Sclerosing sialadenitis of the submandibular gland is rarely an immunoglobulin G4-related disease in the Finnish population.

Chronic sclerosing sialadenitis may represent one of many manifestations of an immunoglobulin G4-related disease. However, existing studies typically consist of small patient cohorts rarely conducted in Western populations. The clinical behavior of chronic sclerosing sialadenitis, including follow-up data, warrants further study. Thus, we aimed to determine whether chronic sclerosing sialadenitis always presents as IgG4-related disease or associates with autoimmune diseases and to determine which additional examinations patients may require. Between 2000 and 2017, 51 patients undergoing submandibular gland resection within the Helsinki University Hospital area were diagnosed with chronic sclerosing sialadenitis. We re-evaluated all specimens and performed immunostaining for IgG4. IgG and CD31 stainings were performed for IgG4-positive specimens. IgG4-related disease diagnosis was defined by the Boston consensus statement criteria. We revised clinical data, distributing a follow-up questionnaire to patients to register symptoms of IgG4-related disease or autoimmune disease during follow-up. The chronic sclerosing sialadenitis criteria were fulfilled in 34 patients, whereby 17 were diagnosed as non-sclerosing chronic sialadenitis. In 19 cases, a sialolith associated with a salivary gland lesion. In total, 12 of 51 cases were recognized as IgG4-positive, while two met the criteria for IgG4-related disease. These two cases belonged to the non-sclerosing chronic sialadenitis group, and both involved other organs. The histopathological features between chronic sclerosing sialadenitis and non-sclerosing chronic sialadenitis overlapped regarding the degree of fibrosis and inflammatory infiltrates. In the Finnish population, chronic sclerosing sialadenitis of the submandibular gland does not appear to present as IgG4-related disease. Non-sclerosing chronic sialadenitis can associate with IgG4-related disease. A histopathological distinction between chronic sclerosing sialadenitis and non-sclerosing chronic sialadenitis is not always unequivocal and the presence of a sialolith does not exclude IgG4-positivity. Therefore, immunostaining for IgG4 should be performed when dense plasma cell infiltration is present in either non-sclerosing chronic sialadenitis or chronic sclerosing sialadenitis.

Chronic stress-induced elevation of IL-1ß in the saliva and submandibular glands of mice.

Psychological stress is involved in the development of various oral diseases. Alterations in the levels of cytokines in the saliva of patients with stress-related oral diseases have been reported. However, the inconsistencies in the results of these studies might be attributed to differences in the local and systemic factors in the oral cavities of the patients. We examined the effect of chronic stress on three major inflammatory cytokines Interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the saliva and salivary glands of chronically stressed mice. Six-week-old C57BL/6 J mice were randomly divided into a control and a stress group. The mice in stress group were exposed to 4 h of stress daily for 10 days and subsequently saliva, as well as the submandibular glands, were collected from both groups. The expression levels of cytokines in the saliva were examined by enzyme-linked immunosorbent assay. The submandibular glands were subjected to histopathological and mRNA expression analyses. IL-1ß was significantly elevated in saliva of the chronic stressed mice. Furthermore, the mRNA expression levels of both IL-1ß and IL-6 were significantly elevated in the submandibular gland of chronic stressed mice. IL-1ß may be a potential salivary biomarker in response to chronic stress in mice.

The G-Protein-Coupled Receptor ALX/Fpr2 Regulates Adaptive Immune Responses in Mouse Submandibular Glands.

Lipoxin receptor (ALX)/N-formyl peptide receptor (FPR)-2 is a G-protein-coupled receptor that has multiple binding partners, including the endogenous lipid mediators resolvin D1, lipoxin A4, and the Ca2+-dependent phospholipid-binding protein annexin A1. Previous studies have demonstrated that resolvin D1 activates ALX/Fpr2 to resolve salivary gland inflammation in the NOD/ShiLtJ mouse model of Sjögren syndrome. Moreover, mice lacking the ALX/Fpr2 display an exacerbated salivary gland inflammation in response to lipopolysaccharide. Additionally, activation of ALX/Fpr2 has been shown to be important for regulating antibody production in B cells. These previous studies indicate that ALX/Fpr2 promotes resolution of salivary gland inflammation while modulating adaptive immunity, suggesting the need for investigation of the role of ALX/Fpr2 in regulating antibody production and secretory function in mouse salivary glands. Our results indicate that aging female knockout mice lacking ALX/Fpr2 display a significant reduction in saliva flow rates and weight loss, an increased expression of autoimmune-associated genes, an up-regulation of autoantibody production, and increased CD20-positive B-cell population. Although not all effects were noted among the male knockout mice, the results nonetheless indicate that ALX/Fpr2 is clearly involved in the adaptive immunity and secretory function in salivary glands, with further investigation warranted to determine the cause(s) of these between-sex differences.

