Autoantibodies in Sjögren's syndrome patients acutely inhibit muscarinic receptor function

Oral Diseases (2012) 18 , 132–139 Objectives: Autoantibodies from the sera of Sjögren’s syndrome patients (SS IgG) have been suggested to inhibit muscarinic receptor function. However, the acute nature of such an inhibitory effect remains controversial. In this study, we investigated the acute effects of SS IgG on muscarinic receptor function in human submandibular gland (HSG) cells. Methods: The effects of autoantibodies on muscarinic receptor function were studied using microspectrofluorimetry, whole‐cell patch clamp, immunofluorescence confocal microscopy, and a co‐immunoprecipitation assay. Results: Carbachol (CCh) was found to consistently increase intracellular calcium concentration ([Ca²⁺]_i) and activate K⁺ current in HSG cells. However, pretreatment of the cells with SS IgG for 5 or 30 min significantly attenuated these responses, with a substantially more prominent effect after 30 min of treatment. Like CCh, adenosine 5′‐triphosphate (ATP) also increased [Ca²⁺]_i and activated K⁺ currents in HSG cells, although pretreatment with SS IgG did not affect the cellular response to ATP. CCh was found to reorganize α‐fodrin in HSG cells in a Ca²⁺‐dependent manner. However, pretreatment with SS IgG prevented the cytoskeletal reorganization of α‐fodrin induced by CCh. Conclusions: SS IgG acutely and reversibly inhibited muscarinic receptor function, thereby inhibiting the Ca²⁺ mobilization necessary for the activation of K⁺ currents and α‐fodrin reorganization in HSG cells.     

Gene‐expression profile and localization of Na+/K+‐ATPase in rat enamel organ cells

The sodium pump Na⁺/K⁺‐ATPase, expressed in virtually all cells of higher organisms, is involved in establishing a resting membrane potential and in creating a sodium gradient to facilitate a number of membrane‐associated transport activities. Na⁺/K⁺‐ATPase is an oligomer of α, β, and γ subunits. Four unique genes encode each of the α and β subunits. In dental enamel cells, the spatiotemporal expression of Na⁺/K⁺‐ATPase is poorly characterized. Using the rat incisor as a model, this study provides a comprehensive expression profile of all four α and all four β Na⁺/K⁺‐ATPase subunits throughout all stages of amelogenesis. Real‐time PCR, western blot analysis, and immunolocalization revealed that α1, β1, and β3 are expressed in the enamel organ and that all three are most highly expressed during late‐maturation‐stage amelogenesis. Expression of β3 was significantly higher than expression of β1, suggesting that the dominant Na⁺/K⁺‐ATPase consists of an α1β3 dimer. Localization of α1, β1, and β3 subunits in ameloblasts was primarily to the cytoplasm and occasionally along the basolateral membranes. Weaker expression was also noted in papillary layer cells during early maturation. Our data support that Na⁺/K⁺‐ATPase is functional in maturation‐stage ameloblasts.     

Follicular dendritic cells confirm lymphoid organization in the minor salivary glands of primary Sjögren’s syndrome

Background: Sjögren’s syndrome (SS) is an autoimmune chronic inflammatory disorder affecting the salivary and lacrimal glands. The aim of this study was to explore immunophenotypic features of chronic inflammatory reactions in the minor salivary glands in patients with primary SS (pSS). Methods: Formalin‐fixed, paraffin‐embedded labial minor salivary gland tissue sections from randomly selected patients with pSS (n = 60) were investigated for the expression of CD21, CD23, CD35 and IgD by immunohistochemistry. Results: Based on the distribution and staining pattern of CD21, CD23, CD35 and IgD in lymphoid aggregates, several stages of chronic inflammatory reactions were observed. In 12/60 (20%) patients, lymphoid infiltrates with germinal centre (GC)‐like features such as extensive networks of CD21‐, CD23‐ and CD35‐positive cells were observed in the minor salivary gland tissue. Smaller networks and /or focal infiltrates with scattered CD21⁺, CD23⁺ and CD35⁺ cells were observed in the remaining 48/60 (80 %) cases. When dividing patients according to the presence (GC+) or the absence (GC?) of GC in the minor salivary glands, the mean focus score was significantly higher in the GC+ patients (P < 0.05). Double staining of the minor salivary glands revealed focal infiltrates with follicular dentritic cell networks and B cells resembling normal GCs in tonsillar tissue. Conclusion: A particular cellular profile was demonstrated in a sub‐group of patients with pSS and could be linked to serological aberrations. These findings warrant further prospective studies.     

