Inhibition of JAK-STAT Signaling by Baricitinib Reduces Interferon-γ-Induced CXCL10 Production in Human Salivary Gland Ductal Cells.

Sjögren's syndrome (SS) is a chronic autoimmune disease targeting salivary and lacrimal glands. C-X-C motif chemokine ligand 10 (CXCL10) expression is upregulated in lip salivary glands (LSGs) of primary SS (pSS) patients, and CXCL10 involved in SS pathogenesis via immune-cell accumulation. Moreover, interferon (IFN)-γ enhances CXCL10 production via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. We investigated the effects of baricitinib, a selective JAK1/2 inhibitor, on both IFN-γ-induced CXCL10 production and immune-cell chemotaxis. We used immunohistochemical staining to determine the expression levels and localization of JAK1 and JAK2 in LSGs of SS patients (n = 12) and healthy controls (n = 3). We then evaluated the effect of baricitinib in an immortalized normal human salivary gland ductal (NS-SV-DC) cell line. Immunohistochemical analysis of LSGs from pSS patients revealed strong JAK1 and JAK2 expression in ductal and acinar cells, respectively. Baricitinib significantly inhibited IFN-γ-induced CXCL10 expression as well as the protein levels in an immortalized human salivary gland ductal-cell clone in a dose-dependent manner. Additionally, western blot analysis showed that baricitinib suppressed the IFN-γ-induced phosphorylation of STAT1 and STAT3, with a stronger effect observed in the case of STAT1. It also inhibited IFN-γ-mediated chemotaxis of Jurkat T cells. These results suggested that baricitinib suppressed IFN-γ-induced CXCL10 expression and attenuated immune-cell chemotaxis by inhibiting JAK/STAT signaling, suggesting its potential as a therapeutic strategy for pSS.

Sialendoscopy With Intraductal Steroid Irrigation in Patients With Sialadenitis Without Sialoliths.

Sialendoscopy has emerged as a safe, effective and minimally invasive technique for management of obstructive and inflammatory salivary gland disease. The aim of our study was to analyze outcomes of sialendoscopy and steroid irrigation in patients with sialadenitis without sialoliths. We performed a retrospective analysis of patients who underwent interventional sialendoscopy with steroid irrigation from 2013 to 2016, for the treatment of sialadenitis without sialolithiasis. Twenty-two patients underwent interventional sialendoscopy with ductal dilation and steroid irrigation for the treatment of sialadenitis without any evidence of sialolithiasis. Conservative measures had failed in all. Eleven patients had symptoms arising from the parotid gland, 4 patients had symptoms arising from the submandibular gland, while 6 patients had symptoms in both parotid and submandibular glands. One patient complained of only xerostomia without glandular symptoms. The mean age of the study group which included 1 male and 21 females was 44.6 years (range: 3-86 years). Four patients had autoimmune disease, while 7 patients had a history of radioactive iodine therapy. No identifiable cause for sialadenitis was found in the remaining 11 patients. The mean follow-up period was 378.9 days (range: 16-1143 days). All patients underwent sialendoscopy with ductal dilation and steroid irrigation. Twelve patients showed a complete response and 9 patients had a partial response, while 1 patient reported no response. Only 3 patients required repeat sialendoscopy. The combination of sialendoscopy with ductal dilation and steroid irrigation is a safe and effective treatment option for patients with sialadenitis without sialoliths refractory to conservative measures. Prospective studies with a larger case series are needed to establish its role as a definitive treatment option.

Cepharanthine Inhibits IFN-γ-Induced CXCL10 by Suppressing the JAK2/STAT1 Signal Pathway in Human Salivary Gland Ductal Cells.

Cepharanthine, a biscolaurine alkaloid isolated from the plant Stephania cephalantha Hayata, has been reported to have potent anti-inflammatory properties. Here, we investigated the effects of cepharanthine on the expression of CXCL10 (a CXC chemokine induced by interferon-gamma [IFN-γ] that has been observed in a wide variety of chronic inflammatory disorders and autoimmune conditions) in IFN-γ-treated human salivary gland cell lines. We observed that IFN-γ-induced CXCL10 production in NS-SV-DC cells (a human salivary gland ductal cell line), but not in NS-SV-AC cells (a human salivary gland acinar cell line). Cepharanthine inhibited the IFN-γ-induced CXCL10 production in NS-SV-DC cells. A Western blot analysis showed that cepharanthine prevented the phosphorylation of JAK2 and STAT1, but did not interfere with the NF-κB pathway. Moreover, cepharanthine inhibited the IFN-γ-mediated chemotaxis of Jurkat T cells. These results suggest that cepharanthine suppresses IFN-γ-induced CXCL10 production via the inhibition of the JAK2/STAT1 signaling pathway in human salivary gland ductal cells. Our findings also indicate that cepharanthine could inhibit the chemotaxis of Jurkat T cells by reducing CXCL10 production.