Submandibular gland-specific inflammaging-induced hyposalivation in the male senescence-accelerated mouse prone -1 line (SAM-P1).

Aging has pronounced effects on mammalian tissues and cells, but the impacts of aging on salivary gland function are relatively unknown. This study aims to evaluate the effects of aging on submandibular gland (SMG) and parotid gland (PG) functions in the male senescence-accelerated mouse. In vivo analysis at the systemic level revealed that salivary secretion induced by pilocarpine, a muscarinic agonist, from the SMG was significantly decreased in aged mice, whereas salivary secretion from the PG was not affected. To evaluate organ-level function, the SMG was perfused with the muscarinic agonists carbachol and calcium ionophore A23187 ex vivo to induce salivary secretion, and decreased saliva production was also observed in the aged SMG. Histological analysis revealed the presence of CD4-positive lymphocytes infiltrating the aged SMG. Furthermore, real-time PCR revealed that the aged SMG exhibited accelerated cell aging, increased levels of the inflammatory cytokine interleukin-6, and decreased mRNA levels of the water channel protein aquaporin-5 (AQP5). In summary, these results demonstrate that SMG function in aged mice was diminished, and that cell senescence, chronic inflammation, and the decreased gene expression of AQP5 are the likely causes of hyposalivation in the SMG of aged mice.

Cutting Edge: A Critical Role of Lesional T Follicular Helper Cells in the Pathogenesis of IgG4-Related Disease.

IgG4-related disease (IgG4-RD) is a newly recognized systemic chronic fibroinflammatory disease. However, the pathogenesis of IgG4-RD remains unknown. To determine the pathophysiologic features of IgG4-RD, we examined T follicular helper (Tfh) cells in lesions and blood from patients with IgG4-RD. Patients with IgG4-related dacryoadenitis and sialadenitis (IgG4-DS) showed increased infiltration of Tfh cells highly expressing programmed death 1 and ICOS in submandibular glands. Tfh cells from IgG4-DS submandibular glands had higher expression of B cell lymphoma 6 and a greater capacity to help B cells produce IgG4 than did tonsillar Tfh cells. We also found that the percentage of programmed death 1hi circulating Tfh cells in IgG4-DS patients was higher than that in healthy volunteers and was well correlated with clinical parameters. Our findings indicate that anomalous Tfh cells in tissue lesions of IgG4-RD have features distinct from those in lymphoid counterparts or blood and potentially regulate local IgG4 production in IgG4-RD.

The synergistic effect of NOD2 and TLR4 on the activation of autophagy in human submandibular gland inflammation.

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.

NFIL3 Expression Distinguishes Tissue-Resident NK Cells and Conventional NK-like Cells in the Mouse Submandibular Glands.

The submandibular salivary gland (SMG), a major site of persistent infection for many viruses, contains a large NK cell population. Using NFIL3-deficient mice, PLZF reporter/fate mapping mice, and mixed bone marrow chimeras, we identified two distinct populations of NK cells in the SMG. Although phenotypically unique, the main population relies on NFIL3, but not PLZF, for development and, therefore, is developmentally similar to the conventional NK cell subset. In contrast, we found that approximately one quarter of the SMG NK cells develop independently of NFIL3. Interestingly, NFIL3-independent SMG tissue-resident NK (trNK) cells are developmentally distinct from liver trNK cells. We also demonstrated that the SMG NK cell hyporesponsive phenotype during murine CMV infection is tissue specific and not cell intrinsic. In contrast, NFIL3-independent SMG trNK cells are intrinsically hyporesponsive. Altogether, our data show that the SMG tissue environment shapes a unique repertoire of NK-like cells with distinct phenotypes.