Oral distress in primary Sjögren's syndrome: implications for health-related quality of life

Enger TB, Palm Ø, Garen T, Sandvik L, Jensen JL. Oral distress in primary Sjögren’s syndrome: implications for health‐related quality of life.
Eur J Oral Sci 2011; 119: 474–480. © 2011 Eur J Oral Sci The aims of the study were to evaluate oral distress in patients with primary Sjögren’s syndrome (pSS) compared with age‐ and sex‐matched Norwegian normative data, to estimate the occurrence of oral symptoms in pSS, and to evaluate the impact of oral distress on health‐related quality of life (HRQoL). The Medical Outcomes Study 36‐Item Short‐Form Health Survey (SF‐36) was used to assess HRQoL, and the Oral Health Impact Profile 14 (OHIP‐14) was used to measure oral distress. Of the 246 pSS patients invited to participate in the study, 177 (72%) responded. Data were analysed for the female participants (n = 163). Significant deviations from normative estimates were found in all OHIP‐14 item results, and the findings indicated a high level of oral distress among the pSS patients. Health‐related quality of life was decreased among pSS patients, with the largest deviations from normative estimates related to general health and role physical. The patients with high levels of oral distress scored significantly lower than patients with low levels of oral distress in five of the SF‐36 subscales, indicating that oral conditions have a marked impact on general quality of life. In conclusion, oral distress in pSS is pronounced and severe, and should receive increased attention with a view to improving the quality of life for these patients.     

Anti-M(3) muscarinic cholinergic autoantibodies from patients with primary Sjögren's syndrome trigger production of matrix metalloproteinase-3 (MMP-3) and prostaglandin E(2) (PGE(2)) from the submandibular glands

Background

We demonstrated that serum immunoglobulin G (IgG) from patients with primary Sjögren's syndrome (pSS), interacting with the second extracellular loop of human glandular M₃ muscarinic acetylcholine receptors (M₃ mAChR), trigger the production of matrix metalloproteinase-3 (MMP-3) and prostaglandin E₂ (PGE₂).

Methods

Enzyme-linked immunosorbent assays (ELISAs) were performed in the presence of M₃ mAChR synthetic peptide as antigen to detect in serum the autoantibodies. Further, MMP-3 and PGE₂ production were determined in the presence of anti-M₃ mAChR autoantibodies.

Results

An association was observed between serum and anti-M₃ mAChR autoantibodies and serum levels of MMP-3 and PGE₂ in pSS patients. Thus, we established that serum anti-M₃ mAChR autoantibodies, MMP-3 and PGE₂ may be considered to be early markers of pSS associated with inflammation. Affinity-purified anti-M₃ mAChR peptide IgG from pSS patients, whilst stimulating salivary-gland M₃ mAChR, causes an increase in the level of MMP-3 and PGE₂ as a result of the activation of phospholipase A₂ (PLA₂) and cyclooxygenase-2 (COX-2) (but not COX-1).

Conclusions

These results provide a novel insight into the role that cholinoceptor antibodies play in the development of glandular inflammation. This is the first report showing that an antibody interacting with glandular mAChR can induce the production of pro-inflammatory mediators (MMP-3/PGE₂).     

Local suppression of IL‐21 in submandibular glands retards the development of Sjögren’s syndrome in non‐obese diabetic mice

J Oral Pathol Med (2012) 41 : 728–735 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.     

Primary Sjögren syndrome in a 2-year-old patient: role of the dentist in diagnosis and dental management with a 6-year follow-up