Epithelial disruptions, but not immune cell invasion, induced secretory dysfunction following innate immune activation in a novel model of acute salivary gland injury.

BACKGROUND: Salivary gland (SG) injurious agents are all translated into loss of salivation (xerostomia). An association has been established between activation of innate immunity and SG injury and dysfunction. However, it remains unclear how the secretory epithelia respond by halting saliva production. METHODS: C57BL/6 submandibular glands (SMGs) were acutely challenged using a single dose of the innate immune stimulant: polyinosinic-polycytidylic acid (poly (I:C)). Secretory capacity of the infected SMGs was substantiated by assessing the flow rate in response to pilocarpine stimulation. Depletion of the acute inflammatory cells was achieved by pre-treating mice with RB6-8C5 depletion antibody. Flow cytometry, histology and immunohistochemistry were conducted to verify the immune cell depletion. Epithelial expression of saliva-driving molecules: muscarinic 3 receptor (M3R), aquaporin 5 water channel (AQP5), Na-K-CL-Cotransporter 1 (NKCC1) and transmembrane member 16A (TMEM16A), was characterized using RT-qPCR and immunohistochemistry. Tight junction (TJ) protein; zonula occludens (ZO-1) and basement membrane (BM) protein; and laminin were assessed by immunohistochemistry. RESULTS: Innate immune challenge prompted dysfunction in the exocrine SGs. Dysregulated gene and protein expression of molecules that drive saliva secretion was substantiated. Aberrant expression of TJ and BM proteins followed innate immune activation. Hyposalivation in the current model was independent of myeloperoxidase (MPO)-positive, acute inflammatory cells. CONCLUSIONS: In this study, we developed a novel injury model of the SGs, featuring acute secretory dysfunction and immediate structural disruptions. Our results ruled out the injurious role of aggressively infiltrating inflammatory cells.

Apoptosis in Early Salivary Gland Duct Morphogenesis and Lumen Formation.

Salivary glands are essential for the maintenance of oral health by providing lubrication and antimicrobial protection to the mucosal and tooth surfaces. Saliva is modified and delivered to the oral cavity by a complex multifunctional ductal system. During development, these ducts form as solid tubes, which undergo cavitation to create lumens. Apoptosis has been suggested to play a role in this cavitation process along with changes in cell polarity. Here, we show that apoptosis occurs from the very earliest stages of mouse salivary gland development, much earlier than previously reported. Apoptotic cells were observed in the center of the first epithelial stalk at early-stage embryonic day 12.5 (E12.5) according to both TUNEL staining and cleaved caspase 3 immunofluorescence. The presumptive lumen space was highlighted by the colocalization of a predictive lumen marker, cytokeratin 7. At E14.5, as lumens start to form throughout the glands, apoptotic expression decreased while cytokeratin 7 remained positive. In vitro inhibition of all caspases in E12.5 and E13.5 salivary glands resulted in wider ducts, as compared with the controls, and a defect in lumen formation. In contrast, no such defect in lumen formation was observed at E14.5. Our data indicate that apoptosis is involved during early stages of gland formation (E12.5 onward) and appears important for shaping the forming ducts.

mRNA expression and localization of LPS-induced ß-defensin isoforms in rat salivary glands.

ß-defensins are small, cationic peptides with broad-spectrum antimicrobial activity that are produced by mucosal epithelia. However, little is known about the expression of ß-defensins in the major salivary glands. The purpose of this study was to characterize expression of rat ß-defensin-1 (RBD-1) and -2 (RBD-2) mRNA within the major salivary glands together with the effect of injection of intraductal lipopolysaccharide (LPS) on that expression. ß-defensin mRNA expression was quantitated by RT-PCR in salivary gland tissues and salivary acinar and striated duct cells collected by laser captured microdissection. RBD-1 and -2 were expressed in the parotid gland, the submandibular gland, and the sublingual gland. ß-defensins were expressed in both the acinar and striated duct cells of the major salivary glands. Intraductal injection of LPS increased expression of RBD-1 and -2 mRNA, which peaked at 12 hrs. These results suggest that salivary cells (acinar and striated duct cells) have the potential to produce ß-defensins.

