Aberrant localization of fusion receptors involved in regulated exocytosis in salivary glands of Sjögren's syndrome patients is linked to ectopic mucin secretion.

Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease that mainly affects tear and salivary glands, whereby SS-patients frequently complain of eye and mouth dryness. Salivary acinar cells of SS-patients display alterations in their cell polarity; which may affect the correct localization and function of proteins involved in regulated exocytosis. Here we determined whether the expression and localization of SNARE proteins (membrane fusion receptors) involved in regulated secretion, such as VAMP8, syntaxin 3 (STX3), STX4 and SNAP-23 were altered in salivary glands (SG) from SS-patients. Additionally, we investigated SNARE proteins function, by evaluating their ability to form SNARE complexes under basal conditions. In SG from SS-patients and control subjects mRNA and proteins levels of SNARE complex components were determined by real-time PCR and Western blotting, respectively. SNARE protein distribution and mucin exocytosis were determined by indirect immunofluorescence. In SS-patients, the expression levels of mRNA and protein for VAMP8, STX4 and STX3 were altered. STX4, STX3, SNAP-23 and VAMP8 relocated from the apical to the basal region of acinar cells. Increased formation of SNARE complexes in a manner independent of external stimuli for secretion was detected. Mucins were detected in the extracellular matrix (ECM). Presence of mucins in the ECM, together with the observed alterations in SNARE protein localization is indicative of ectopic exocytosis. In the context of SS, such aberrantly localized mucins are likely to favor a pro-inflammatory response, which may represent an important initial step in the pathogenesis of this disease.

Decreased salivary sulphotransferase activity correlated with inflammation and autoimmunity parameters in Sjogren's syndrome patients.

OBJECTIVES: To determine the expression and enzymatic activities of sulphotransferases involved in mucin hyposulphation in labial salivary glands (LSGs) from SS patients and to correlate sulphotransferase activity with clinical parameters such as secretion, inflammation and serology. METHODS: LSG from 31 SS patients and 31 control subjects were studied. Relative mRNA and protein levels of Gal3-O-sulphotransferases (Gal3STs) and ß1,3-galactosyltransferase-5 (ß3GalT5) were determined by quantitative RT-PCR and western blotting, respectively. Enzymatic activities were quantified using radioactively labelled donor substrates and specific acceptor substrates. Products were purified by chromatography. Spearman's correlation analysis was used to compare data. RESULTS: The levels of Gal3ST activity were significantly decreased in SS patients, without changes in mRNA and protein levels, while the enzymatic activities of glycosyltransferases involved in mucin glycosylation were similar in both groups. An inverse correlation was observed between Gal3ST activity and glandular function measured by scintigraphy, but not with unstimulated salivary flow. Gal3ST activity was inversely correlated with focus score, TNF-α levels and presence of the autoantibodies Ro/SS-A and La/SS-B. CONCLUSION: The decrease in sulphotransferase activity provides an explanation for mucin hyposulphation observed in the LSGs from SS patients. The decrease in Gal3STs activity was not a consequence of reduced gene expression, but probably due to alterations in the enzyme activity regulation. Interestingly, the levels of sulphotransferase activity detected correlated well with secretory function, inflammation and serology. Finally, we postulate that pro-inflammatory cytokines induced by autoantibodies, such as Ro/SS-A and La/SS-B in SS patients, may modulate Gal3ST activity, thereby altering mucin quality and leading to mouth dryness.

Changes in Rab3D expression and distribution in the acini of Sjögren's syndrome patients are associated with loss of cell polarity and secretory dysfunction.

