MUC1/SEC and MUC1/Y overexpression is associated with inflammation in Sjögren's syndrome.

OBJECTIVES: To evaluate the expression and localization of MUC1/SEC and MUC1/Y isoforms in labial salivary glands (LSG) from Sjögren's syndrome patients (SS patients), as well as their in vitro expression induced by cytokines. SUBJECTS AND METHODS: Labial salivary gland from 27 primary SS patients and 22 non-SS sicca subjects were studied. Relative MUC1/SEC and MUC1/Y mRNA levels were determined by qPCR and protein levels by Western blotting. Induction of mucin mRNAs was assayed in vitro. Immunohistochemistry was used for localization. RESULTS: Relative MUC1/SEC and MUC1/Y mRNA and protein levels were significantly higher in LSG from SS patients. These mRNAs were induced by cytokines. MUC1/SEC and MUC1/Y were detected in acini apical region of control LSGs, and significant cytoplasmic accumulation was observed in acini of SS patients. MUC1/Y localized in acinar nuclei and cytoplasm of inflammatory cells of LSG from SS patients. A strong positive correlation was observed between cellular MUC1/SEC levels and glandular function determined by scintigraphy. CONCLUSIONS: We show for the first time that MUC1/SEC and MUC1/Y are expressed in LSG of both SS patients and non-SS sicca subjects. The observed overexpression and aberrant localization of MUC1/SEC and MUC1/Y and their induction by pro-inflammatory cytokines may favor the perpetuation of the inflammatory environment that disrupts the salivary glandular homeostasis in SS patients.

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. Sjögren's 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 Sjögren's 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 Sjögren's 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 Sjögren's syndrome and will contribute to abnormalities of mucins within the extracellular matrix. Our ability to understand Sjögren's syndrome and develop viable therapeutic options will depend on defining these events of epithelial cell biology.

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

Sjögren's 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.

Severe alterations in expression and localisation of {alpha}6{beta}4 integrin in salivary gland acini from patients with Sjogren syndrome.

OBJECTIVES: In salivary glands from patients with Sjögren syndrome, overexpression of laminins 1 and 5 and disorganisation of the acinar basal lamina have been reported. Laminin 5 mediates association of the basal lamina with epithelial cells by forming adhesion complexes upon interaction with alpha6beta4 integrin. In the present work, mRNA and protein levels of alpha6beta4 integrin were determined and its localisation in salivary glands evaluated in patients with Sjögren syndrome. METHODS: Salivary glands of 12 patients with Sjögren syndrome and 8 controls were studied. The mRNA and protein levels of alpha6beta4 were determined by semiquantitative reverse transcriptase (RT)-PCR and western blot analysis, respectively. The subcellular localisation of alpha6beta4 and laminin were evaluated by confocal microscopy. RESULTS: In patients, no significant differences in alpha6 and beta4 mRNA levels were detected. However, beta4 integrin protein levels were significantly lower, whereas, changes in alpha6, were highly variable. In controls, alpha6beta4 was detected in the basolateral and basal surface of serous and mucous acini, respectively. In patients, alterations in alpha6beta4 distribution were particularly dramatic for acini with strong basal lamina disorganisation. alpha6beta4 was also detected in the cytoplasm and lateral plasma membrane in serous and mucous acini. CONCLUSION: Mild alterations in the basal lamina correlated with lateral redistribution of alpha6beta4 integrin and the formation of new cell-cell adhesions that help maintain acinar organisation and promote cell survival. Conversely, in cases with severe basal lamina alterations, lateral alpha6beta4 redistribution was no longer sufficient to maintain acinar cell survival. Thus, maintenance of equilibrium between cell-cell and cell-basal lamina attachment is required to sustain gland cell survival.

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-Lewis(a) 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-Lewis(a) antigen levels were lower in patients. The number of sulfo-Lewis(a) 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 Sjogren'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.

Cell-enlargement-related polypeptides are induced via beta(1)-adrenoceptors in mouse parotids.