Rapamycin delays salivary gland atrophy following ductal ligation.

Salivary gland atrophy is a frequent consequence of head and neck cancer irradiation therapy but can potentially be regulated through the mammalian target of rapamycin (mTOR). Excretory duct ligation of the mouse submandibular gland provokes severe glandular atrophy causing activation of mTOR. This study aims to discover the effects of blocking mTOR signaling in ligation-induced atrophic salivary glands. Following 1 week of unilateral submandibular excretory duct ligation: gland weights were significantly reduced, 4E-BP1 and S6rp were activated, and tissue morphology revealed typical signs of atrophy. However, 3 days following ligation with rapamycin treatment, a selective mTOR inhibitor, gland weights were maintained, 4E-BP1 and S6rp phosphorylation was inhibited, and there were morphological signs of recovery from atrophy. However, following 5 and 7 days of ligation and rapamycin treatment, glands expressed active mTOR and showed signs of considerable atrophy. This evidence suggests that inhibition of mTOR by rapamycin delays ligation-induced atrophy of salivary glands.

Epiregulin is critical for the acinar cell regeneration of the submandibular gland in a mouse duct ligation model.

Acinar cell regeneration from tubular structures has been reported to occur in duct-deligated salivary glands. However, the detailed process of acinar cell regeneration has not been clarified. We have developed a mouse duct ligation model to clarify the mechanisms underlying acinar cell regeneration, and we analyzed the epidermal growth factor receptor (EGFR) and epidermal growth factor (EGF) ligands using the model. We studied these ligands expressions in the course of acinar cell regeneration using immunohistochemistry and RT-PCR methods. In the duct-ligated portion of the submandibular gland (SMG) that underwent atrophy, newly formed acinar cells were observed arising from the tubular structures after the release of the duct obstruction. The constitutive expression of EGFR was observed by immunohistochemistry in both the duct-ligated and duct-deligated animals as well as in normal controls. The EGFR phosphorylation detected on the tubular structures after duct ligation paralleled the acinar cell regeneration. RT-PCR showed an increase in the epiregulin and heparin-binding EGF levels from day 0 to day 3 after the release of the duct obstruction. The EGF level was increased only after day 7. In vitro, cultured cells isolated from ligated SMGs proliferated and produced EGF ligands following the addition of epiregulin to the culture medium. These findings suggest that the tubular structures localized in an atrophic gland are the source of acinar cell regeneration of the salivary gland. The induction of EGF ligands, in particular epiregulin, may play an important role in acinar cell regeneration in this model.

Radioprotective effect of vitamin E in parotid glands: a morphometric analysis in rats.

The aim of this study was to evaluate the radioprotective effect of vitamin E on rat parotid glands by morphometric analysis. Sixty male rats were divided into 5 groups (n=6): control, in which animals received olive oil solution; olive oil/irradiated, in which animals received olive oil and were irradiated with a dose of 15 Gy of gamma radiation; irradiated, in which animals were irradiated with a dose of 15 Gy gamma radiation; vitamin E, which received α-tocopherol acetate solution; vitamin E/irradiated, which received α-tocopherol acetate solution before irradiation with a dose of 15 Gy gamma rays. Half of the animals were euthanized at 8 h, and the remaining at 30 days after irradiation. Both parotid glands were surgically removed and morphometric analysis of acinar cells was performed. Data were subjected to two-way ANOVA and Tukey's test (α=0.05). Morphometric analysis showed a significant reduction in the number of parotid acinar cells at 30 days in olive oil/irradiated and irradiated groups. In groups evaluated over time a significant reduction was shown at 30 days in olive oil/irradiated and irradiated groups, indicating that ionizing radiation caused tissue damage. The vitamin E/irradiated group presented more acinar cells than the irradiated group, but no statistically significant difference was observed (p>0.05). In conclusion, vitamin E seems to have failed as a radioprotective agent on acinar cells in rat parotid glands.