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.

Interaction between innate immunity and Ro52-induced antibody causes Sjögren's syndrome-like disorder in mice.

OBJECTIVES: Autoantibodies reactive with Ro52 are often found in sera of patients with Sjögren's syndrome (SS). This study was undertaken to investigate the role of Ro52-induced immune responses in pathogenesis of SS. METHODS: New Zealand Mixed (NZM) 2758 mice were immunised with Ro52 in alum adjuvant. Control mice were immunised either with maltose-binding protein or injected with alum alone. Mice were monitored for anti-Ro52 antibody, sialoadenitis and pilocarpine-induced salivation. Antibody binding to salivary gland (SG) cells was analysed in vivo and in vitro by immunofluorescence. Sera from immunised mice were passively transferred into untreated or alum injected NZM2758 mice. RESULTS: By day 30 post-immunisation, Ro52 immunised mice generated immunoprecipitating anti-Ro52 antibodies and they had the maximum drop in saliva production. Both Ro52 immunised and control mice showed evidence of mild sialoadenitis. However, only Ro52 immunised mice had antibody deposition in their SG. Passive transfer of Ro52-immune sera induced SG dysfunction in recipient mice, only if the recipients were primed with alum. In vitro, antibodies from Ro52-immune sera were internalised by a SG cell line and this uptake was inhibited by cytochalasin D treatment. CONCLUSIONS: Our data show for the first time that antibodies induced by Ro52 are capable of inducing SG dysfunction, and that this phenomenon is dependent on the activation of innate immunity. The mouse model described in this study implies that autoantibody deposition in the SG might be an important step in the induction of xerostomia and pathogenesis of SS.

Male-specific submaxillary gland protein, a lipocalin allergen of the golden hamster, differs from the lipocalin allergens of Siberian and Roborovski dwarf hamsters.

BACKGROUND: An increasing number of asthma cases upon exposure to hamsters and anaphylactic reactions following hamster bites are being reported, but the allergens responsible are still poorly characterized. In the Golden hamster, male-specific submaxillary gland protein (MSP), a lipocalin expressed in a sex- and tissue-specific manner in the submaxillary and lacrimal glands, is secreted in the saliva, tears and urine. The purpose of this study was to determine if MSP is an allergen, to identify IgE-reactive proteins of different hamster species and to analyse potential cross-reactivities. METHODS: Fur extracts were prepared from four hamster species. Hamster-allergic patients were selected based on a history of positive IgE-test to hamster epithelium. The IgE-reactivity of patients' sera was investigated by means of immunoblot and ELISA. IgE-reactive proteins in fur extracts and the submaxillary gland were identified using anti-MSP antibodies, Edman sequencing or mass spectrometry. MSP was purified from Golden hamster and recombinant MSP was expressed in E. coli. RESULTS: Four patients had IgE-antibodies against 20.5-kDa and 24-kDa proteins of Golden hamster fur extract, which were identified as MSP. IgE-reactive MSP-like proteins were detected in European hamster fur extract. Three patient sera showed IgE-reactive bands at 17-21 kDa in Siberian and Roborovski hamster fur extracts. These proteins were identified as two closely related lipocalins. Immunoblot inhibition experiments showed that they are cross-reactive and are different from MSP. CONCLUSION: MSP lipocalin of the Golden hamster was identified as an allergen, and it is different from the cross-reactive lipocalin allergens of Siberian and Roborovski hamsters. Our findings highlight the need for specific tools for the in vitro and in vivo diagnosis of allergy to different hamster species.

Intake of indigestible carbohydrates influences IgA response and polymeric Ig receptor expression in the rat submandibular gland.

