Antimicrobial peptide hCAP‐18/LL‐37 protein and mRNA expressions in different periodontal diseases

Oral Diseases (2010) 17 , 60–67 Objective: To investigate the levels of antimicrobial peptide hCAP‐18/LL‐37 protein and mRNA expression in gingival tissues with different periodontal disease. Materials and methods: Ten patients with generalized aggressive periodontitis, 10 patients with chronic periodontitis, and 10 healthy controls were included in this study. Periodontal parameters including probing depth, clinical attachment level, plaque index, and papilla bleeding index were assessed in study subjects. hCAP‐18/LL‐37 mRNA analysis by RT‐PCR and immunohistochemistry were performed in 19 samples provided enough RNA in terms of concentration and integrity. Results: This study demonstrated that hCAP‐18/LL‐37 was a product of neutrophils. Tissue samples of chronic periodontitis patients had significantly higher immunostaining of hCAP‐18/LL‐37 on neutrophils infiltrating in both epithelium and connective tissue compared with controls. hCAP‐18/LL‐37 mRNA expression levels in tissue samples of chronic periodontitis patients seemed to be upregulated compared with controls. While two generalized aggressive periodontitis patients showed downregulated hCAP‐18/LL‐37 mRNA expression levels, one generalized aggressive periodontitis patient showed slightly increased hCAP‐18/LL‐37 mRNA level compared with controls. Conclusions: hCAP‐18/LL‐37 has an important role in innate response during periodontal inflammation. Local deficiency in hCAP‐18/LL‐37 might be a confounding effect in the pathogenesis of generalized aggressive periodontitis.     

Quantitative changes in amylase activity in the salivary glands, pancreas, saliva, and serum after administration of isoproterenol, pilocarpine, and acetylcholine

Amylase activity in various tissues--i.e., submandibular/sublingual and parotid glands, the pancreas, saliva, and serum--in rats was measured after injection of isoproterenol, pilocarpine, and acetylcholine. All agents reduced amylase activity in the parotid gland and increased the enzyme activity in the submandibular/sublingual glands, in saliva and serum.     

Antimicrobial peptide LL-37 and its pro-form,hCAP18, in desquamated epithelial cells of human whole saliva

The antimicrobial peptide LL-37 is active against oral bacteria and has been demonstrated to be present in human saliva, but its distribution in different fractions of saliva is not known. LL-37 is formed from its intracellular pro-form, hCAP18, in an extracellular enzymatic reaction catalyzed by proteinase 3 and kallikrein 5. Here, we prepared cell-containing and cell-free fractions of unstimulated human whole saliva by centrifugation after depolymerization of mucins with dithiothreitol, and measured the levels of hCAP18/LL-37 in these fractions using ELISA. Cellular expression of hCAP18/LL-37 was determined by western blotting and immunocytochemistry. The ELISA analyses demonstrated that both cells and cell-free saliva contained hCAP18/LL-37. Western blot analysis of cell-pellet homogenates showed a strong band corresponding to hCAP18 at the correct molecular weight and a weak band corresponding to LL-37. Phase-contrast and light microscopy revealed that the cells consisted of desquamated epithelial cells. These cells expressed cytoplasmic immunoreactivity for hCAP18/LL-37. The peripheral part of the cytoplasm, corresponding to the plasma membrane, was particularly rich in hCAP18/LL-37 immunoreactivity. No immunoreactivity was observed after omission of the primary antibody. We conclude that desquamated epithelial cells of human whole saliva contain antimicrobial hCAP18/LL-37, suggesting that these cells may take part in the innate immune system by harboring and releasing these peptides.     

The contribution of oral minor mucous gland secretions to the volume of whole saliva in man

To determine the contribution of minor mucous gland secretions to total saliva by a direct method, flow rates of both unstimulated and sour lemon drop (SLD)-stimulated saliva were initially determined in 15 subjects. The right and left lingual nerves were then anaesthetized to halt submandibular and sublingual secretion, and both parotid ducts were cannulated. The only remaining saliva in the mouth was that secreted by minor salivary glands. Unstimulated and SLD-stimulated minor mucous gland secretions were then collected and the median percentage contributions to whole saliva were calculated to be 8 and 7 per cent, respectively. Comparable results were obtained on 3 subjects using an indirect method similar to that of Schneyer (1956). With the left parotid duct cannulated, subjects maintained a constant, SLD-stimulated, left parotid flow rate of 1 ml/min and the remaining mixed saliva was collected to determine its flow rate. The right parotid and the submandibular and sublingual glands were then also cannulated and the flow rate from these glands determined whilst that from the left parotid was maintained at 1 ml/min. The contribution from minor mucous glands was the difference between the flow rate of mixed saliva and the combined flow rate from the right parotid, submandibular and sublingual glands.     

