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Background: Sialadenitis is a nonneoplastic disease that causes salivary dysfunction. Autophagy may be involved in helping protect salivary function when the salivary gland is impaired; this process is primarily activated by sensors of innate immunity, such as Toll-like receptors and nucleotide-binding oligomerization domain (NOD)-like receptors. The role of these pattern recognition receptors (PRRs) in the regulation of salivary gland tissue defense and homeostasis has been underappreciated. This study hypothesized that NOD2 and TLR4 have a synergistic effect on the activation of autophagy in human submandibular gland (HSG) inflammation., Methods: Submandibular gland inflammation was modeled by treating HSG cell lines in vitro with muramyl dipeptide (MDP) and lipopolysaccharide (LPS) for 24 hours. The mRNA and protein expression of NOD2, TLR4 and autophagy-related proteins (ATG5, LC3, Beclin1) were evaluated by real-time PCR and Western blot. Immunohistochemistry and double immunofluorescence were used to analyze the presence, distribution and colocalization of the aforementioned indicators in HSG tissues., Result: The mRNA and protein expression of autophagy-related proteins were significantly increased in HSG cells costimulated with LPS and MDP for 24 hours. NOD2, TLR4 and the autophagy-related proteins were also highly expressed in residual acini and dilated ducts of chronic submandibular sialadenitis tissues. In addition, PRRs and autophagy markers were obviously colocalized in chronic submandibular sialadenitis tissues and HSG cells., Conclusion: TLR4 and NOD2 have unique expression sites in salivary glands, and they may synergistically activate autophagy in salivary glands under conditions of inflammation., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The aim of this study was to verify the validity of IL-21 local suppression in submandibular glands of preventing the development of Sjögren's syndrome in non-obese diabetic (NOD) mice and figure out the mechanism., Methods: IL-21 levels in submandibular glands were suppressed by ductal cannulation of IL-21 shRNA lentivirus. Then, saliva flow rates (SFR) and histopathologic changes of submandibular glands were measured to assess the severity of disease development. Real-time PCR, flow cytometry, and immunohistochemistry were used to detect the changes of T helper cells and related cytokines., Results: The reduction in SFRs in NOD mice was significantly alleviated from 9 to 17 weeks of age along with the suppression of IL-21 in submandibular glands. Lymphocytic infiltration was also milder than control NOD mice. Moreover, the lower level of IL-21 led to the down-regulation of follicular helper T (Tfh) cells., Conclusions: Local suppression of IL-21 in submandibular glands could retard the development of Sjögren's syndrome in NOD mice. IL-21 might contribute to the development of B-cell disorder in Sjögren's syndrome via Tfh cells pathway., (© 2012 John Wiley & Sons A/S.)

Wegener's granulomatosis is one of the anti-cytoplasmic autoantibodies (ANCA)-associated vasculitides. Although it typically affects the lungs and kidneys, the head and neck are also involved in most cases but the site usually affected is the upper airway. However, there are 35 cases with well-documented clinicopathological data in which Wegener's granulomatosis manifested in the major salivary glands, most commonly the parotid. Twenty-four patients presented with salivary signs and symptoms, in eight of whom there was no other presenting manifestation. These signs and symptoms may mimic infection or neoplasia and laboratory investigations, including ANCA serology and histopathology, may be non-specific; thus, in 21 of the 35 patients (60%) there was a delay in diagnosis. Amongst the 21 were 11 of the 14 (78.6%) patients who presented with unilateral parotid disease and three of the five who died. Three other patients suffered permanent pulmonary, two renal and five facial nerve damage. This article reviews the literature on major salivary gland involvement in Wegener's granulomatosis, which should be considered in the differential diagnosis of major salivary gland disease particularly if commoner causes have been excluded. A detailed medical history, and persistently inconclusive laboratory tests, could provide the clues that enable prompt diagnosis., (© 2012 John Wiley & Sons A/S.)

Background: The maintenance mechanisms of peripheral tolerance by CD4(+)CD25(+) T cells before the development of sialoadenitis in secondary Sjögren's syndrome (sSS) are not well understood. The aim of the present study is to examine the effect of reduction of CD4(+)CD25(+) T cells on the development of sialoadenitis during the early life in female NZB x NZWF(1) (B/WF(1)) mice, a model for human sSS., Methods: Female B/WF(1) mice at 3 days after birth were treated with either anti-mouse CD4(+)CD25(+) T cells rat IgG(1) monoclonal antibody (mAb) or Rat IgG(1)(control). At 25 weeks of age, autoantibodies against nucleus and cytoplasm of ductal epithelial and myoepithelial cells, and histpathology of submandibular glands were examined in the mAb-treated and control groups. Also the development of anti-Ro/SS-A antibodies was examined until 25 weeks of age in both groups., Results: The mAb-treated group showed severe lesions with the development of autoantibodies compared to the control group., Conclusions: The present results suggest that peripheral CD4(+)CD25(+) T cells may, at least in part, contribute to down-regulate the development of sialoadenitis in submandibular glands of lupus-prone female B/WF(1) mice during their early life.

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.

Expression of transgenes from adenoviral vectors is short-lived in salivary glands, in part because of immune responses to the virus and/or transgene product. Previous studies demonstrated that depletion of macrophages with multilamellar liposomes containing clodronate (Cl2MBP) increases adenoviral-mediated transgene expression in the liver. This technique was tested in salivary glands. Rats were treated with Cl2MBP-liposomes or control liposomes by femoral vein, intraperitoneal, or carotid artery injections. Thereafter, a recombinant adenovirus, AdCMVluciferase, was instilled intraductally in submandibular glands (SMGs), or delivered to the liver via femoral vein injection. Marked depletion (>94%) of liver macrophages and increased levels of luciferase activity in the liver (45-fold higher than controls) were present in animals receiving Cl2MBP-liposomes. In contrast, the same treatment never depleted more than 41% of SMG macrophages nor increased luciferase activity in SMGs, regardless of the route of administration. In conclusion, while macrophage depletion with Cl2MBP-liposomes is associated with markedly increased adenoviral-mediated transgene expression, this strategy was ineffective for salivary glands.

A human monoclonal antibody, BM2, was produced by a hybridoma line generated by fusion of lymph node cells from a patient with squamous cell carcinoma (SCC) of the tongue with human B-lymphoblastoid cell line HO-323. BM2, an IgM class antibody, was reactive with all of the SCC cell lines tested. Frozen sections of normal and malignant tumor specimens were investigated to examine the reactivity of BM2 towards them. All 35 oral SCC specimens reacted with BM2. Normal stratified squamous epithelium showed positive staining in basal cells, but no staining was seen in other layers of the stratified epithelium, simple epithelium, and tissues of nonepithelial origin. Ductal basal cells of normal salivary gland also showed positive staining. Western blotting and immunoprecipitation analysis revealed that BM2 recognized 52 kDa membrane-associated protein. BM2 may therefore be a useful tool for biological and clinical studies of SCC.