HBD) and consecutive SS, SLE and rheumatoid arthritis (RA) patients. Cross-admixture ApoCell-phagocytosis experiments were also performed using phagocytes from HBD or patients, and apoptotic cells pretreated with whole sera or purified serum IgG derived from patients or HBD. RESULTS: Compared to HBD, approximately half of SS and SLE patients studied (but not RA) manifested significantly reduced ApoCell-phagocytosis (p<0.001) and MB-phagocytosis (p<0.003) by blood-borne phagocytes that correlated inversely with disease activity (p≤0.004). In cross-admixture assays, healthy monocytes showed significantly reduced ApoCell-phagocytosis when fed with apoptotic cells that were pretreated with sera or purified serum IgG preparations from SS and SLE patients (p<0.0001, compared to those from HBD or RA). Such aberrant effect of the SS and SLE sera and IgG preparations correlated linearly with their content of IgG antibodies against apoptotic cells (p≤0.0001). Phagocytic dysfunction maybe also present in certain SS and SLE patients, as supported by deficient capacity of MDM for ApoCell-phagocytosis and MB-phagocytosis under patients' serum-free conditions. CONCLUSION: Similarly to SLE, efferocytosis is frequently impaired in SS and is primarily due to the presence of inhibitory IgG anti-ApoCell antibodies and secondarily to phagocytes' dysfunction.

Phenotypical analysis of lymphocytes with suppressive and regulatory properties (Tregs) and NK cells in the papillary carcinoma of thyroid.

CONTEXT: The immune system seems to play a key role in preventing metastasis and recurrence of thyroid cancer. T regulatory lymphocytes (Tregs) and natural killer (NK) cells play an important role in the dysfunction of the host immune system in cancer patients. OBJECTIVE: We investigated thyroid gland infiltration by Tregs and NK cells in patients with papillary thyroid cancer (PTC) and thyroid nodular goiter (TNG). The correlation between the extent of the disease and the lymphocytic infiltration of Tregs and NK cells was examined. DESIGN, SETTING, AND PARTICIPANTS: A total of 65 patients with PTC, 25 with TNG, and 50 healthy controls were studied. Blood and tissue samples from 28 patients with PTC and 13 with TNG and blood samples from the healthy controls were analyzed for T4 (CD3(+)CD4(+)), T8 (CD3(+)CD8(+)), NK (CD3(-)CD16(+)CD56(+)), and CD4(+)CD25(+)CD127(-/low) Tregs by flow cytometry (FC). Tissue samples were also analyzed for Foxp3(+) Tregs by immunohistochemistry. RESULTS: Tregs showed greater infiltration in thyroid tissue of PTC patients compared with patients with TNG (P < 0.0009 for FC and P < 0.0001 for immunohistochemistry); FC analysis of blood samples showed no difference between the groups. Flow cytometry analysis showed significantly increased NK cells in PTC tissue compared with TNG tissue (P = 0.037), whereas blood samples showed no difference. CD4(+) and CD8(+) T cells did not differ in blood and tissue samples. Increased Tregs tissue infiltration was positively correlated with advanced disease stage (P < 0.0026), whereas NK infiltration was negatively correlated (P < 0.0041). CONCLUSION: Tregs and NK cells may be important regulators of thyroid cancer progression.

Cellular microRNAs (miRNAs) and Sjögren's syndrome: candidate regulators of autoimmune response and autoantigen expression.

MicroRNAs (miRNAs) are small non-coding RNA molecules that suppress gene expression at post-transcriptional level. miRNAs are considered as fine-tuning regulators of diverse biological processes, including the development and function of the immune system. Emerging data have implicated the deregulated expression of certain miRNAs or miRNA networks in the pathogenesis of autoimmune diseases. Sjögren's syndrome (SS) is a common chronic autoimmune disease, characterized by destruction and dysfunction of the exocrine glands (predominantly of the salivary and lachrymal glands). Humoral autoimmune responses observed in the disease, primarily target Ro/SSA and La/SSB ribonucleoproteins, whilst aberrantly increased expression of these autoantigens has been described in the salivary glands (SG) and the salivary gland epithelial cells (SGEC) of SS patients. Comparative array analysis of miRNA expression in the SGs of SS and control subjects had revealed distinctive miRNA signatures in SS patients, associated with glandular inflammation and dysfunction. Furthermore, the expression analysis of miRNAs that are predicted to target Ro/SSA and La/SSB autoantigens revealed differential expression of certain miRNAs in the SG tissues, SGECs and peripheral blood mononuclear cells (PBMC) of SS patients and controls. Although these association data implicate miRNAs in SS pathogenesis, thorough functional studies are needed to delineate their role in disease.