Secretory IgA in the saliva is essential for protection from mucosally transmitted pathogens and maintaining homeostasis at mucosal surfaces of the oral cavity. Expression of submandibular gland polymeric Ig receptor (pIgR) is essential for IgA secretion. In the present study, we investigated the influence of indigestible carbohydrates on IgA production in the salivary gland and saliva. Five-week-old rats were fed a fibre-free diet (control), or a diet with 5 % (w/w) fructo-oligosaccharide (FOS) or a combination of 2·5 % (w/w) polydextrose (PDX) and 2·5 % (w/w) lactitol for 21-d. IgA concentrations in the caecal digesta, submandibular gland tissue, and saliva in the FOS and PDX+lactitol diet groups were significantly higher than those in the control group (P< 0·05). The increase in IgA in the submandibular gland tissue was confirmed using immunohistochemical analysis. However, the IgA concentrations of serum did not differ between the FOS or PDX+lactitol groups and the control group (P= 0·5). In the FOS and PDX+lactitol groups, the pIgR mRNA (pIgR/ß-actin) expression level in the submandibular gland tissue was significantly higher than that in the control group (P< 0·05). The present study suggests that indigestible carbohydrates play an important role in the increase in IgA concentrations in the submandibular gland tissue, saliva, and caecal digesta.

Different stages of primary Sjogren's syndrome involving lymphotoxin and type 1 IFN.

Primary Sjögren's syndrome (pSS) is a complex autoimmune disease starting in the salivary and lacrimal glands and continuing to involve the lungs and kidneys with the eventual development of lymphoma. Many studies have emphasized the role of type 1 IFN (IFN-α) and lymphotoxin α (LTα) in the pathogenesis of the disease. The present studies were designed to delineate the role of IFN-α in pSS using an animal model, the IL-14α (IL14αTG) transgenic mouse. IL14αTG mice lacking the type 1 IFNR (IL14αTG.IFNR(-/-)) had the same submandibular gland and lacrimal gland injury as did the IL14αTG mice, but they lacked the later parotid gland and lung injury. Development of lymphoma was delayed in IL14αTG.IFNR(-/-) mice. The switch from IgM to IgG autoantibodies as well as the increase in serum IgG2a seen is IL14αTG mice was inhibited in IL14αTG.IFNR(-/-) mice. Production of LTα was identified in both IL14αTG mice and IL14αTG.IFNR(-/-) mice at the time that salivary gland injury was occurring. These and previous studies suggest a model for pSS that separates the disease into several stages: 1) initial injury to the submandibular and lacrimal glands via an environmental insult and LTα; 2) amplification of local injury via the production of type 1 IFN; injury to the parotid glands, lungs, and kidneys is seen; 3) progression of systemic inflammation with the eventual development of large B cell lymphoma. Understanding these different stages will help to develop strategies for treatment of patients with pSS based on the status of their disease.

STIM1 and STIM2 protein deficiency in T lymphocytes underlies development of the exocrine gland autoimmune disease, Sjogren's syndrome.

Primary Sjögren's Syndrome (pSS) is an autoimmune disease involving salivary and other exocrine glands that leads to progressive lymphocytic infiltration into the gland, tissue damage, and secretory defects. The mechanism underlying this disease remains poorly understood. Here we report that mice with T-cell-targeted deletion of Stromal Interaction Molecule (STIM) 1 and STIM2 [double-knockout (DKO)] mice develop spontaneous and severe pSS-like autoimmune disease, displaying major hallmarks of the disease. In DKO mice, diffuse lymphocytic infiltration was seen in submandibular glands, a major target of pSS, by age 6 wk, progressing to severe inflammation by age 12 wk. Sjögren's syndrome-specific autoantibodies (SSA/Ro and SSB/La) were detected in the serum, and progressive salivary gland destruction and loss of fluid secretion were also seen. Importantly, we report that peripheral blood mononuclear cells as well as lymphocytic infiltrates in submandibular glands from patients with pSS demonstrated significant reductions in STIM1 and STIM2 proteins. Store-operated calcium entry was also reduced in peripheral blood mononuclear cells from pSS patients compared with those from healthy controls. Thus, deficiency of STIM1 and STIM2 proteins in T cells, and consequent defects in Ca(2+) signaling, are associated with salivary gland autoimmunopathy in DKO mice and pSS patients. These data reveal a previously unreported link between STIM1 and STIM2 proteins and pSS.

T helper 2 and regulatory T-cell cytokine production by mast cells: a key factor in the pathogenesis of IgG4-related disease.