Radioprotective Effect of Vitamin E in Parotid Glands: a Morphometric Analysis in Rats

The aim of this study was to evaluate the radioprotective effect of vitamin E on rat parotid glands by morphometric analysis. Sixty male rats were divided into 5 groups (n=6): control, in which animals received olive oil solution; olive oil/irradiated, in which animals received olive oil and were irradiated with a dose of 15 Gy of gamma radiation; irradiated, in which animals were irradiated with a dose of 15 Gy gamma radiation; vitamin E, which received α-tocopherol acetate solution; vitamin E/irradiated, which received α-tocopherol acetate solution before irradiation with a dose of 15 Gy gamma rays. Half of the animals were euthanized at 8 h, and the remaining at 30 days after irradiation. Both parotid glands were surgically removed and morphometric analysis of acinar cells was performed. Data were subjected to two-way ANOVA and Tukey's test (α=0.05). Morphometric analysis showed a significant reduction in the number of parotid acinar cells at 30 days in olive oil/irradiated and irradiated groups. In groups evaluated over time a significant reduction was shown at 30 days in olive oil/irradiated and irradiated groups, indicating that ionizing radiation caused tissue damage. The vitamin E/irradiated group presented more acinar cells than the irradiated group, but no statistically significant difference was observed (p>0.05). In conclusion, vitamin E seems to have failed as a radioprotective agent on acinar cells in rat parotid glands.

O objetivo neste estudo foi avaliar o efeito radioprotetor da vitamina E sobre glândulas parótidas de ratos por meio de análise morfométrica. Sessenta ratos machos foram divididos em cinco grupos: controle, no qual os animais receberam solução de óleo de oliva; óleo de oliva irradiado, em que os animais receberam óleo de oliva e foram irradiados com uma dose de 15 Gy de radiação gama; irradiado, em que os animais foram irradiados com uma dose de 15 Gy de radiação gama; vitamina E, no qual receberam solução de acetato α-tocoferol; vitamina E irradiado, os quais receberam solução de acetato de α-tocoferol antes da irradiação com uma dose de 15 Gy de radiação gama. Metade dos animais foi eutanasiada em 8 h, e o restante aos 30 dias após a irradiação. Ambas as glândulas parótidas foram removidas cirurgicamente e análise morfométrica das células acinares foi realizada. Os dados foram submetidos à Análise de Variância com 2 fatores e teste de Tukey (α=0,05). A análise morfométrica mostrou uma redução significativa no número de células acinares da glândula parótida aos 30 dias nos grupos óleo irradiado e irradiado. Nos grupos avaliados ao longo do tempo uma redução significativa foi mostrada aos 30 dias nos grupos óleo irradiado e irradiado, indicando que a radiação ionizante causou danos teciduais. O grupo vitamina E/irradiado apresentou mais células acinares que o grupo irradiado, mas diferença estatisticamente significante não foi observada. Em conclusão, a vitamina E parece ter fracassado como um agente radioprotetor nas células acinares das glândulas parótidas de ratos.

Capsaicin regulates the NF-κB pathway in salivary gland inflammation.

Salivary gland epithelial cells (SGEC) release several cytokines that play important roles in the inflammatory process. In this study, we examined whether capsaicin can modulate cytokine release in SGEC. After cells were stimulated with polyinosinic-polycytidylic acid [poly(I:C)] or lipopolysaccharide (LPS), mRNA transcript and protein levels were detected by reverse-transcriptase-polymerase chain-reaction (RT-PCR), real-time PCR, and enzyme-linked immunosorbent assay (ELISA). These findings demonstrated that the increases in TNFα and IL-6 mRNA transcripts were highest at 3 hrs and 1 hr after incubation with poly(I:C) and LPS, respectively. Pre-treatment of the cells with 10 µµ capsaicin, however, significantly inhibited mRNA transcripts and its protein levels. The simultaneous application of 10 µµ capsazepine with capsaicin did not block the inhibitory effect of capsaicin. Furthermore, the inhibitory effect of capsaicin was also shown in primary cultured cells from TRPV1(-/-) mice. We found that both poly(I:C) and LPS induced IκB-α degradation and phosphorylation, which resulted in NF-κB activation, and capsaicin inhibited this NF-κB pathway. These results demonstrate that SGEC release pro-inflammatory cytokines mediated by TLR, and capsaicin inhibits this process through the NF-κB pathway. This study suggests that capsaicin could potentially alleviate inflammation in salivary glands.