International Journal of Paediatric Dentistry 2011; 21: 471–475 Background. Primary Sjögren syndrome is a rare autoimmune disease, especially in children, mainly affecting girls (77%), and usually diagnosed around 10 years of age. Diagnosis during childhood is difficult, especially because of the diversity of the clinical presentation and difficulty obtaining reliable history data, accounting for a higher frequency of underdiagnosed cases. Differential conditions should be considered, especially the ones that promote xerostomia, such as diabetes, ectodermal dysplasia, rheumatoid arthritis, scleroderma, systemic lupus erythematosus, sarcoidosis, lymphoma, HIV and HTLV infection. Conditions associated with parotid enlargement should also be excluded, including juvenile recurrent parotitis (JRP), sialadenosis, sarcoidosis, lymphoma, infectious parotitis caused by streptococcal and staphylococcal infections, viral infections (paramyxovirus, Epstein–Barr virus, cytomegalovirus, and parvovirus), and diffuse infiltrative lymphocytosis syndrome (associated with HIV infection), and rare congenital conditions, such as polycystic parotid disease. Case report. A paediatric female patient was referred to our clinic for dental treatment complaining about dry mouth, oral discomfort, and dysphagia. The patient presented five of the required criteria to establish the diagnosis of pSS, including ocular symptoms, oral symptoms, evidence of keratoconjunctivitis sicca, focal sialadenitis confirmed by minor salivary gland biopsy, and evidence of major salivary gland involvement. Our patient did not have positive SS‐A and SS‐B autoantibodies. According to the literature, about 29% of individuals with pSS can present seronegativity for SS‐A (anti‐Ro) antibodies and about 33% can present seronegativity for SS‐B (anti‐La) antibodies. Conclusion. To the best of our knowledge, this is the youngest patient reported in the scientific English literature with pSS. Primary Sjögren syndrome has a wide clinical and immunologic spectrum and may progress with increased morbidity. Clinicians must be aware of the development of pSS in such an early age and exclude all possible differential findings to provide early diagnosis and treatment.     

Submandibular secretion in the dog after intraluminal injections of amiloride

The effects of amiloride on flow rate, osmolarity, Na⁺, K⁺ and Cl^? concentrations were studied in the submandibular glands of dogs. The drug was injected in a retrograde fashion into the duct system of the gland, which was subsequently stimulated to secrete with pilocarpine. In concentrations of 10^?4 and 10^?3 M, amiloride increased the Na⁺ concentration but did not significantly alter the K⁺ concentration of saliva. A slight decrease in Cl^? concentration was observed at high flow rates after 10^?4 M amiloride, but otherwise the drug did not significantly affect the Cl^? concentration of stimulated salivary secretion. The diuretic has a measurable mucosal (luminal) effect on Na⁺ transport in dog submandibular gland, which is similar to that reported in amphibian epithelia. Its lack of effect on K⁺ excretion differs from findings reported in other epithelial structures, where it has a marked K⁺-sparing effect. The drug increased the salivary osmolarity in a fashion similar to Na⁺, a finding that suggests a dissociation between Na⁺ and water transport in salivary duct epithelium.     

Primary Sjögren''s syndrome: salivary gland function and clinical oral findings

OBJECTIVE: To evaluate salivary gland function, saliva composition and oral findings in patients with primary Sjogren's syndrome (pSS) subdivided into patients with and without focus score ≤1 (FS) and/or antibodies to SSA/SSB (AB) as well as in healthy controls. SUBJECTS AND METHODS: Unstimulated (UWS) and chewing stimulated (SWS) whole saliva, and stimulated parotid saliva (SPS) were collected in 16 patients fulfilling the European classification criteria for pSS subdivided into those with FS and/or AB (n= 8) and those without FS and AB (n= 8), and in age-matched (n= 14) and young healthy controls (n= 13).UWS and SWS were analysed for Na⁺ and K⁺.SPS was analysed for Na⁺, K⁺, statherin, and proline-rich proteins (PRPs).Sicca symptoms, DMFT/DMFS, plaque (PI) and gingival (GI) scores, periodontal pocket depth (PPD), and mucosal status were recorded. RESULTS: The young healthy controls had lower UWS as compared to the aged controls (P= 0.03).However, the aged controls had higher DMFT/DMFS (P < 0.001) and PI, GI and PPD (P < 0.01).Patients with FS and/or AB generally had lower saliva secretory rates than patients without FS and/or AB (P= 0.01 for UWS and SPS) and age-matched healthy controls (P= 0.001). There was no significant difference in the content of Na⁺ and K⁺, statherin and PRPs between groupS. Patients with FS and/or AB had the highest frequency of oral mucosal changes and higher DMFT/DMFS than patients without FS and/or AB and healthy controls (P < 0.01).However, PI, GI, and PPD did not differ significantly. CONCLUSION: Patients with FS and/or AB had lower salivary secretory rates, higher DMFT/DMFS, and more oral mucosal changes than patients without FS and/or AB.Additionally, data suggest that salivary gland function in healthy individuals do not decrease with age.     

