Sergio Aguilera

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 Sjögrens 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.

Sjögren's syndrome and the epithelial target: A comprehensive review

The most difficult component in our understanding of human autoimmunity remains a rigorous dissection of etiological events. Indeed, the vast literature on autoimmune diseases focuses on the inflammatory response, with the hope of developing drugs that reduce inflammation. However, there is increasing recognition that understanding the immunobiology of target tissues will also have direct relevance to disease natural history, including breach of tolerance. Sjögrens syndrome is essentially an epitheliitis and there are major changes to normal architectural salivary organization. We propose that loss of homeostasis is the initial event that precipitates inflammation and that such inflammatory response includes not only the adaptive response, but also an intense innate immune/bystander response. To understand these events this review focuses on the architecture, phenotype, function and epithelial cell organization. We further submit that there are several critical issues that must be defined to fully understand epithelial cell immunobiology in Sjögrens syndrome, including defining epithelial cell polarity, cell-cell and cell to extracellular matrix interactions and a variety of chemical and mechanical signals. We also argue that disruption of tight junctions induces disorganization of the apical pole of salivary acinar cells in Sjögrens syndrome. In addition, there will be a critical role of inflammatory cytokines in the apico-basal relocation of tight junction proteins. Further, the altered disorganization and relocation of proteins that participate in secretory granule formation are also dysregulated in Sjögrens syndrome and will contribute to abnormalities of mucins within the extracellular matrix. Our ability to understand Sjögrens syndrome and develop viable therapeutic options will depend on defining these events of epithelial cell biology.

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

Primary Sjögrens 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.

Reduced sulfation of muc5b is linked to xerostomia in patients with Sjögren syndrome

Objectives: MUC5B contains sulfated and sialylated oligosaccharides that sequester water required for moisturising the oral mucosa. Xerostomia, in patients with Sjögren syndrome, is generally associated with reduced quantities, rather than altered properties, of saliva. Here, we determined the amount of MUC5B (mRNA and protein) as well as sulfation levels in salivary glands of patients with normal or altered unstimulated salivary flow. Localisation of MUC5B and sulfated MUC5B, as well as total levels sulfated groups were determined and compared with acini basal lamina disorganisation. Patients and methods: In all, 18 patients with normal or altered unstimulated salivary flow and 16 controls were studied. MUC5B mRNA and protein were evaluated in salivary glands by semiquantitative RT-PCR and Western blot analysis. MUC5B sulfation was determined by Western blotting. MUC5B and sulfo-Lewisa antigen localisation were assessed by immunohistochemistry. The total amount of sulfated oligosaccharides was determined microdensitometrically. Results: No significant differences were detected in MUC5B mRNA and protein levels between controls and patients, while sulfo-Lewisa antigen levels were lower in patients. The number of sulfo-Lewisa positive mucous acini was reduced in patients but no correlation was observed between lower levels of sulfation and unstimulated salivary flow. Microdensitometric data confirmed the presence of reduced sulfated oligosaccharides levels in mucous acini from patients with highly disorganised basal lamina. Conclusion: Disorganisation of the basal lamina observed in patients with Sjögren syndrome may lead to dedifferentiation of acinar mucous cells and, as a consequence, alter sulfation of MUC5B. These changes are suggested to represent a novel mechanism that may explain xerostomia in these patients.

Basal lamina disorganisation of the acini and ducts of labial salivary glands from patients with Sjögren’s syndrome: association with mononuclear cell infiltration

Objective: To study the expression of laminin and type IV collagen as biomarkers of the organisation of the basal lamina of acini and ducts in labial salivary glands from patients with Sjögren’s syndrome, and to relate this organisation to inflammatory cell invasion of acini and ducts. Methods: Immunohistochemistry for laminin and type IV collagen was undertaken on sections of labial salivary glands from 30 patients with Sjögren’s syndrome, 10 control subjects, and 24 controls with chronic sialoadenitis. Immunohistochemistry reaction, alterations to cell morphology, and the presence of inflammatory cells in acini and ducts were evaluated and scored using a semiquantitative method. Results: Changes in the expression of laminin and type IV collagen in the basal lamina of acini and ducts of labial salivary glands from patients with Sjögren’s syndrome were more pronounced than in labial salivary glands from control groups. A remarkable characteristic was the disorganisation of the basal lamina in the labial salivary glands in Sjögren’s syndrome. The pattern of immunoreactivity of the basal lamina of other structures (for example, blood vessels) did not change. In Sjögren’s syndrome, invasion of cytotoxic T lymphocytes was only observed in acini and ducts which had a disorganised basal lamina. Conclusions: The high state of disorganisation of the basal lamina of acini and ducts could allow invasion of cytotoxic T lymphocytes in Sjögren’s syndrome, contributing to cell death and ductal hyperplasia.