[Intraductal application of Levovist® in salivary glands of animals]. / Intraduktale Applikation von Levovist® in Speicheldrüsenausführungsgänge am Tiermodell.

BACKGROUND: Several methods are well established for the imaging of salivary glands. Excluding the invasive method sialendoscopy all other methods show the salivary duct system inadequately. The aim of this study is to demonstrate a method to visualize the salivary duct system by B-mode ultrasound. MATERIAL AND METHODS: In 10 parotid glands of common pig cadavers the ultrasound contrast agent Levovist (®), which is galactose stabilized by palmitic acid was applied into the main salivary ducts while simultaneously performing a transcutaneous B-mode ultrasound. RESULTS: In all cadavers a visualization of the salivary duct system could be achieved by the application of Levovist (®) because of contrast enhancement. This effect arises as a result of an increased reflection of ultrasound waves on the surface of the microbubbles contained in the contrast agent. CONCLUSION: A reproducible visualization of the salivary duct system with B-mode ultrasound is possible by an intraductal application of an ultrasound contrast agent. In future this could be established as a reliable and fast method for imaging of the salivary ducts without ionizing radiation for the patient.

Effect of estrogen therapy, soy isoflavones, and the combination therapy on the submandibular gland of ovariectomized rats.

The objective of this study was to evaluate the effect of estrogen deficiency, estrogen therapy, and soy isoflavones on the salivary glands in female rats. Ninety-six animals were ovariectomized, and 24 were sham-operated. Among the ovariectomized rats, 24 received 17ß-estradiol; 24 received isoflavone extract; 24 received a combination therapy of both; and 24 received water as placebo. The submandibular glands were histomorphometrically analyzed. As a result, the ANOVA test revealed that the hormonal deficiency affected the acini and the ducts of ovariectomized rats, reducing their percentage compared to the sham group. All treatments caused an increase in ducts and acini compared to the placebo group. It was concluded that the estrogen deficiency may be related to salivary gland function due to a reduction in the quantity of salivary acini and ducts secondary to ovariectomy. The estrogen therapy, soy isoflavone therapy, and the combination of both are effective in reducing the effects of ovariectomy on the salivary glands.

Maintenance of paracellular barrier function by insulin-like growth factor-I in submandibular gland cells.

Insulin-like growth factor-I (IGF-I) is expressed in salivary glands. We examined the effects of IGF-I on cell number, the expression and distribution of tight junction proteins and the paracellular barrier function in cells derived from rat submandibular glands. When those cells were cultured in medium containing 10% foetal bovine serum (FBS) or IGF-I, the number of cells was comparable at 10 days. However, in the presence of inhibitor of IGF-I receptors, the number of cells cultured with FBS only was clearly reduced. The tight junction proteins occludin and claudin-3 were similarly detected by Western blotting in cells cultured with IGF-I or FBS. Immunostaining revealed that occludin and another tight junction protein (ZO-1) were similarly localized at intracellular junctions of cells cultured with IGF-I or FBS. The barrier functions were evaluated by transepithelial resistance (TER) and by FITC-dextran permeability. The TER values and FITC-dextran permeability of cells cultured with IGF-I or FBS were comparable. These observations suggest that IGF-I contributes to the maintenance not only of the cell number of salivary gland cells but also of their paracellular barrier function via the expression and distribution of tight junction proteins.

[Alpha-1-blockers (alfuzosin) for obstructive salivary gland diseases]. / Les Alpha-1-Bloquants (alfuzosine) en pathologie salivaire obstructive.