Characterization of a microsomal fraction from rabbit parotid gland: Partial separation of alkaline phosphatase from Na,K-ATPase

Subfractionation of the 140,000 g_max pellet from the rabbit parotid gland revealed 3 fractions. Both linear and discontinuous sucrose-density gradients were used to separate a region of high-density particles with considerable mitochondria, a middle region rich in Na⁺,K⁺-ATPase (sodium-potassium activated adenosine triphosphatase) and a low-density (top) region with elevated alkaline phosphatase. The top and middle fractions appeared to represent subfractions of plasma-membrane enriched membranes. Treatment of these 2 gradient fractions with low concentrations of sodium dodecyl sulphate confirmed that the middle fraction was preferentially enriched in Na⁺,K⁺-ATPase. Thus, alkaline phosphatase-enriched membranes are at least partially separable from Na⁺,K⁺-ATPase-enriched membranes. Separation of alkaline phosphatase from Na⁺,K⁺-ATPase may be an important purification step in future work involving isolation of plasma membranes of secretory cells, as both alkaline phosphatase and Na⁺,K⁺-ATPase have been used as plasma-membrane markers in the parotid gland. The present findings demonstrate that considerable caution should be exercised in defining the purity of plasma-membrane fractions from the parotid gland in view of the heterogeneity of the plasma-membrane enriched fraction.     

Calcium-stimulated ATPase of dog submandibular gland

A Ca^2?-stimulated ATPase present in a microsomal fraction prepared from canine submandibular glands was investigated. The Ca²⁺ concentration for half maximal activation of the enzyme was about 0.3 mM. Addition of Mg²⁺ to incubation media containing Ca²⁺ decreased the ATPase activity. The presence of neither Na⁺ nor K⁺ is required for Ca²⁺-activation of the enzyme. Also, Ca²⁺ will not substitute for Mg²⁺ in the Mg²⁺-dependent (Na⁺ + K⁺)-ATPase reaction. The Ca²⁺-activation was not appreciably affected by ouabain (10^?4M), but was inhibited by about 50 per cent by 5 × 10^?3M ethacrynic acid. These studies provide a possible enzymatic basis for the calcium uptake by salivary gland microsomes that has been reported by other workers.     

Sialochemical markers of salivary gland involvement with Sjögren''s syndrome secondary to rheumatoid arthritis and primary biliary cirrhosis

Abstract: Sjögren’s syndrome is an autoimmune condition affecting the lacrimal and salivary glands and can be associated with rheumatoid arthritis and primary biliary cirrhosis. Parotid salivas collected from patients and normal controls were analysed for lactoferrin, IgA and beta₂‐microglobulin (measured by ELISA), and cystatin (measured by a enzyme inhibition assay). Output data provided less variable means, whilst expressing results as a proportion of the total protein provided greater specificity as markers for Sjögren’s syndrome. Levels of specificity for IgA, lactoferrin and beta₂‐microglobulin were all high (100, 95 and 100%, respectively). Sensitivity levels of these markers (but not cystatin) tended to be similar for Sjögren’s syndrome secondary to primary biliary cirrhosis (IgA, 25%; lactoferrin, 63%; and beta₂‐microglobulin, 50%), compared to Sjögren’s syndrome secondary to connective tissue diseases such as rheumatoid arthritis (IgA, 50%; lactoferrin, 86%; and beta₂‐microglobulin; 38%).     

Fcγ receptors mediate internalization of anti-Ro and anti-La autoantibodies from Sjögren''s syndrome and apoptosis in human salivary gland cell line A-253

Background: The presence of serum anti‐Ro and anti‐La autoantibodies directed against the ribonucleoproteins Ro and La has been associated with Sjögren’s syndrome (SS), an autoimmune rheumatic disease that targets salivary and lachrymal glands. There is increasing evidence of the direct involvement of autoantibodies in the pathogenesis of tissue injury and correlation of their presence with clinical manifestations in SS. The focus of this work was to explore the cellular apoptotic pathway triggered by binding and penetration of anti‐Ro and anti‐La autoantibodies in human salivary gland cell line A‐253 and to identify the membrane receptors through which anti‐Ro and anti‐La could exert their effect. Methods: Anti‐Ro and anti‐La autoantibodies were purified from IgG fractions, obtained from eleven healthy volunteers and patients with primary Sjögren’s syndrome, using Sepharose 4B‐Ro and Sepharose 4B‐La affinity columns. Flow cytometry, RT‐PCR, western blot and confocal microscopy analysis were used to visualize the FCγRI, FCγRII and FCγRIII receptors on the A‐253 cell membrane. DNA laddering and western blot analysis of caspases activation were studied to evaluate in A‐253 cells treated with anti‐Ro and anti‐La autoantibodies. Results: The results yeilded the evidence of the presence of members of the Fcγ receptors (FcγRs) family on the cell membrane of the human salivary gland cell line A‐253. Furthermore, we demonstrated that, in the A‐253 cell line, anti‐Ro and anti‐La autoantibodies can access the cells probably through Fcγ receptors, and trigger apoptotis. Conclusions: We conclude that anti‐Ro and anti‐La autoantibodies have pathogenic effects that could depend on binding to Fcγ receptors.     