OBJECTIVE: Oral and ocular dryness are frequent and serious symptoms of Sjögren's syndrome (SS) that reflect problems in secretion due to glandular dysfunction. Exocytosis, an important process in the secretory pathway, requires the participation of Rab family GTPases. This study was undertaken to analyze the expression and localization of Rab3D and Rab8A and to examine their correlation with acinar cell polarity and glandular secretory function. METHODS: Nineteen patients with SS and 17 controls were evaluated. Levels of Rab3D and Rab8A messenger RNA (mRNA) and protein were determined by real-time polymerase chain reaction and Western blotting. Subcellular localization of proteins was determined by indirect immunofluorescence analysis. RESULTS: In patients with SS, total Rab3D protein levels decreased significantly, while mRNA levels remained unchanged. For Rab8A, no changes in either mRNA or protein levels were detected. In serous acini of labial salivary glands from patients with SS, the following 4 patterns of Rab3D staining were distinguishable: severely decreased, distribution throughout the cytoplasm, distribution throughout the cytoplasm combined with loss of nuclear polarity, and normal apical localization. Basal localization of Rab8A was not modified. Rab3D changes were accompanied by apicobasolateral redistribution of ezrin, loss of nuclear polarity, thicker Golgi stacks, and mucin 7 accumulation in the cytoplasm. Finally, low Rab3D protein levels correlated with alterations in scintigraphy measurements. CONCLUSION: Our findings indicate that Rab3D regulates the exocytosis of many components critical for the maintenance of oral physiology. Hence, the changes observed in Rab3D expression and distribution are likely to contribute to the decrease in or loss of saliva components (i.e., mucins), which may explain the variety of oral and ocular symptoms associated with SS.

Disruption of tight junction structure in salivary glands from Sjögren's syndrome patients is linked to proinflammatory cytokine exposure.

OBJECTIVE: Disorganization of acinar cell apical microvilli and the presence of stromal collagen in the acinar lumen suggest that the labial salivary gland (LSG) barrier function is impaired in patients with Sjögren's syndrome. Tight junctions define cell polarity and regulate the paracellular flow of ions and water, crucial functions of acinar cells. This study was undertaken to evaluate the expression and localization of tight junction proteins in LSGs from patients with SS and to determine in vitro the effects of tumor necrosis factor alpha (TNFalpha) and interferon-gamma (IFNgamma) on tight junction integrity of isolated acini from control subjects. METHODS: Twenty-two patients and 15 controls were studied. The messenger RNA and protein levels of tight junction components (claudin-1, claudin-3, claudin-4, occludin, and ZO-1) were determined by semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting. Tight junction protein localization was determined by immunohistochemistry. Tight junction ultrastructure was examined by transmission electron microscopy. Isolated acini from control subjects were treated with TNFalpha and IFNgamma. RESULTS: Significant differences in tight junction protein levels were detected in patients with SS. ZO-1 and occludin were strongly down-regulated, while claudin-1 and claudin-4 were overexpressed. Tight junction proteins localized exclusively to apical domains in acini and ducts of LSGs from controls. In SS patients, the ZO-1 and occludin the apical domain presence of decreased, while claudin-3 and claudin-4 was redistributed to the basolateral plasma membrane. Exposure of isolated control acini to TNFalpha and IFNgamma reproduced these alterations in vitro. Ultrastructural analysis associated tight junction disorganization with the presence of endocytic vesicles containing electron-dense material that may represent tight junction components. CONCLUSION: Our findings indicate that local cytokine production in LSGs from SS patients may contribute to the secretory gland dysfunction observed in SS patients by altering tight junction integrity of epithelial cells, thereby decreasing the quality and quantity of saliva.

Aberrant localization of ezrin correlates with salivary acini disorganization in Sjogren's Syndrome.

OBJECTIVES: To analyse whether the alterations in the structure and organization of microvilli in salivary acinar cells from SS patients are linked to changes in the expression and/or cellular localization of ezrin. METHODS: Salivary gland (SG) acini from controls and SS patients were used to evaluate ezrin expression by western blot and localization of total and activated (phospho-Thr567) ezrin by IF and EM. RESULTS: In acini from control labial SGs, ezrin was located predominantly at the apical pole and to a lesser extent at the basal region of these cells. Conversely, in acini extracts from SS patients, ezrin showed significantly elevated levels, which were accompanied with localization mostly at the basal region. Moreover, F-actin maintained its distribution in both the apical region and basolateral cortex; however, it was also observed in the acinar cytoplasm. Phospho-ezrin (active form) was located exclusively at the apical pole of acinar cells from control subjects and abundantly located at the basal cytoplasm in SS samples. These results were confirmed by immunogold studies. CONCLUSIONS: The decrease of ezrin and phospho-ezrin at the apical pole and the cytoplasmic redistribution of F-actin suggest an altered interaction between the F-actin-cytoskeleton and plasma membrane in SS patient acini, which may explain the microvilli disorganization. These alterations could eventually contribute to SG hyposecretion in SS patients.

