The human host defense peptide, LL-37, is an important player in the first line of defense against invading microorganisms. LL-37 and its precursor, hCAP18, have been detected in unstimulated whole saliva but no reports showing hCAP18/LL-37 in isolated, parotid, and/or submandibular/sublingual saliva have been presented. Here, we measured the levels of hCAP18/LL-37 in human parotid and submandibular/sublingual saliva and investigated the expression of hCAP18/LL-37 in parotid and submandibular gland tissue. Parotid and submandibular/sublingual saliva was collected from healthy volunteers, and the levels of hCAP18/LL-37 in saliva were analyzed by dot blot, ELISA, and western blotting. Cellular expression of hCAP18/LL-37 in human parotid and submandibular glands was investigated by immunohistochemistry. Immunoreactivity for hCAP18/LL-37 was detected in both parotid and submandibular/sublingual saliva of all individuals. The concentration of hCAP18/LL-37 was similar in parotid and submandibular/sublingual saliva, and was determined by densitometric scanning of each dot and normalization to the total protein concentration of each sample, and by ELISA. Double immunohistochemistry revealed that intravascular neutrophils of both parotid and submandibular glands express hCAP18/LL-37. For the first time, we demonstrate hCAP18/LL-37 in isolated human parotid and submandibular/sublingual saliva and expression of hCAP18/LL-37 in glandular intravascular neutrophils, indicating that neutrophils of the major salivary glands contribute to the LL-37 content of whole saliva.

Background and ObjectiveThe aim of this study was to assess the impact of 1,25-dihydroxyvitamin D3 (vitamin D3) on osteogenic and inflammatory properties of human periodontal ligament (PDL) cells and investigate underlying mechanisms. Material and MethodsHuman PDL cells, obtained from four subjects, were stimulated with vitamin D3 for 4-48h. The bone markers osteopontin and osteocalcin and proinflammatory cytokine/chemokine expression was determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Cytokine and chemokine expression was determined after stimulation with the inflammation promoter lipopolysaccharide (LPS) in the presence or absence of vitamin D3. Alkaline phosphatase activity was assessed using p-nitrophenylphosphate substrate. ResultsTreatment with 30ng/mL of vitamin D3, corresponding to an optimal plasma concentration of vitamin D, for 24h had no effect on PDL cell number and morphology but increased PDL cell osteopontin and osteocalcin mRNA expression by about 70 and 40%, respectively, and, moreover, treatment with vitamin D3 for 48h enhanced PDL cell alkaline phosphatase activity by about two times showing that vitamin D3 exerts pro-osteogenic effects in human PDL cells. Stimulation with LPS (1g/mL) for 4h increased PDL cell interleukin (IL)-6 cytokine and chemokine ligand 1 (CXCL1) chemokine mRNA expression several fold. The LPS-induced increase in IL-6 and CXCL1 transcripts was attenuated by vitamin D3 (30ng/mL). Treatment with vitamin D3 (3-300ng/mL) for 24h reduced the LPS-evoked increase in PDL cell IL-6 protein by about 50%. Vitamin D3 (30ng/mL) had no effect on LPS-induced IL-1 and MCP-1 mRNA expression. ConclusionsVitamin D3 promotes osteogenic differentiation but also downregulates inflammation promoter-induced IL-6 cytokine and CXCL1 chemokine expression in human PDL cells, suggesting that vitamin D3 both stimulates bone regeneration and antagonizes inflammation in human periodontal tissue.