The role of intrinsic epithelial activation in the pathogenesis of Sjögren's syndrome.

Sjögren's syndrome (SS) is a chronic autoimmune disorder that is characterized by dysfunction and destruction of the exocrine glands. Exocrinopathy is associated with periductal mononuclear cell infiltrates in the affected exocrine glands and B-cell hyperreactivity. Epithelial cells are thought to play an important pathogenetic role, as suggested by the occurrence of infiltrating lesions in various epithelial tissues (described as autoimmune epithelitis) as well as the increased epithelial expression of several inflammatory proteins in the histopathologic lesions of patients. The application of long-term cultured non-neoplastic salivary gland epithelial cell (SGEC) lines has permitted the more explicit investigation of the role of these cells in the pathophysiology of SS. These studies have revealed the inherent capacity of SGEC to induce and promote chronic inflammatory reactions, as corroborated by the constitutive or inducible expression of various molecules implicated in innate and acquired immune responses. Furthermore, significantly increased constitutive expression of several molecules has been observed in SGEC lines derived from SS patients, as compared to those obtained from disease control patients. This fact strongly indicates the operation of intrinsic activation mechanisms in the epithelia of SS patients and further supports the active participation of these cells in the pathogenesis of the disorder.

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.

Salivary gland epithelial cells (SGEC): carriers of exquisite B7-2 (CD86) costimulatory molecules.

Costimulatory molecules are cell-surface glycoproteins that can direct, modulate and fine tune immune responses. B7-2(CD86) costimulatory molecules are considered as major regulators of T cell responses, acting by appropriate interactions with the stimulatory CD28 or inhibitory CTLA-4 receptors found on T cells. Although their expression is thought to be restricted in lymphoid cells, evidence raised during the last decade show their expression in other types of cells, including human non-neoplastic salivary gland epithelial cells (SGEC). The expression of B7-2 molecules by SGECs requires special attention, due to their unique expression pattern and distinctive binding properties. Thus, SGECs express three B7-2 alternate transcripts that encode the full-length protein, the soluble form and a truncated membrane-bound molecule, that lacks the IgV-like counter-receptor binding domain and has a negative regulatory role. A similar pattern of expression is observed in monocytes, but not in several other types of cells, including dendritic cells. Furthermore, the full-length B7-2 molecules in SGEC display unique binding properties, denoted by the functional interaction with CD28 receptor, but reduced binding of the negative regulator CTLA-4. These distinctive features suggest the tight regulation of B7-2 molecules expression and indicate the key immunoregulatory role of SGECs.

Functional estrogen receptors alpha and beta are expressed in normal human salivary gland epithelium and apparently mediate immunomodulatory effects.

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.

Functional expression of the alpha 2-macroglobulin receptor CD91 in salivary gland epithelial cells.

CD91 molecule is a multifunctional receptor of alpha 2-macroglobulin, heat-shock proteins and calreticulin. CD91 has been implicated in cross-presentation of peptides chaperoned by these proteins to MHC molecules, thus eliciting antigen-specific immune responses. Hence, CD91 is considered as a major regulator of innate and acquired immune responses. Herein, we show that CD91 molecules are expressed by human salivary gland epithelial cells (SGEC), as indicated by immunohistochemical studies in minor salivary gland biopsy tissues (n = 21) as well as by the analyses of human long-term cultured non-neoplastic SGEC lines (n = 11) and the neoplastic HSG cell line. In these cell lines CD91 expression was evaluated by RT-PCR, flow cytometry and confocal microscopy. Standard internalization assays revealed that HSG and SGECs are capable to bind and internalize the CD91 ligand alpha 2-macroglobulin. This internalization is specific, as attested by inhibition studies using unlabeled alpha 2-macroglobulin and a blocking antibody against human CD91 receptor. Conclusively, our findings indicate that SGEC functionally express CD91 receptor, suggesting that this pathway might be involved in the presentation of exogenous antigens in SGEC.

A novel B7-2 (CD86) splice variant with a putative negative regulatory role.