Gene expression and chromosomal location for susceptibility to Sjögren's syndrome.

Primary Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease affecting mainly the exocrine glands. Its physio-pathology is poorly understood and most of the knowledge has been related to the inflammatory component. The aim of this work was to evaluate gene expression profiling in fractions enriched in epithelial cells from labial salivary glands (LSGs) of patients with primary SS and identify chromosomal regions harboring susceptibility genes expressed in epithelial cells. A combined approach of gene expression and genome-wide association study was used. Enriched epithelial cell fractions were obtained from LSGs of patients and controls. Amplified total RNA was labeled and hybridized to 10K cDNA microarrays. Results were normalized and subjected to statistical and functional analysis. A genome-wide microsatellite screen at 10 cM resolution (393 markers) was performed. In salivary gland-epithelial cells from patients 528 genes were expressed differentially in comparison to controls. Pathways not previously linked to disease were found to be altered. Twenty-eight and 15 genes associated with apoptosis were up-regulated and down regulated, respectively. Interferon-related genes, most of which participated in interferon signaling, were also found to be up-regulated. From the genome-wide screen, 6 markers showed evidence of highly significant association with the disease. Of these, five loci harbor genes differentially expressed in patients LSG-epithelial cells. Our results show that in enriched gland-epithelial cells of pSS, both pro-apoptotic/anti-apoptotic and interferon signaling inhibition/stimulation balances may occur. Genes found over-expressed in epithelial cells are candidates for disease susceptibility.

Mechanotransduction and epigenetic control in autoimmune diseases.

Differentiation of epithelial cells is required to define tissue architecture and appropriate function of these cells is associated with a specific pattern of gene expression. DNA methylation, post-translational modification of histones and chromatin remodeling are nuclear mechanisms implicated in epigenetic control of gene expression. All factors relevant to tissue differentiation, including cell adhesion and shape, extracellular stimuli and transcriptional control, modulate gene expression and, thus, some of them are likely to impact on nuclear mechanisms of epigenetic control. The epithelial cells of salivary glands from Sjögren's syndrome patients display alterations in cell adhesion and shape. In this review, we summarize how these alterations are thought to lead to chromatin remodeling and, in doing so, bring about changes in transcriptional patterns. Additionally, we discuss how mechanotransduction in cells with impaired structural organization is implicated in modifying gene expression in these patients.

Involvement of specific laminins and nidogens in the active remodeling of the basal lamina of labial salivary glands from patients with Sjögren's syndrome.

OBJECTIVE: To investigate remodeling of the basal lamina of labial salivary glands (LSGs) from patients with Sjögren's syndrome (SS) by analyzing the expression of specific components that participate in its assembly and attachment to acinar and ductal cells. METHODS: Two groups of SS patients with similar levels of remnant glandular tissue but with low and high levels of interacinar fibrosis, respectively, were studied. The expression of laminin alpha1, alpha4, and gamma2 chains and nidogens was examined at the messenger RNA (mRNA) and protein levels. Nidogens 1 and 2 were also studied in situ by immunohistochemistry. RESULTS: Increases in the amount of mRNA and protein for both the processed and unprocessed laminin gamma2-chain were more pronounced in patients with low interacinar fibrosis. Increases in the protein levels of laminin alpha1 and alpha4 chains were observed in patients with low interacinar fibrosis, but not in those with high interacinar fibrosis. Nidogen mRNA and protein levels were similar in SS patients and controls. Interestingly, high levels of nidogen degradation were observed in patients with low interacinar fibrosis. Nidogens were readily detected by immunofluorescence in the basal lamina of the capillaries and stroma in SS patients, but were less apparent in the basal lamina of the acini and ducts. CONCLUSION: These results suggest that the basal lamina of LSGs from patients with SS is undergoing active remodeling, such that alterations are less evident in patients who have advanced morphologic signs of the disease (high interacinar fibrosis). Nidogen proteolysis might account for the disorganization of the basal lamina that is typically observed in LSGs from SS patients, assuming that cleavage impairs their ability to crosslink type IV collagen and laminin networks.