Strontium ranelate (SrRan) is the active component of drugs currently used for reducing the risk of fractures in patients suffering from osteoporosis. Despite extensive use, the underlying mechanisms of action of Sr2+ are not fully understood. In the present study, we assess the impact of SrCl2 on human osteoblast activity and proliferation. Cultures of the human osteoblast-like cell line MG63 were treated for 72 h in presence of 0.1 mM, 1 mM, 5 mM and 10 mM SrCl2 or vehicle, used in control groups. Cells were counted manually using a Bürker chamber. Total protein content was determined by colorimetric analysis performed by a microplate reader using Bio-Rad protein assay. Alkaline phosphatase (ALP) activity was determined enzymatically and normalized to total protein content in each sample. Cell viability was assessed using the MTT assay. Treatment with 5 mM SrCl2 for 72 h enhanced total MG63 cell protein content by 37% compared to controls (p<0.01). A lower concentration (0.1 mM) of SrCl2 had no effect on total protein. Incubation with 5 mM SrCl2 for 72 h increased MG63 cell number by 38% compared to controls (p<0.001). The SrCl2-induced increase in cell number was associated with enhanced (+14% compared to controls, p<0.05) cell viability. Treatment with a higher concentration (10 mM) of SrCl2 enhanced cell number similar to 5 mM SrCl2 (+54% compared to controls, p<0.05). Treatment with 0.1 or 5 mM SrCl2 for 72 h had no effect (p>0.05) on MG63 cell ALP activity, while 1 mM SrCl2 reduced ALP activity as well as total protein content by about 25% compared to controls (p<0.05). The current results demonstrate that treatment with SrCl2 for 72 h, at concentrations higher than 1 mM promotes cell proliferation in human osteoblast-like cells, suggesting that Sr2+ may enhance bone formation through this mechanism.

Objective. Periodontal ligament (PDL) cells produce IL-6 upon stimulation with inflammation promoters, but the signaling pathways involved have not been characterized. This study investigates underlying mechanisms behind regulation of PDL cell IL-6 production by E. coli and P. gingivalis LPS. Materials and methods: Human PDL cells, endothelial cells and monocytes were stimulated with E. coli or P. gingivalis LPS in the presence or absence of pharmacological agents in order to disclose pathways involved in LPS signaling. Gene expression and cellular protein levels were assessed by quantitative real-time PCR and ELISA, respectively. Results. Stimulation with LPS from E. coli (1 µg/ml) for 24 h enhanced PDL cell IL-6 expression several fold, demonstrated both on transcript and protein levels, but P. gingivalis LPS (1–5 µg/ml) had no effect. TLR2 mRNA was more highly expressed than TLR4 transcript in PDL cells. Treatment with the non-selective nitric oxide synthase inhibitor L-NAME (100 µM) reduced E. coli LPS-induced PDL cell IL-6 by 30%, while neither aminoguanidine (10 µM), an inhibitor of inducible nitric oxide synthase, nor estrogen (17β-estradiol, 100 nM) influenced IL-6. Treatment with the glucocorticoid dexamethasone (1 µM) totally prevented the E. coli LPS-induced PDL cell IL-6. In endothelial cells, neither E. coli LPS nor P. gingivalis LPS promoted IL-6 production. In monocytes, serving as positive control, both E. coli and P. gingivalis LPS stimulated IL-6. Conclusions. E. coli LPS but not P. gingivalis LPS stimulates PDL cell IL-6 production through a glucocorticoid-sensitive mechanism involving nitric oxide formation, probably via endothelial nitric oxide synthase.