Preliminary investigation of the vitamin D pathway in periodontal connective tissue cells

1 Background

The vitamin D pathway, from toll‐like receptor activation to human cationic antimicrobial protein (hCAP‐18/LL‐37) generation, has been identified in monocytes and keratinocytes. This study aimed to investigate the vitamin D pathway in human gingival fibroblasts (hGFs) and human periodontal ligament cells (hPDLCs) and to provide preliminary evidence of its role in periodontal immune defense.

2 Methods

Primary cultures of hGFs and hPDLCs were stimulated with 1,25‐dihydroxy vitamin D₃ and 25‐hydroxy vitamin D₃, with or without Porphyromonas gingivalis lipopolysaccharide. CYP27B1 RNA interference and vitamin D receptor (VDR) antagonism were also used for reverse proof. The mRNA expression of hCAP‐18/LL‐37, VDR, interleukin (IL)‐6, IL‐8, and monocyte chemotactic protein‐1 were detected using real‐time polymerase chain reaction. The LL‐37 concentrations were measured using enzyme‐linked immunosorbent assay.

3 Results

In hGFs and hPDLCs, 25‐hydroxy vitamin D₃ and 1,25‐dihydroxy vitamin D₃ induced hCAP‐18/LL‐37 expression, which was further increased by Porphyromonas gingivalis lipopolysaccharide. If the function of CYP27B1 or VDR was blocked, the induction was significantly weakened. IL‐8 and monocyte chemotactic protein‐1 mRNA expression could be suppressed by the vitamin D pathway.

4 Conclusion

These findings suggest that the vitamin D pathway exists in hGFs and hPDLCs and plays an important role in immune defense in periodontal soft tissues.     

Ultrastructural localization of microliths in salivary glands of cat

Although microliths occur in normal human salivary glands and may be an aetiological factor of sialadenitis, little is known of their natural history. In an attempt to remedy this, we investigated a large archival collection of normal and experimental feline parotid, submandibular and sublingual salivary glands. In submandibular and sublingual glands, microliths were detected ultrastructurally in: all types of acinar secretory cells; myoepithelial cells; ductal cells; lumina; intercellular spaces; basement membrane; stroma; macrophages; multinuclear giant cells; and neutrophils. Microliths were not detected ultrastructurally in parotid glands. Microliths appear to form in acinar cells during autophagy and in stagnant secretory material in lumina. Microliths appear to be removed by secretion in the saliva, discharge from cells laterally and basally, and engulfment by macrophages. There appears to be a turnover of microliths, which possibly is upset by secretory inactivity with a resulting accumulation that leads to localized obstruction and sialadenitis.     

Relative secretory contributions of the three major salivary glands of the rat in response to substance P and super-sensitivity

In vivo salivation in the rat in response to a range of intravenous doses of substance P was studied. The ducts of the parotid, submandibular and sublingual glands were cannulated. The secretory threshold dose of substance P, in microgram/kg, was 0.05-0.1 in the submandibular glands, 0.2 in the parotid glands and 0.2-0.5 in the sublingual glands. The maximal secretory response in all three types of glands was obtained at a dose level of 5-10 micrograms/kg. The total amount of saliva secreted at this dose level from the three pairs of glands was calculated to about 300 mg; the submandibular glands were responsible for 65 per cent, the parotid glands for 32 per cent and the sublingual glands for 3 per cent. Parasympathetic decentralization but not sympathetic denervation caused the sublingual glands to develop a super-sensitivity to substance P. The secretory effect of substance P was not exerted via cholinergic, alpha-adrenergic or beta-adrenergic receptors.     

The effect of X-ray irradiation on the function and saliva composition of rat parotid and submandibular/ sublingual glands

Radiation therapy to the head and neck area frequently causes severe salivary gland dysfunction and xerostomia. Morphological studies of irradiated salivary glands have suggested that the submandibular/sublingual gland may be less radiosensitive than the parotid gland. The purpose of this study was to evaluate the effect of radiation on major salivary gland functions in rats with radiation-induced xerostomia. The effect of salivary gland irradiation on salivary function was examined in specific pathogen-free Sprague-Dawley rats. The animals were irradiated with a single exposure of either 22 Gy or 32 Gy. Stimulated saliva excretion time was measured for the parotid and submandibular/ sublingual glands, and the total protein in saliva was analysed. Our results showed that the saliva flow rate and protein concentration of parotid saliva were significantly reduced in the 32 Gy-irradiated rats.     