Induction of cell and gland enlargement (growth-in-size) and induction of a group of secretory polypeptides (polypeptides C-G) seem to occur in close relationship in mouse parotid glands stimulated chronically by the nonselective beta-adrenergic agonist isoproterenol. To determine whether beta(1), beta(2), or both subtypes of beta-adrenergic receptors are involved in those responses, dose-dependency studies were carried out during a 7-day period of daily stimulations to assess the relative abilities of the selective beta-adrenergic agonists dobutamine (beta(1)) and salbutamol (beta(2)) to induce polypeptides C-G and growth-in-size. The relative abilities of the selective beta-adrenoceptor antagonists atenolol (beta(1)) and I.C.I. 118.551 (beta(2)) to interfere with the induction of both responses by chronic treatment with the various beta-adrenergic agonists were also studied. Parotid growth-in-size was assessed by evaluating wet weight, whole protein content, and light microscopy histology. The presence of polypeptides C-G was evaluated after SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. Under these experimental conditions, dobutamine was found to be at least one order of magnitude more potent than salbutamol at inducing growth-in-size. Dobutamine was also found to be clearly stronger than salbutamol as an inducer of polypeptides C-G. On the other hand, atenolol was more effective than I.C.I. 118.551 at preventing the induction of polypeptides C-G and growth-in-size by isoproterenol, dobutamine, or salbutamol. Taken together, these results suggest that in mouse parotid glands, polypeptides C-G and growth-in-size are induced preferentially via adrenergic receptors of the beta(1)-subtype.

Differential expression of matrix metalloproteinases in labial salivary glands of patients with primary Sjögren's syndrome.

OBJECTIVE: To determine the enzymatic activity and cellular localization of matrix metalloproteinases (MMPs) 2, 3, and 9 in labial salivary glands from patients with different degrees of severity of primary Sjogren's syndrome (primary SS). METHODS: Gelatinase activity was determined by zymography and quantified by densitometry. The specificity of MMPs was determined using protease inhibitors and chelators, as well as activators of the latent forms of these enzymes. The cellular localization of MMPs was carried out using monoclonal antibodies that recognize their latent and active forms. RESULTS: Labial glands from control subjects and patients showed gelatinase activity for MMP-2 and MMP-9. Activation studies revealed that both enzymes were predominantly present in their latent forms. The highest levels of MMP-9 activity were detected in patients with severe, active, primary SS (except for patients with severe clinical symptoms for extended periods) and correlated with structural and functional glandular changes. MMP-2 activity was almost the same in patients and controls. MMPs were detected by immunolocalization only in acinar and ductal cells and were homogeneously distributed throughout patients' glands. MMP-2 and MMP-9 expression paralleled their gelatinase activity. MMP-3, detectable only with immunologic methods, was absent in control subjects but abundantly expressed in patients. Importantly, MMP protein levels in acinar and ductal cells were independent of either the presence or the proximity of mononuclear infiltrate cells. CONCLUSION: MMP-3 and MMP-9 expression, as well as MMP-9 catalytic activity, were increased in tissue samples from SS patients in a manner that correlated with the severity of the disease. Most important, increased MMP activity stemmed from exocrine epithelial cells and was not due to infiltrating lymphocytes. Thus, changes in salivary glands as a consequence of proteolysis may lead to severe glandular destruction.

Transcriptional and posttranscriptional control in synthesis of growth marker polypeptides in mouse parotids.

The chronic daily administration of isoproterenol provokes in mouse parotid glands the induction and progressive accumulation of a family of secretory polypeptides named polypeptides C, D, E, F, and G (polypeptides C-G). These polypeptides, which seem to be part of the family of proline-rich proteins, have been considered as molecular markers of the growth-in-size response in the mouse parotid acinar cells. In the present study, two pharmacological approaches were used to determine whether the induction and the postsecretory reappearance of polypeptides C-G may be distinguished from each other. First, actinomycin D, a transcriptional inhibitor, was found to interfere with the induction by isoproterenol but not with the postsecretory reappearance. Second, pilocarpine, a secretagogue that was found to be a very weak inducer of polypeptides C-G, was able to provoke secretion and then reappearance of the whole group of isoproterenol-induced polypeptides. Accordingly, these data suggest that the induction of polypeptides C-G is dependent on transcriptional activity and that it is unrelated to secretion stimulation. By contrast, the postsecretory reappearance of polypeptides C-G may occur even when transcriptional activity is inhibited and it would be related to the secretory activity.