IgG4-related disease is a systemic disorder with unique clinicopathological features and uncertain etiological features and is frequently related to allergic disease. T helper 2 and regulatory T-cell cytokines have been reported to be upregulated in the affected tissues; thus, the production of these cytokines by T helper 2 and regulatory T cells has been suggested as an important factor in the pathogenesis of IgG4-related disease. However, it is not yet clear which cells produce these cytokines in IgG4-related disease, and some aspects of the disorder cannot be completely explained by T-cell-related processes. To address this, we analyzed paraffin-embedded sections of tissues from nine cases of IgG4-related submandibular gland disease, five cases of submandibular sialolithiasis, and six cases of normal submandibular gland in order to identify potential key players in the pathogenesis of IgG4-related disease. Real-time polymerase chain reaction analysis confirmed the significant upregulation of interleukin (IL)4, IL10, and transforming growth factor beta 1 (TGFß1) in IgG4-related disease. Interestingly, immunohistochemical studies indicated the presence of mast cells expressing these cytokines in diseased tissues. In addition, dual immunofluorescence assays identified cells that were double-positive for each cytokine and for KIT, which is expressed by mast cells. In contrast, the distribution of T cells did not correlate with cytokine distribution in affected tissues. We also found that the mast cells were strongly positive for IgE. This observation supports the hypothesis that mast cells are involved in IgG4-related disease, as mast cells are known to be closely related to allergic reactions and are activated in the presence of elevated non-specific IgE levels. In conclusion, our results indicate that mast cells produce T helper 2 and regulatory T-cell cytokines in tissues affected by IgG4-related disease and possibly have an important role in disease pathogenesis.

E-cadherin promotes accumulation of a unique memory CD8 T-cell population in murine salivary glands.

The salivary glands are important effector sites for IgA-mediated humoral immunity to protect oral surfaces. Within murine submandibular glands (SMG), we identified a memory CD8 T-cell population that exhibited a unique cell-surface phenotype distinct from memory CD8 T cells in spleen but similar to memory T cells resident in the intraepithelial lymphocyte compartment of the intestinal mucosa. In mice immune to lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus(VSV), virus-specific memory CD8 T cells with this unusual phenotype were present in SMG at remarkably high frequencies. LCMV-specific memory CD8 T cells in SMG showed potent functional activities in vivo, including cytokine-induced bystander proliferation, antigen-triggered IFNγ production, and viral clearance. Adoptive transfer experiments further revealed that the capacity to accumulate in SMG decreased during CD8 T-cell differentiation and that SMG CD8 T cells were poorly replenished from the circulation, indicating that they were tissue-resident. Moreover, they preferentially relocalized within their tissue of origin after adoptive transfer and antigen rechallenge, thus revealing an imprinted differentiation status. Accumulation of memory CD8 T cells within SMG did not require local antigen presentation but was promoted by the epithelial differentiation molecule E-cadherin intrinsically expressed by these CD8 T cells. This finding extends the epithelial-restricted function of E-cadherin to an impact on lymphocyte accumulation within epithelial tissues.

Autoantibodies in primary Sjögren's syndrome patients induce internalization of muscarinic type 3 receptors.

OBJECTIVES: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by lymphocyte infiltration into the salivary and lachrymal glands, leading to dry mouth and eyes. The presence of functional autoantibodies against muscarinic type 3 receptor (M3R) has been reported in pSS patients. However, the pathological role of anti-M3R autoantibodies in pSS salivary dysfunction remains controversial. METHODS: Purified IgGs were obtained from normal (control) and primary SS patients' sera (pSS IgG). Internalization of M3R and clathrin was analyzed by biochemical assay and immunofluorescence confocal microscopy using human submandibular gland (hSMG) cells. Cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) was measured by microspectrofluorimetry. RESULTS: Incubation of hSMG cells with pSS IgG (1mg/ml) significantly decreased M3R expression levels at the membrane. Carbachol-induced [Ca(2+)](i) transients (CICTs) in these cells were also inhibited by pSS IgG. In contrast to pSS IgG, control IgG had no effect on both the M3R expression level and CICTs. We found that binding of pSS IgG to M3R induces phosphorylation of the receptor, and that the pSS IgG-induced M3R internalization is prevented by the lysosomal inhibitor, chloroquine. In addition, pSS IgG decreased membrane clathrin expression, which was inhibited by atropine. Our immunofluorescence study further confirmed that pSS IgG induces a co-localization of M3R with clathrin and subsequent internalization of M3R. CONCLUSION: pSS IgG induces internalization of M3R partly through a clathrin-mediated pathway. The results suggest M3R internalization as a potential mechanism to explain the exocrinopathy seen in pSS patients.