Morphological and biochemical changes in the rat parotid gland after compensatory and isoproterenol-induced enlargement

Enlargement of parotid glands can be induced in rats by treatment with isoproterenol (ISP) or by removal of the submandibular and sublingual glands. In this study, morphological changes in the enlarged parotid glands and qualitative changes in secreted proteins were examined in rats that had been treated with ISP for 10 days or that had been partially sialoadenectomized by removal of the submandibular/sublingual glands 2 weeks prior to killing. After ISP treatment or salivary gland ablation, secretory cells were enlarged and contained enlarged secretory granules that stained differently from granules in normal glands. Isoproterenol treatment induced the greatest enlargement of cells and granules. Even though gland structure was altered in both experimental groups, electrophoresis of saliva showed that submandibular/sublingual gland ablation did not lead to significant qualitative changes in secreted proteins, while ISP treatment induced major changes in the pattern of secreted protein. The results suggest that compensatory enlargement of the parotid glands and changes after ISP treatment are induced by stimulation of different regulatory pathways.     

Salivary alpha-amylase and lysozyme levels: a non-invasive technique for measuring parotid vs submandibular/sublingual gland activity

Cannulation procedures have shown that alpha-amylase is produced primarily by the parotid gland, whereas lysozyme is produced mainly by the submandibular and sublingual glands. In this study, the ratio of lysozyme to alpha-amylase was determined in whole human saliva following challenge with various gustatory and mechanical stimuli. Comparison of this ratio with the immediately preceding "baseline" value, and knowledge of the salivary glandular origin of these enzymes, gives an indication of the level of activation of these differing glands. This methodology obviates the need for invasive cannulation techniques. The findings also show that strong taste stimuli, such as salt, activate the submandibular/sublingual glands more as compared to the parotid gland.     

Expression of muscarinic receptor subtypes in salivary glands of rats, sheep and man

In rat parotid, submandibular and sublingual glands and in ovine parotid and in human labial glands, the expression of muscarinic receptor subtypes was examined by immunoblotting and immunohistochemistry. Functional correlates were searched for in rat salivary glands. In the rat submandibular and sublingual glandular tissues clear signals of muscarinic M1 and M5 receptors could be detected in the immunoblotting and vague bands for muscarinic M3 and, in particular for, M4 receptors. The rat parotid gland differed. In this gland, the signal was less obvious for the muscarinic M1 receptor, and further, muscarinic M4 receptors appeared more strongly marked than in the submandibular glands. The results from the immunohistochemistry could be interpreted as the muscarinic M4 receptors are located on nerve fibres, since the outer layer of lobuli were densely stained. Intraglandular vessels in the rat submandibular and parotid glands showed expression of M3 receptors. In contrast to the parotid gland, the submandibular vessels also expressed M1 and M2 receptors. Occasionally M5 receptors appeared in the arteries and veins also. The functional studies in the rat confirmed muscarinic M1 receptor mediated secretion in the submandibular gland. Since the M1 receptor blockade did not affect submandibular blood flow, indirect vascular effects could not in total explain the secretory inhibition. Also in the human labial glands, muscarinic M1, M3 and M5 receptors occurred. No or low amounts of muscarinic M2 and M4 receptors could be detected. In patients with Sj?gren-like symptoms an up-regulation of M3, M4 and M5 receptors was apparent in the labial glands. In ovine parotid glands all receptors could be detected, but constantly with vague bands for muscarinic M2 receptors. In conclusion, muscarinic M1 receptors seem to be expressed in seromucous/mucous glands. A secretory effect by muscarinic M5 receptors is not to be excluded, since they were expressed in all the glands examined. However, other functions, such as promotion of inflammation, cell growth and proliferation are possible as well.     