B7-2 (CD86) costimulatory molecules are pivotal for the regulation of T cell responses. In this study, a novel human B7-2 alternate transcript (termed B7-2C) is described. This transcript is characterized by the deletion of exon 4 that encodes the IgV-like counter-receptor binding domain of the B7-2 protein (full-length; B7-2A). B7-2C was detected as mRNA and cell surface protein in human non-neoplastic salivary gland epithelial cells and monocytes, but not in fibroblasts, T cells, B cells, dendritic cells, and several epithelial tumor cell lines. In monocytes, B7-2C protein expression was found to be significantly down-regulated following activation. The analysis of Chinese hamster ovary (CHO) single-transfected (CHO-B7-2C) and double-transfected (CHO-B7-2A/B7-2C) cell lines had indicated that cell surface B7-2C expression is by itself unable to provide T cell costimulation, but inhibits the transmission of costimulatory signals via B7-2A (by 23-69%). Such inhibition was found to depend on the relative cell surface expression of B7-2A and B7-2C proteins, as it occurred in CHO-B7-2A/B7-2C transfectants with significantly lower B7-2A to B7-2C ratios (1.0-3.5), compared with those with unaffected B7-2A-mediated costimulatory function (10.0-19.5). Our findings suggest that B7-2C is expressed by monocytes, as well as by nonimmune cells with potential Ag-presenting capacity (such as salivary gland epithelial cells). The expression of B7-2C on certain B7-2A-expressing cells appears to represent a mechanism for the fine tuning of B7-2A-mediated costimulatory signals, possibly through the interruption of B7-2A clustering required for the productive interaction between B7-2A and cognate receptors.

The role of epithelial cells in the pathogenesis of Sjögren's syndrome.

Sjögren's syndrome (SS) is a chronic autoimmune exocrinopathy that is characterized by periductal mononuclear cell infiltrates in the affected exocrine glands. Epithelial cells are thought to play an important pathogenetic role, as suggested by the occurrence of infiltrating lesions in various epithelial tissues (described as autoimmune epithelitis) as well as the increased epithelial expression of several inflammatory proteins in the histopathologic lesions of patients. In the recent decade, the application of long-term cultured nonneoplastic salivary gland epithelial cell (SGEC) lines has permitted the more explicit analysis of the role of these cells in SS pathophysiology. In such studies, cultured SGEC have been demonstrated to express constitutively or inducibly various molecules that are implicated in innate and acquired immune responses, a fact that underscores the inherent capacity of these epithelial cells to induce and promote chronic inflammatory reactions. Furthermore, the parallel analysis of SGEC lines obtained from SS patients and disease controls has revealed the significantly increased constitutive expression of several molecules in cells derived from SS patients. This fact strongly suggests the operation of intrinsic activation mechanisms in the epithelia of patients and further supports the active participation of epithelia in SS pathogenesis.

Salivary gland epithelial cells: a new source of the immunoregulatory hormone adiponectin.

OBJECTIVE: Adiponectin is an adipocytokine that displays insulin-sensitizing and immunoregulatory properties. Adipocyte development in association with fibrosis is frequently detected in primary Sjögren's syndrome lesions, connoting a healing process. The aim of this study was to examine the expression of adiponectin in minor salivary gland biopsy specimens obtained from patients with primary SS and controls. METHODS: The expression of adiponectin in minor salivary gland biopsy specimens and in long-term-cultured non-neoplastic salivary gland epithelial cell (SGEC) lines obtained from patients with primary SS and control subjects was examined, using immunohistochemistry and immunoblotting, respectively. The expression of adiponectin, adiponectin receptor 1 (AdipoR1), and AdipoR2 messenger RNA (mRNA) by SGECs was investigated by reverse transcription-polymerase chain reaction. RESULTS: Immunohistochemical analysis for adiponectin revealed positive staining of adipocytes from primary SS lesions as well as ductal epithelial cells from both patients with primary SS and controls. All of the SGEC lines tested were shown to express adiponectin, AdipoR1, and AdipoR2 mRNA, whereas adiponectin protein expression was detected by immunoblotting in SGECs from patients with primary SS but not in those from controls. The analysis of concentrated culture supernatants also revealed increased adiponectin expression by SGECs from patients with SS compared with controls. CONCLUSION: Our findings provide novel evidence that adiponectin is produced by SGECs. The high constitutive expression of adiponectin by SGECs from patients with primary SS is likely attributable to aberrant activation of these cells. Although the significance of adiponectin expression remains unknown, it is possible that adiponectin functions in an autocrine manner, as suggested by concurrent expression of the relevant receptors.