INTRODUCTION: Alpha-blockers are used in urology to treat stenosis and lithiasis. The pathophysiology is similar in salivary glands. We had for aim to assess the safety and effectiveness of an alpha-blocker (Alfuzosin) in patients with ductal stenosis, allergic pseudo-parotitis or sialolithiasis after lithotripsy. PATIENTS AND METHODS: Three hundred and fifty-two patients were included, 194 of whom presented with sialolithiasis fragmented by extracorporeal lithotripsy (112 parotidic and 82 submandibular). Sixty-nine presented with ductal stenosis, and 89 with allergic pseudo-parotitis. This retrospective study lasted 3 years (January 2005 to January 2008) with a mean follow-up of 33 months (18 months to 4 years). Male patients were given 2.5mg tid of the alpha-blocker Alfuzosin and female patients 2.5mg bid for 3 to 24 months. After 6 months and up to 2 years of treatment, patients were assessed every 3 months by US and with a questionnaire on symptoms. RESULTS: Results were similar in male and female patients. Eighty percent of patients with colic-like pain due to stenosis reported a significant improvement after treatment. 78.6% of patients with allergic pseudo-parotitis felt they had improved and noted a sharp decrease of pruritus. Sixty-seven of the patients with residual parotid lithiasis after extracorporeal lithotripsy presented with less ductal lithiasis and fragments were evacuated more rapidly in the two months following lithotripsy. Forty-two percent of the patients treated for residual submandibular lithiasis reported a significant functional improvement and faster evacuation of fragments. Twelve patients out of 352 (3.4%) reported adverse effects. The incidence of orthostatic hypotension was 2.2%. DISCUSSION: A significant improvement of symptoms was observed in patients treated with Alfuzosin for obstructive salivary gland diseases. The drug was well tolerated. These preliminary results are good in terms of effectiveness and inocuity. They should be confirmed with a prospective controlled study.

Androgens and integrins in salivary glands in Sjogren's syndrome.

OBJECTIVE: Laminin alpha1-chain normally induces intercalated duct progenitors to differentiate to acinar cells through integrin (INT) alpha1ss1 and alpha2ss1 receptors. Maintenance of acinar cells is impaired in Sjögren's syndrome (SS), which is also characterized by low levels of serum and salivary androgens. We hypothesized that androgens normally support salivary gland remodeling by upregulating either laminin alpha1 chain or its cellular alpha1 or alpha2 INT subunit-containing receptors. METHODS: Intercalated duct and acinar human salivary gland (HSG) cells and labial salivary gland (LSG) biopsies from healthy controls and patients with SS were cultured without or with sex steroids. Laminin alpha1 chain and INT alpha1 and alpha2 subunits were studied using quantitative reverse-transcription real-time polymerase chain reaction and INT alpha1 and alpha2 subunits using immunofluorescence staining. RESULTS: INT alpha1-subunit and alpha2-subunit messenger RNA (mRNA) levels were increased in intercalated duct and acinar cells by DHEA and testosterone. In contrast, laminin alpha1-chain mRNA levels were not affected. The upregulating effect of DHEA on INT subunits was also seen at the protein level. DHEA also increased mRNA levels of both INT subunits in healthy but not SS LSG. CONCLUSION: Androgens increased INT alpha1 and alpha2 subunits in tubuloepithelial cells and in healthy LSG, but in SS salivary glands this androgen regulation was defective, which is likely to contribute to defective outside-in signaling, acinar atrophy, and ductal cell hyperplasia.

Peptidergic modulation of serotonin and nerve elicited responses of the salivary duct muscle in the snail, Helix pomatia.

In the present study, the ability of a range of endogenous neuropeptides to modulate neuromuscular transmission was examined in the salivary duct neuromuscular preparation of the terrestrial snail, Helix pomatia. Immunohistochemical and physiological techniques were used to localize the neuropeptides (GSPYFVamide, CARP, FMRFamide and APGWamide) and to investigate whether contractions elicited by the stimulation of the salivary nerve or by exogenously applied 5-HT are subject to peptidergic modulation. All of the neuropeptides studied decreased the tonus by a direct action on the muscle fibers in a concentration dependent manner in a range of 10(-9) to 10(-6)M. Neuropeptides distinctly affected the 5-HT evoked contraction or relaxation and GSPYFVa and APGWa decreased also the amplitude of contractions elicited by the stimulation of the salivary nerve. All four neuropeptides facilitated the relaxation phase providing further evidence for the postsynaptic action of neuropeptides. Low Ca(2+)/high Mg(2+) saline abolished the nerve-elicited contractions, however the denervated muscle retained the ability to contract due to the mobilization of the Ca(2+) from intracellular stores. It was concluded, that peptides belonging to different peptide families exerted their effects through pre- and postsynaptic mechanisms. The modulatory effect of neuropeptides can be assigned to the partial co-localization of 5-HT and neuropeptides in the nerves innervating muscles of the salivary duct, as it was demonstrated by double-labeling immunohistochemistry. A double origin of the 5-HTergic innervation was demonstrated, including efferents originating from both the cerebral and visceral ganglia.

Spontaneous oscillations in intracellular Ca(2+) concentration via purinergic receptors elicit transient cell swelling in rat parotid ducts.