Immunochemical analysis of high molecular-weight human salivary mucins (MG1) using monoclonal antibodies

Using four Mabs with different specificities for salivary mucins, an ELISA has been developed in which human whole saliva, glandular salivas, salivary protein fractions and purified, high molecular-weight, mucin fractions (MG1) isolated from human submandibular and sublingual glandular tissues have been immunochemically analysed. All four Mabs reacted with MG1s. Three of them reacted with the purified, low molecular-weight salivary mucins (MG2). None was reactive with parotid saliva. MG1 preparations isolated from submandibular and sublingual glandular tissues of one and the same individual displayed different patterns of reactivity with these Mabs, indicating that they differ immunochemically. Analysis of the MG1s in salivas derived from individual salivary glands showed differences in immunochemical composition. These results indicate that the MG1 fraction in human whole saliva consists of several immunochemically different species.     

Expression of podoplanin in the mouse salivary glands

OBJECTIVE: Podoplanin is one of the most highly expressed lymphatic-specific genes. Here, we report the distribution of cells expressing podoplanin in mouse salivary glands. DESIGN: We immunohistochemically investigated the distribution of cells expressing podoplanin in mouse major salivary glands by laser-scanning microscopy. The expression of endothelial cell marker PECAM-1 was tested to discriminate lymphatic endothelium from salivary gland cells, and myoepithelial cells were identified by an antibody for P-cadherin. RESULTS: The podoplanin expression was rarely found in acini of the parotid gland but clearly found at the basal portion of acini in the submandibular and sublingual glands. The number of portion reacted with anti-podoplanin is greater in the sublingual gland than in the submandibular gland. The expression was also found at the basal portion of ducts in all major salivary glands. The P-cadherin expression was rarely found in acini of the parotid gland but found in acini of the sublingual gland and on ducts in parotid and sublingual glands, corresponding to the area of podoplanin expression. CONCLUSIONS: It was suggested that the acinar and myoepithelial cells in the salivary glands have the ability to express podoplanin, and that the expression may be concerned with the mucous saliva excretion.     

Alpha-D-mannosidase activity in human saliva and rate of production of enzymes by parotid and submandibular glands

The aim of the investigation was to study alpha-D-mannosidase activity in samples of whole, parotid and submandibular saliva and the rate of production of alpha-D-mannosidases by parotid and submandibular glands. alpha-D-mannosidase activity was determined spectrophotometrically and spectrofluorimetrically. alpha-D-mannosidase activity was found at three different pH optima in whole saliva, around pH 4.8, 6.1 and 6.9. In parotid and submandibular saliva there were two different pH optima, around pH 4.8 and 6.1. The rate of production of alpha-D-mannosidase by parotid glands varied between 0.48 and 13.66 U/min and by submandibular glands between 0.25 and 2.52 U/min. The present study shows that two of the three alpha-D-mannosidases found in whole human saliva are produced by parotid and submandibular glands.     

Physostigmine-induced salivary secretion in the rat

The purpose of this study was to see if physostigmine, a reversible cholinesterase inhibitor, affects the secretion and composition of saliva of the major salivary glands of the rat. Low doses of physostigmine did not elicit secretion. At higher doses there was significant flow from the parotid and submandibular glands within 5 min; however, no sublingual secretion was observed. The submandibular flow rate was highest for the first 5 min, then declined rapidly. The parotid flow rate initially was one-fifth of the maximum submandibular rate and then gradually decreased. The concentrations of Ca, Na and K of physostigmine-induced parotid saliva, and the Na of submandibular saliva, were similar to those with carbachol stimulation. The Ca and K concentrations of submandibular saliva were significantly higher than with carbachol or parasympathetic stimulation, and resembled those of alpha-adrenergic stimulation. The protein concentrations of physostigmine-evoked saliva from both glands were similar. The amylase activity of physostigmine-evoked parotid saliva was much higher than that of carbachol or parasympathetic stimulation. Physostigmine-evoked secretion was completely blocked by atropine, a cholinergic antagonist, and by reserpine, partially blocked by phentolamine, an alpha-adrenergic antagonist and not affected by surgical sympathectomy. Morphologically, physostigmine resulted in a moderate decrease in the number of acinar, but not ductal, secretory granules of both the parotid and submandibular glands, while the sublingual gland was unaffected. Numerous patches of parotid acini also developed vacuoles or vesicles. These results suggest that physostigmine-induced salivary secretion is mediated primarily by direct effects on cholinergic and alpha-adrenergic receptors.     