Reproducibility of biopsy grade in Sjögren''s syndrome

Abstract: The purpose of this study was to examine the reproducibility of biopsy grades at various tissue depths in Sjögren’s syndrome. The biopsy grades of 38 minor salivary gland biopsies were examined at 6 μm, 50 μm, 100 μm, 150 μm, 200 μm, and 250 μm tissue depths. Tissue sections were stained with routine hematoxylin and eosin, graded I–IV, and compared with the initial “baseline” biopsy grade. The majority of the biopsies showed a wide range of grade variability at all depths. No tissue depth was consistently reproducible for any grade (P0.41, 0.64, 0.91, and 0.20, respectively). The difference between baseline grades and grades of deeper sections was sufficient to impact the diagnosis of Sjögren’s syndrome in approximately 60% of the biopsies (P<0.001). The overall result of this study suggests that examination of multiple sections of minor salivary gland biopsies is advisable to improve the reliability of the grade when evaluating Sjögren’s syndrome.     

Intracellular Ca2+ responses and cell volume regulation upon cholinergic and purinergic stimulation in an immortalized salivary cell line

Aure MH, Røed A, Kanli Galtung H. Intracellular Ca ²⁺ responses and cell volume regulation upon cholinergic and purinergic stimulation in an immortalized salivary cell line. Eur J Oral Sci 2010; 118: 237–244. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci The water channel aquaporin 5 (AQP5) seems to play a key role in salivary fluid secretion and appears to be critical in the cell volume regulation of acinar cells. Recently, the cation channel transient potential vanilloid receptor 4 (TRPV4) was shown to be functionally connected to AQP5 and also to cell volume regulation in salivary glands. We used the Simian virus 40 (SV40) immortalized cell line SMG C10 from the rat submandibular salivary gland to investigate the effect of ATP and the neurotransmitter analogue carbachol on Ca²⁺ signalling and cell volume regulation, as well as the involvement of TRPV4 in the responses. We used fura‐2‐AM imaging, cell volume measurements, and western blotting. Both carbachol and ATP increased the concentration of intracellular Ca²⁺, but no volume changes could be measured. Inhibition of TRPV4 with ruthenium red impaired both ATP‐ and carbachol‐stimulated Ca²⁺ signals. Peak Ca²⁺ signalling during hyposmotic exposure was significantly decreased following inhibition of TRPV4, while the cells’ ability to volume regulate appeared to be unaffected. These results show that in the SMG C10 cells, simulation of nervous stimulation did not induce cell swelling, although the cells had intact volume regulatory mechanisms. Furthermore, even though Ca²⁺ signals were not needed for this volume regulation, TRPV4 seems to play a role during ATP and carbachol stimulation.     

Salivary gland hypofunction induced by activation of innate immunity is dependent on type I interferon signaling