Secretory character of a group of isoproterenol-induced polypeptides in mouse parotid glands.

The secretory nature of the isoproterenol-induced mouse parotid polypeptides C, D, E, F, and G (molecular weights 64,000, 61,000, 51,500, 38,000, and 37,000, respectively) is documented. Polypeptides C, D, E, F, and G, accumulated in response to successive daily stimulations with isoproterenol, were detected in a fraction enriched in hypertrophic parotid acinar cells. These cells, characterized by an increased content of cytoplasmic granules, maintain a secretory responsiveness to isoproterenol, which has been evidenced by light microscopy, enzymatic analysis, and unidimensional SDS-polyacrylamide gel electrophoresis. Thus, a parallelism in the loss and recovery of both secretory granules, alpha-amylase and polypeptides C, D, E, F, and G, was observed. Moreover, after secretion stimulation, polypeptides C, D, E, F, and G were detected in the fluid collected directly from parotid gland cannulation. Given the secretory character of polypeptides C, D, E, F, and G, mechanisms explaining both their progressive accumulation along the chronic administration of isoproterenol, as well as their progressive disappearance observed after suspending that treatment, are discussed.

Nucleolar changes induced by isoproterenol in mouse acinar parotid cells.

This paper deals with the analysis of the nucleolar morphology and distribution of the nucleolar component (i.e. fibrils and granules) in normal and isoproterenol-treated parotid acinar cells of mice. Normally nucleoli present both components intermingled, but at 2 h treatment a considerable decrease of the fibrillar area is detected by a silver staining method. Normal nucleolar characteristics are recovered after eight hours treatment. In hypertrophic cells nucleolar size is considerably increased, but the ratio total nucleolar area/fibrillar area is similar to control cells as shown by a stereometrical computerized analysis. Rounded nucleolar bodies occur in these nuclei which do not seem to be dependent on the modifications induced by the drug.

Changes in the polypeptide composition related to the growth response in chronically isoproterenol-stimulated mouse parotid glands.

The administration of isoproterenol induces DNA-synthesis mitosis and growth (increase in size) responses in mouse parotid glands. Both responses were uncoupled by means of daily stimulations with isoproterenol in such a way that the DNA-synthesis mitosis response was observed during the first 4 days only, whereas the growth response was continuous since the first stimulation until about day 12. In parallel to the chronic stimulation by isoproterenol, drastic changes in the polypeptide composition of parotid glands were observed. These modifications, consisting basically of the reduction in content of a couple of major poly peptides (polypeptides A and B) together with the reciprocal massive accumulation of five new polypeptides (polypeptides C, D, E, F and G), were also progressive and continuous along the chronic stimulation by isoproterenol, even after the disappearance of the DNA-synthesis mitosis response. Thus, a relationship between specific changes in the mouse parotid content of polypeptides A, B, C, D, E, F and G and the isoproterenol-induced growth response, rather than with the DNA-synthesis mitosis response, is suggested. The correlation is firmly supported by the progressive recovery of the normal polypeptide composition upon suspending isoproterenol treatment, which allows parotid glands to return to normal size parameters.

Phosphatase activity in Trypanosoma cruzi. Phosphate removal from ATP, phosphorylated proteins and other phosphate compounds.

T. cruzi epimastigotes have a lysosomal acid phosphatase (pH 4.0) and acid and alkaline phosphatases (pH 5.5 and 8.0) localized in the cytosolic fraction. The levels of the lysosomal acid phosphatase increase with the age of the cultures, but the cytosolic phosphatases decline after the logarithmic phase of growth. The lysosomal phosphatase preferentially hydrolyses low mol. wt phosphate esters; whereas, the cytosolic alkaline phosphatases primarily act on phosphorylated proteins, and both the cytosolic acid and alkaline phosphatases on uridine nucleotide derivatives. The parasite also contains a microsomal glucose 6-phosphatase, and ATPases (Mg2+ and Ca2+-activated) derived from plasma membranes and mitochondria.