Increased acinar damage of salivary glands of patients with Sjögren's syndrome is paralleled by simultaneous imbalance of matrix metalloproteinase 3/tissue inhibitor of metalloproteinases 1 and matrix metalloproteinase 9/tissue inhibitor of metalloproteinases 1 ratios.

OBJECTIVE: Previous findings in labial salivary glands (LSGs) from patients with Sjögren's syndrome (SS) suggest that increased activity and expression of matrix metalloproteinase 9 (MMP-9) and MMP-3 trigger the destruction of acinar structures in these glands. Tissue inhibitors of matrix metalloproteinases (TIMPs) tightly control MMP activity, and TIMP expression is an important modulator of effects attributed to MMPs. This study was undertaken to investigate the correlation between the balance of MMPs/TIMPs in the LSGs of SS patients and the degree of inflammatory infiltration and acinar structure integrity. METHODS: Three groups of SS patients classified according to focus score and residual tissue were studied. The expression of MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 was examined at the messenger RNA and protein levels. The ratio of MMP/TIMP expression (R value) was calculated. Focus score and acinar structure were evaluated by histologic analysis. RESULTS: In SS patients the MMP-3/TIMP-1 ratio was higher than 1 and the MMP-9/TIMP-1 ratio was much higher than 1 whereas the MMP-2/TIMP-2 ratio nearly equaled 1, suggesting elevated proteolytic activity due mainly to MMP-9. R values were independent of the focus score of inflammatory cells, but correlated well with the dramatic changes observed in morphologic integrity of acini, as revealed mainly by the lack of nuclear polarity. Acinar changes were more evident when R values for both MMP-9/TIMP-1 and MMP-3/TIMP-1 were higher. CONCLUSION: This study provides evidence that an altered balance between MMPs and their inhibitors is associated with acinar damage. Since salivary gland acinar cells express both MMPs and TIMPs, these cells may play an important role in extracellular matrix destruction and in the LSG pathophysiology in SS.

Enhanced degradation of proteins of the basal lamina and stroma by matrix metalloproteinases from the salivary glands of Sjögren's syndrome patients: correlation with reduced structural integrity of acini and ducts.

OBJECTIVE: To determine the effect of matrix metalloproteinase (MMP) activity from the labial salivary glands (LSGs) of Sjögren's syndrome (SS) patients on proteins of the extracellular matrix (ECM) that form the basal lamina and stroma, and to compare this effect with the structural integrity of acini and ducts as well as the functionality of the LSGs. METHODS: Gelatinase activity was determined by zymography. The digestion pattern of extracellular matrix (ECM) macromolecules was detected by gel electrophoresis and quantified by densitometry. The structural integrity of acini and ducts was evaluated by light and electron microscopy. Secretory function was evaluated by measuring unstimulated salivary flow and by scintigraphy. RESULTS: LSG extracts showed increased levels of proteolytic activity toward purified proteins of the basal lamina (laminin and type IV collagen) and stroma (types I and III collagen and fibronectin). Enhanced degradation was most evident for fibronectin, laminin, and type IV collagen. Analysis of the ultrastructure of the acinar and ductal basal lamina revealed abnormalities ranging from disorganization to disappearance of this ECM structure. These changes were paralleled by an important loss of microvilli on the apical surface, as well as decreased unstimulated salivary flow. Interestingly, the results were similar in LSGs from all SS patients, regardless of the proximity of infiltrating mononuclear cell foci. CONCLUSION: Our observation that the proteolytic action of MMPs toward ECM macromolecules is increased in SS patients provides a rationale for understanding the dramatic changes in the structural organization observed in the basal lamina and apical surface of acini in these patients. The results provide new evidence that acinar and ductal cells from the LSGs of SS patients display a molecular potential, with increased capacity to markedly disorganize their ECM environment and, thus, damage their architecture and functionality.