Menelaos N. Manoussakis

Expression of B7 costimulatory molecules by salivary gland epithelial cells in patients with Sjögren's syndrome

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 Sjögrens 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.

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 Sjögrens 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.

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

Sjögrens 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.

CD40 on salivary gland epithelial cells: high constitutive expression by cultured cells from Sjögren's syndrome patients indicating their intrinsic activation.

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 and maintenance of lymphocytic infiltrates of patients.

Activation of epithelial and myoepithelial cells in the salivary glands of patients with Sjögren's syndrome: high expression of intercellular adhesion molecule‐1 (ICAM.1) in biopsy specimens and cultured cells

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. Sjogrens 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.

Detection of Coronary Artery Lesions and Myocardial Necrosis by Magnetic Resonance in Systemic Necrotizing Vasculitides

OBJECTIVE Myocardium and coronary arteries can occasionally be affected in patients with systemic necrotizing vasculitides; however, such involvement has not been systematically assessed using cardiovascular magnetic resonance imaging (MRI). METHODS Magnetic resonance angiography and contrast-enhanced MRI were applied for the assessment of coronary arteries (the left anterior descending [LAD], left circumflex [LCx], and right coronary artery [RCA]) and myocardium, respectively, in 39 patients with vasculitis who were asymptomatic for cardiac disease (16 with microscopic polyangiitis [MPA], 11 with Wegeners granulomatosis [WG], 9 with Churg-Strauss syndrome [CSS], and 3 with polyarteritis nodosa [PAN]). Data were compared with age-matched disease-control patients with rheumatoid arthritis (n = 20) or systemic lupus erythematosus (n = 13), and with healthy control individuals with normal coronaries (n = 40). RESULTS Patients with MPA, WG, and PAN (but not with CSS) were found to display significantly increased maximal diameters of coronary arteries compared with healthy controls (for MPA and WG; P < 0.001 for LAD and RCA, and P < 0.01 for LCx) and with both disease-control groups (for only MPA; P < 0.01 for LAD and RCA, and P < 0.05 for LCx). Fusiform coronary aneurysms were detected in patients with MPA (4/16) and PAN (2/3), whereas coronary ectasias were evident in patients with MPA (14/16) and WG (2/11). The presence of myocardial necrosis (by assessment of late gadolinium-enhanced images) was identified only in patients with MPA (2/16) and CSS (3/8 studied). CONCLUSION Cardiovascular MRI assessment of patients with systemic vasculitis revealed coronary ectatic disease in the majority of patients with MPA and PAN, as well as in several patients with WG. Myocardial necrosis can be detected in MPA and CSS.

Functional Expression of a Costimulatory B7.2 (CD86) Protein on Human Salivary Gland Epithelial Cells that Interacts with the CD28 Receptor, but Has Reduced Binding to CTLA4

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.

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. Sjögrens 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.

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.

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 Sjogrens 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.