Background: Activation of innate immunity through polyinosinic:polycytidylic acid [poly(I:C)] causes acute salivary gland hypofunction. As a major consequence of poly(I:C) treatment is type I interferon (IFN) production, this study was undertaken to investigate their role in salivary gland dysfunction. Methods: Different strains of mice deficient in either interferon alpha receptor (IFNAR1^?/?) or IL‐6^?/?, or IL‐10^?/?, or EBI3^?/? were treated with poly(I:C). Salivary gland function was determined by measuring pilocarpine‐induced saliva volume. Gene expression levels were measured by real‐time PCR. Ca²⁺ mobilization studies were performed using ex‐vivo acinar cells. Results: A single injection of poly(I:C) rapidly induced salivary gland hypofunction in wild‐type B6 mice (41% drop in saliva volumes compared to PBS‐treated mice). In contrast, the loss of function in poly(I:C)‐treated IFNAR^?/? mice was only 9.6%. Gene expression analysis showed reduced levels of Il‐6, Il‐10, and Il‐27 in submandibular glands of poly(I:C)‐treated IFNAR^?/? mice. While salivary gland dysfunction in poly(I:C)‐treated IL‐10^?/? and EBI3^?/? mice was comparable to wild‐type mice, the IL‐6^?/? mice were more resistant, with only a 21% drop in function. Pilocarpine‐induced Ca²⁺ flux was significantly suppressed in acinar cells obtained from poly(I:C)‐treated wild‐type mice. Conclusions: Our data demonstrate that a combined action of type I IFNs and IL‐6 contributes toward salivary gland hypofunction. This happens through interference with Ca²⁺ mobilization within acinar cells. Thus, in acute viral infections and diseases like Sjögren’s syndrome, elevated levels of type I IFNs and IL‐6 can directly affect glandular function.     

Human β‐defensin‐3 up‐regulates cyclooxygenase‐2 expression and prostaglandin E2 synthesis in human gingival fibroblasts

Chotjumlong P, Khongkhunthian S, Ongchai S, Reutrakul V, Krisanaprakornkit S. Human β‐defensin‐3 up‐regulates cyclooxygenase‐2 expression and prostaglandin E ₂ synthesis in human gingival fibroblasts. J Periodont Res 2010; 45: 464–470. © 2010 John Wiley & Sons A/S Background and Objective: Oral epithelial cells express three antimicrobial peptide human β‐defensins (hBDs) that have previously been demonstrated to exert proinflammatory effects on various immune cells. We wanted to examine whether hBDs could induce cyclooxygenase‐2 (COX‐2) expression and prostaglandin E₂ (PGE₂) synthesis in non‐immune cells, such as human gingival fibroblasts. Material and Methods: Cultured fibroblasts were treated with different concentrations of hBD‐1, ‐2, ‐3 or interleukin‐1β, as a positive control, for various times, in the presence or absence of NS‐398, a specific COX‐2 inhibitor. The levels of COX‐1 and COX‐2 mRNA expression were analyzed using RT‐PCR and real‐time PCR. Whole cell lysates were analyzed for COX‐1 and COX‐2 protein expression by western blotting. Cell‐free culture supernatants were assayed for PGE₂ levels by ELISA. The lactate dehydrogenase assay was performed to determine the cytotoxicity of hBDs. Results: Ten and 40 μg/mL of hBD‐3 up‐regulated COX‐2 mRNA and protein expression, consistent with COX‐2 up‐regulation by interleukin‐1β, whereas hBD‐1 and hBD‐2 did not. However, COX‐1 mRNA and protein were constitutively expressed. The time‐course study revealed that hBD‐3 up‐regulated COX‐2 mRNA and protein expression at 6 and 12 h, respectively. Consistent with COX‐2 up‐regulation, 10 and 40 μg/mL of hBD‐3 significantly increased PGE₂ levels in cell‐free culture supernatants (p < 0.05), and this was inhibited by NS‐398 in a dose‐dependent manner. Neither of the hBD concentrations tested in this study was toxic to the cells. Conclusion: These findings indicate that epithelial human β‐defensin‐3 functions as a proinflammatory mediator in controlling arachidonic acid metabolism in underlying fibroblasts.     

