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.

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.

Distribution of aquaporins in the colon of Octodon degus, a South American desert rodent.

Octodon degus is a desert rodent of northern Chile, adapted to survive with a limited supply of water. This rodent has a high degree of fecal dehydration, related to colon water absorption. With the hypothesis that aquaporins (AQPs) might be present in the colon epithelium of O. degus and involved in fluid absorption, we studied colon water absorption in vivo and the distribution of AQPs and Na(+) transporters by immunocytochemistry. AQP-1 was found in apical and basolateral membranes of surface-absorptive and crypt epithelial cells. AQP-8 was found in the cytoplasm of enterocytes of surface colon. AQP-3 immunolabeling, on the other hand, was absent from the epithelium but present in a subepithelial fibroblast layer, pericryptal cells, and muscularis mucosae. The hydration state did not modify the amount of immunostaining for any of the AQPs. Colon water absorption was markedly decreased by the mercurial agent p-chloromercuribenzenesulfonic acid and was not affected by water deprivation. The NHE3 isoform of Na(+)/H(+) exchanger and alpha-1 subunit of the Na(+)-K(+)-ATPase were found in apical and basolateral membranes of surface-absorptive cells, respectively. These results suggest that colon water absorption is mostly transcellular and mediated by water channels like AQP-1. Apical Na(+)/H(+) exchanger and basolateral Na(+)-K(+)-ATPase in surface cells could be part of the Na(+) absorption pathway. It is hypothesized that this transport is necessary to provide an osmotic gradient for water absorption. The roles of AQP-8 and AQP-3 in water absorption remain to be established.