Alterations in type I hemidesmosome components suggestive of epigenetic control in the salivary glands of patients with Sjögren's syndrome

Objective Acinar cells in the salivary glands of patients with Sjogrens syndrome (SS) display severe alterations in anchorage to the basal lamina. Bioinformatics analysis of the BP230 gene sequence has revealed the presence of CpG islands that might be involved in epigenetic control of gene expression, and methylation of the BP230 promotor region may be implicated as an epigenetic control mechanism in salivary gland damage. Thus, the present study was undertaken to evaluate the protein BP230, as well as proteins BP180, α6β4 integrin, and cytokeratin-18, for their expression levels, localization, and ability to form hemidesmosome adhesion complexes. Methods Eighteen patients with primary SS and 14 healthy control subjects were studied. Levels of messenger RNA (mRNA) and protein were measured by reverse transcription–polymerase chain reaction and Western blotting, respectively. BP230 methylation was determined by methylation-sensitive polymerase chain reaction. Protein complexes were analyzed by immunoprecipitation and assessed for localization by immunofluorescence. Results In patients with SS as compared with controls, BP230 mRNA levels were decreased while protein levels were increased, and the gene promotor region was hypermethylated. Augmented proteolysis of BP180 was detected, since levels of linear IgA disease fragment 1 were increased. The complex-forming ability of BP230, BP180, α6β4 integrin, and cytokeratin-18 was maintained in patients with SS, in contrast to that in controls. BP230 and BP180 colocalized at the basal membrane of acinar cells, and cleavage of BP180 coincided with a loss of colocalization. Conclusion The decrease in BP230 mRNA levels may be explained by gene hypermethylation. We postulate that local epigenetic modifications of BP230 are produced in response to factors present in the damaged salivary glands of patients with SS. Additionally, the paradoxical increase in BP230 protein levels and the formation of both normal and altered adhesion complexes may help avoid cell death induced by the loss of anchorage.

Oral dryness in Sjögren's syndrome patients. Not just a question of water

Sjögrens syndrome (SS) is a chronic autoimmune disease of undefined etiology. Patients with this syndrome suffer from severe alterations in both the quality and quantity of saliva and tears, due to impaired function of the relevant exocrine glands. Prevalent symptoms experienced by SS-patients include a persistent dry mouth sensation (xerostomia) and dry eyes (keratoconjunctivitis sicca). Water content of saliva depends of acetylcholine levels, glandular innervation, M3R signaling, calcium tunneling and water release, among other factors. However, unstimulated salivary flow correlates only poorly with symptoms of mouth dryness, raising the question as to which other components of saliva may be involved in mouth dryness experienced by SS-patients? Salivary mucins are glycoproteins characterized by the presence of large oligosaccharide side chains attached to the protein backbone. These molecules are key saliva components that are required to sequester water and thereby moisturize, as well as lubricate the oral mucosa. In the labial salivary glands of SS patients, morphological and functional alterations are detectable that affect the maturation and trafficking of salivary mucins. In this review, we will focus the discussion on these aspects of reduced salivary flow and decreased quality of salivary mucins, since they are likely to be responsible for xerostomia in SS-patients.

Changes in Rab3D Expression and Distribution in the Acini of Sjogren's Syndrome Patients Are Associated With Loss of Cell Polarity and Secretory Dysfunction

OBJECTIVE Oral and ocular dryness are frequent and serious symptoms of Sjögrens 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.

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

Sjögrens 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.

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 Sjögrens 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.