Autonomic receptors in the developing submandibular glands of neonatal rats

The functional maturation of the gland during the neonatal period involves specific temporal sequences in the appearance of autonomic membrane receptors and associated changes in the secretory response to receptor stimulation. The density and affinity of cholinergic muscarinic and of α- and β-adrenergic receptors were determined in the glands of 1-, 7-, 14- and 21-day-old rats, using the radioligands [³H]-quinuclidinyl benzylate, [³H]-prazosin and [³H]-dihydroalprenolol for the measurement of muscarinic cholinergic, α₁-adrenergic and β-adrenergic receptors, respectively. The density of binding sites followed similar developmental courses, whether expressed as $\text{pmol}\text{g}$ tissue or $\text{pmol}\text{g}$ protein. The densities ($\text{pmol}\text{g}$ protein) of β-adrenergic and muscarinic receptors were low in 1-day-old animals, but increased rapidly; adult levels were reached by 2 and 3 weeks of age, respectively. α₁-Adrenergic receptor binding was barely detectable at birth, increased slightly during the first week, dramatically by 14 days and approached adult levels by 21 days. The number of receptors per gland for these 3 autonomic receptor-binding sites increased 50 – 100-fold during the first 3 weeks of postnatal development. The affinities (K_D) for each of the three ligands did not differ significantly with age. Surprisingly, the α₂-adrenergic receptor density ($\text{[}{}^3\text{H]-p-}\text{aminoclonidine}$ binding) was high in 1-day-old animals and increased significantly during the first 2 weeks of life. The binding declined after 3 weeks and was nearly undetectable by 6 weeks. Frozen submandibular glands had markedly lower α₂-adrenergic binding compared to fresh glands. These findings suggest that each of the autonomic receptors in rat submandibular glands follows its own specific developmental pattern, and that the appearance of individual receptor types may correlate with the ability of the developing gland to respond to specific stimulants. A K⁺-release response can be elicited by epinephrine at 2 weeks of age, when α₁ receptor binding sites appear, while both the receptor binding sites and the response to muscarinic agonists are present at birth. As β-adrenergic receptor-binding sites may develop simultaneously with isoproterenol-stimulated adenylate-cyclase activity, it seems that receptor density is important in this response. The functional significance of the high density of α₂-adrenergic receptors in the submandibular gland of neonatal animals is not clear.     

Carbachol improves secretion in the early phase after rabbit submandibular gland transplantation

Oral Diseases (2010) 16 , 351–359 Objectives: To investigate the changes in the muscarinic receptor signaling pathway with submandibular gland (SMG) transplantation and whether carbachol improves secretion in transplanted SMGs. Materials and methods: SMG autotransplantation was performed in a rabbit model. Carbachol (1 μM) was infused into the transplanted glands from postoperative day 1–7. The expression of the M1 and M3 muscarinic receptors, aquaporin‐5 (AQP5), and phosphorylated extracellular signal‐regulated kinase 1/2 (p‐ERK1/2) was measured by RT‐PCR, immunoblotting or immunofluorescence. The content of inositol 1, 4, 5‐trisphosphate (IP₃) was measured by radioimmunoassay. Results: Salivary flow of the transplanted SMGs was decreased after transplantation. As well, the expressions of M1 and M3 receptors and their downstream signaling molecules, IP₃, p‐ERK1/2 and AQP5, were all reduced. Atrophy of acinar cells was shown in transplanted glands. However, all these alterations were reversed after carbachol treatment for 7 days. Furthermore, carbachol directly increased the mRNA expression of AQP5 and phosphorylation of ERK1/2 in cultured neonatal rabbit SMG cells. Conclusion: A lack of acetylcholine and downregulation of the muscarinic receptor signaling pathway is involved in the early hypofunction of transplanted SMGs. Carbachol treatment could be a new therapeutic strategy to improve secretion and prevent the obstruction of Wharton’s duct in the early phase after SMG transplantation.     

Loss of PKCδ results in characteristics of Sjögren's syndrome including salivary gland dysfunction

Oral Diseases (2011) 17 , 601–609 Objectives: Chronic infiltration of lymphocytes into the salivary and lacrimal glands of patients with Sjögren’s Syndrome (SS) leads to destruction of acinar cells and loss of exocrine function. Protein kinase C‐delta (PKCδ) is known to play a critical role in B‐cell maintenance. Mice in which the PKCδ gene has been disrupted have a loss of B‐cell tolerance, multiple organ lymphocytic infiltration, and altered apoptosis. To determine whether PKCδ contributes to the pathogenesis of SS, we quantified changes in indicators of SS in PKCδ?/? mice as a function of age. Salivary gland histology, function, the presence of autoantibodies, and cytokine expression were examined. Materials and methods: Submandibular glands were examined for the presence of lymphocytic infiltrates, and the type of infiltrating lymphocyte and cytokine deposition was evaluated by immunohistochemistry. Serum samples were tested by autoantibody screening, which was graded by its staining pattern and intensity. Salivary gland function was determined by saliva collection at various ages. Results: PKCδ?/? mice have reduced salivary gland function, B220+ B‐cell infiltration, anti‐nuclear antibody production, and elevated IFN‐γ in the salivary glands as compared to PKCδ+//+ littermates. Conclusions: PKCδ?/? mice have exocrine gland tissue damage indicative of a SS–like phenotype.