Using multiphoton microscopy, we established that rat parotid ductal cells exhibit spontaneous oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). These oscillatory Ca(2+) responses were observed during continuous perfusion with a physiological salt solution at 37 degrees C in the absence of calcium mobilizing agonist stimulation. The timing and patterns of these spontaneous Ca(2+) oscillations varied among individual ductal cells, and the average number of Ca(2+) responses in a single responding ductal cell was 2.1 in a 10-min recording period. High-speed scanning (0.6 s/image) revealed that most spontaneous elevations in [Ca(2+)](i) were initiated at the luminal side of ductal cells and spread toward the basal side within 2 s. Electron microscopic analysis after Ca(2+) imaging indicated that spontaneously oscillating ducts contained numerous granules at the luminal side, which is characteristic of granular ducts. These Ca(2+) oscillations were completely blocked by the purinergic receptor inhibitors 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS) and suramin but were not blocked by the muscarinic antagonist atropine or the alpha-adrenergic antagonist phentolamine. Simultaneous observation of fura-2 fluorescence and differential interference contrast (DIC) images showed that spontaneous elevations of [Ca(2+)](i) were well correlated with changes in shape of ductal cells. Using a plasma membrane fluorescence probe, SynaptoGreen C4, we found that the changes in DIC images reflected spontaneous cell swelling of ductal cells. Our findings present the possibility that purinergic receptors mediate spontaneous Ca(2+) oscillations in parotid ductal cells and regulate electrolyte reabsorption from the primary saliva in the resting state.

Activation of innate immune responses through Toll-like receptor 3 causes a rapid loss of salivary gland function.

BACKGROUND: Recent studies have demonstrated the expression of Toll-like receptor 3 (TLR3) in salivary glands and epithelial cell lines derived from Sjögren's syndrome (SS) patients. As viral infections are considered to be a trigger for SS, in this study we investigated whether in vivo engagement of TLR3 affects salivary gland function. METHODS: Female New Zealand Black/WF1 mice were repeatedly injected with polyinosinic:polycytidylic acid [poly(I:C)]. TLR3 expression within submandibular glands was studied using immunohistochemistry. RNA levels of inflammatory cytokines in the submandibular glands were determined by real time polymerase chain reaction. Pilocarpine induced saliva volume was used as an index of glandular function. RESULTS: Immunohistochemical analysis of submandibular glands showed TLR3 expression in epithelium of serous and mucous acini, granular convoluted tubules, and ducts. Poly(I:C) treatment rapidly up-regulated the mRNA levels of type I interferon (IFN) and inflammatory cytokines in the submandibular glands. One week after treatment, the saliva volumes in poly(I:C) treated mice were significantly reduced in comparison with the phosphate-buffered saline (PBS) treated mice. Hematoxylin and eosin staining showed that salivary gland histology was normal and lymphocytic foci were not detected. Glandular function recovered after poly(I:C) treatment was stopped. CONCLUSIONS: Our results demonstrate that engagement of TLR3 within the salivary glands results in a rapid loss of glandular function. This phenomenon is associated with the production of type I IFN and inflammatory cytokines in the salivary glands. Restoration of glandular function suggests that for viral etiology of SS, a chronic infection of salivary glands might be necessary.

Immunocytochemical study of granular duct cells with a hormonally enhanced granular cell phenotype in the mouse parotid gland.

In the parotid glands (PGs) of intact male mice (12 weeks of age, ICR strain), immunofluorescence labels for a true tissue kallikrein, mK1, and for nerve growth factor (NGF) were recognized through the subluminal edges of the striated duct (SD) segments and interlobular duct segments. Because of their small size, secretory granules were not detectable by light microscopy in any of the duct cells. Full-fledged granular cells, containing large secretory granules that were visible by light microscopy, were induced in the SD segments of male mice after the injection of 5alpha-dehydrotestosterone (DHT) and triiodothyronine (T(3)), given either alone or in combination every other day for 2 weeks. A stronger effect was detected in the mice that were concomitantly injected with DHT and T(3), and more abundant, fully developed granular cells appeared in the SD segments of these mice. These full-fledged granular cells were immunoreactive for mK1, NGF, and epidermal growth factor, but not for renin. The present results indicate that some of the SD cells with small granules in the mouse PG can develop a granular cell phenotype, producing more kinds of growth factors, as a result of the actions of androgen and thyroid hormone.