Levels of parotid and submandibular/sublingual salivary immunoglobulin A in response to experimental gingivitis in humans

Salivary secretory IgA (s-IgA) is considered to act as an important first line of defense mechanism in the oral cavity. It has therefore been suggested that an increased antigenic load would induce an increase in salivary IgA production. This study investigated the pure glandular levels of salivary IgA in parotid and submandibular/sublingual (SM/SL) saliva during plaque accumulation leading to experimental gingivitis. Starting from regular oral hygiene, 14 healthy, nonsmoking men refrained from all oral hygiene measures for 12 days. On days –2, 0, 3, 6, and 12 a plaque index, a bleeding index, and unstimulated and stimulated saliva from the parotid and the SM/SL glands were measured. Salivary IgA was quantified using a sandwich ELISA. All subjects developed gingivitis as measured by a bleeding index. Compared to baseline the salivary flow rate was increased on day 12. Regarding the secretion rate of IgA there was a statistically significant increase in stimulated parotid saliva but not SM/SL saliva compared to baseline after 6 and 12 days without oral hygiene. No significant changes were observed for the concentration of IgA during the trial. Thus, in healthy subjects with regular oral hygiene the development of plaque induced gingivitis is associated with increased salivary gland output and increased total IgA output levels in stimulated parotid saliva but not in SM/SL saliva.     

Trefoil factor family 3 expression in the oral cavity

This study examined the expression, in oral keratinocytes and in the major and minor salivary glands, of Trefoil factor family 3 (TFF3) peptide. Trefoil factor family 3 messenger RNA (mRNA) and peptide were detected in cultures of normal oral keratinocytes by quantitative real‐time polymerase chain reaction (PCR) and western blotting, respectively. Trefoil factor family 3 was found, by immunohistochemical analyses, to be expressed in the basal layers of the oral mucosal epithelium. In salivary glands, immunohistochemical staining showed that TFF3 peptide expression was strongest in the mucous acini of the submandibular and the small salivary glands. Serous cells in the same glands showed weak staining. In the parotid gland, many serous acini showed weak positive staining, while other areas did not. In all glands examined, the intercalated, striated, and collecting ducts were moderately TFF3‐positive. Double immunostaining confirmed that mucous (MUC5B positive) cells were moderately or strongly positive for TFF3 and that some serous (MUC7 positive) cells showed restricted TFF3 expression, mostly in a granular pattern. The prevalence of the TFF3 peptide in the salivary secretions of healthy volunteers was detected by western blotting of saliva from minor salivary glands (four of five) and the parotid gland (one of five) and of mixed submandibular/sublingual saliva (five of five). In conclusion, the submandibular and small salivary glands appear to be the major producers of oral TFF3, but duct cells of all glands and keratinocytes of the oral mucosa may also contribute as sources of TFF3 in the oral cavity.     

Salivary glands: applied anatomy and clinical correlates.

The major salivary glands include the paired parotid, submandibular and sublingual glands. Salivary glands act as accessory digestive glands and produce a secretion referred to as saliva. Saliva has lubricating, cleansing, digestive and antimicrobial properties. The parotid is the largest salivary gland and saliva is secreted into the mouth via the parotid duct (Stensen's duct). The submandibular gland lies inferior to the body of the mandible and is susceptible to sialolithiases. Drainage is via the duct of the submandibular gland (Wharton's duct) into the floor of the mouth on either side of the lingual frenulum. The sublingual glands are situated under the mucosa in the floor of the mouth, on the sides of the tongue. These glands are in relationship to important nerves in the surrounding tissue. Disease processes such as chronic intraparenchymal sialolithiasis and neoplastic changes frequently mandate surgical removal of the underlying salivary gland. Detailed, applied knowledge of anatomy on a regional basis is required to avoid inadvertent nerve damage during surgery and resulting litigation.     

Salivary LL-37 secretion in individuals with Down syndrome is normal

Antimicrobial peptides play an important role in the innate immune response. Deficiency in salivary LL-37 antimicrobial peptide has been implicated in periodontitis in patients with morbus Kostman syndrome. Down syndrome is associated with periodontitis, diminished salivary flow, and salivary immunoglobulin deficiency. In the present study, levels of LL-37 and its hCAP18 precursor were measured in saliva samples from young individuals with Down syndrome and compared with levels in those from age-matched healthy controls. LL-37 and human cathelicidin antimicrobial protein (hCAP18) were detected in whole but not in parotid saliva. hCAP18 was more abundant than LL-37. The concentrations of salivary hCAP18 and LL-37 were found to be higher in individuals with Down syndrome than in healthy controls, but their secretion rates were similar. We concluded that, while the adaptive immunity of individuals with Down syndrome is impaired at the oral mucosa, the secretion rate of the LL-37 component of the innate immune system is normal.