IgG4-related sialadenitis is rare: histopathological investigation of 129 cases of chronic submandibular sialadenitis.

AIMS: The incidence of IgG4-related sialadenitis among cases of chronic sialadenitis is unknown, and so we investigated the presence of IgG4 plasma cells in 129 specimens from an archival collection of consecutive cases of chronic submandibular sialadenitis collected from 1969 to 1989 that had been previously extensively characterized. METHODS AND RESULTS: Immunohistology revealed that only three of the 129 specimens contained areas over the threshold for IgG4-related sialadenitis of 50 IgG4 plasma cells per high-power field, and these cells were part of a non-specific chronic inflammatory infiltrate associated with ducts that had contained sialoliths. The infiltrate of IgG4 plasma cells in the series was significantly positively related to the total infiltrate of inflammatory cells, fibrosis, atrophy, lymphoid germinal centres and sialoliths. CONCLUSIONS: IgG4-related sialadenitis is rare and was not found in the present series. The IgG4 plasma cells that were present in the glands were part of a non-specific chronic inflammatory infiltrate.

[Effect of yangyin yiqi huoxue recipe on immune balance of Th1/Th2 in serum and submaxillary glands of NOD mice with Sjogren's syndrome].

OBJECTIVE: To observe the effect of Yangyin Yiqi Huoxue Recipe (YYHR) on expression of interferon-gamma (IFN-gamma), IL-2, IL-4, IL-10, and immune balance of Th1/Th2 in serum and submaxillary glands of non-obese diabetic (NOD) mice with Sjogren's syndrome (SS), and to explore its mechanisms. METHODS: Totally 32 NOD mice were randomly into 4 groups, i.e., the model group, the Chinese medicine group [CM, administered with YYHR at the dose of 0.4 mL by gavage (100 g/kg)], the Western medicine group [WM group, administered with hydroxychloroquine 0.4 mL by gavage (60 mg/kg)], and the combined group [administered with YYHR (50 g/kg) and hydroxychloroquine (60 mg/kg) 0.4 mL by gavage], 8 mice in each group. Eight Balb/C mice were recruited as the normal control group. Mice were sacrificed to withdraw blood after 8 weeks. The submaxillary gland was excised. Serum levels of IFN-gamma, IL-2, IL-4, and IL-10 were detected by ELISA. Protein expression of IFN-gamma, IL-2, IL-4, and IL-10 in submaxillary glands was detected by SP method. RESULTS: Compared with the normal group, levels of IFN-gamma, IL-2, IL-4, and IL-10 in serum and submaxillary glands all increased in the model group (P < 0.05). Levels of IFN-gamma and IL-10 in serum in the CM group and the combined group were lower than those of the WM group (P < 0.05). Serum levels of IFN-gamma and IL-2 in submaxillary glands in combined group were lower than those of the WM group (P < 0.05). The ratio of IFN-gamma/IL-4 in serum and submaxillary glands in the model group were higher than that of the rest groups (P < 0.05). Besides, it was the nearest to that of the normal group. CONCLUSIONS: YYHR could decrease the levels of Th1 and Th2 related cytokines and the ratio of IFN-gamma/IL-4 in serum and submaxillary glands of NOD mice with SS. It could achieve therapeutic effects through adjusting immune balance of Th1/Th2 in SS mice.

Lipoxin A4 inhibits immune cell binding to salivary epithelium and vascular endothelium.

Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1ß- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sjögren's syndrome (SS).

Local suppression of IL-21 in submandibular glands retards the development of Sjögren's syndrome in non-obese diabetic mice.

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.