Östrogen är ett kvinnligt könshormon som även har andra effekter. Östrogen verkar i cellkärnan genom att binda till en östrogenreceptor (ER), som finns i två olika typer, ERα och ERβ. Parodontit (tandlossning) är en inflammationssjukdom som drabbar tandens fäste som svar på bakterier som normalt finns i munhålan. I cellmembranet hos vissa bakterier finns en molekyl, LPS, som fungerar retande och startar inflammationen genom att binda till en mottagarmolekyl, som sitter på vita blodkroppar, men också till vävnadsceller som till exempel PDL celler. Effekten blir att cellerna börjar producera ämnen, bland annat cytokiner och kemokiner, som får fler vita blodkroppar att komma till platsen och inflammationen är då ett faktum. Sedan tidigare finns ett flertal studier som visar ett samband mellan förändringar i östrogennivåer och tandlossning men mekanismerna är inte kända. Det övergripande syftet med studierna i denna avhandling var att undersöka ERs betydelse i parodontiet.*Studie I: LPS får PDL-celler att drastiskt öka produktionen av inflammationsproteinerna, IL-6och MCP-1 men påverkar inte PDL-cellers normala funktioner, vilket betyder att LPS specifikt stimulerar produktionen av inflammationsproteiner i dessa celler.*Studie II: För att studera effekten av östrogen i parodontiet opererades de östrogenproducerandeäggstockarna bort på en grupp av honmöss. Tändernas fästenivå hos denna grupp jämfördes med en kontrollgrupp med normal östrogenproduktion. Efter sex veckor visade det sig inte vara någon skillnad på tandfästet mellan de två grupperna.*Studie III: Östrogen påverkar PDL-cellers produktion av flera olika inflammationsproteiner, men mönstret är komplext. Ett protein som stimulerar rekrytering av vita blodkroppar (kemokin) hämmas av östrogen medan en annan kemokin förblir oförändrad . För ett tredje kemokin skilde produktionen sig åt beroende på vilket genetiskt ursprung cellerna hade. Detta tyder på att östrogen verkar både pro- och anti-inflammatoriskt och att det genetiska ursprungetkan påverka östrogens funktion.*Studie IV: I tandköttet (gingivan) är ERβ är den dominerande östrogenreceptorn. Mönstret gårigen både på odlade celler och på vävnadsprover från patienter. Vid höga doser av östrogen minskar förmågan att dela sig hos gingivala epitelceller. Studien visar att östrogen verkar genom ERβ i tandköttet och att höga östrogenhalter minskar gingivala epitelcellers förmåga att dela sig.*Studie V: PDL-celler producerar olika mängder av inflammationsproteinet IL-6, beroende på vilket LPS de behandlas med. LPS från den parodontitassocierade bakterien, P. gingivalis orsakar ingen IL-6 produktion i PDL-celler medan LPS från tarmbakterien E. coli ökar IL-6 produktionen med cirka 30 gånger. När enzymen, som behövs vid bildandet av kväveoxid(NO), blockerades minskade IL-6 produktionen som svar på E. coli LPS med 30% vilket indikerar att NO är inblandat i IL-6 produktionen. Sammanfattningsvis visar studierna att östrogen, sannolikt via ERβ, påverkar parodontiets celler på flera olika sätt. Östrogen utövar både effekter som kan tolkas som skyddande (minskning av produktionen av inflammationsproteiner) men också effekter som kan innebära reducerat skydd (t ex hämning av gingivala epitelcellers celldelning). Studierna bidrar med nykunskap om den biologiska betydelsen av östrogen i parodontiet.

The main functions of estrogen are associated with reproduction. However, estrogen has been shown to be of functional importance also in non-classic target organs. Previous studies, especially epidemiologicand clinical ones, have addressed estrogen’s influence on periodontitis, suggesting that estrogen has a beneficial effect, but the biological mechanisms have not been identified. Estrogen exerts genomic effects in the target cells by binding to the nuclear receptors, estrogen receptor (ERs), ERα and ERβ. The expression of the two subtypes of ERs varies depending on the tissue. The overall objectives of this thesis were to study the functional importanceof estrogen receptors in the periodontium with special focus on inflammation, and stimulators of inflammation and their signaling pathways. The thesis is based on the following five papers.In Paper I, effects of estrogen on E. coli LPS-induced PDL cell production of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and C-reactive protein (CRP) are assessed, by usingELISA. Furthermore, effects of LPS and estrogen on the normal characteristics of the PDL cell such as collagen synthesis and cell proliferation is determined by using L-[3H]proline incorporationand measurement of DNA synthesis, respectively. Key findings: E.coli LPS stimulates PDL cell IL-6 and MCP-1 production but has no effect on the normal physiological properties of PDL cells. LPSinducedIL-6 and MCP-1 is not reversed by estrogen suggestingthat estrogen has no anti-inflammatory effect in these experiments.In Paper II, we investigate the effects of ovariectomy and aging on tooth attachment in female mice by using morphometric analysis. Key findings: Withdrawal of female sex hormone production byovariectomy has no effect on alveolar bone height and apical termination of the junctional epithelium. In a second series of experiments these parameters are similar in mice sacrificed at 8-26 weeks of age, suggesting that tooth attachment is preserved with age in mice within a period of six months. In Paper III, the objective is to investigate the regulation of CCL2/MCP-1, CCL3/MIP-1α, and CCL5/RANTES chemokines by estrogen in human PDL cells by determining mRNA transcript levels (using quantitative real-time PCR) and protein levels (usingELISA). Key findings: A physiological concentration of estrogen reduces the expression of CCL3 mRNA by about 40% compared to PDL cells treated with LPS alone. In contrast, inter-individual differences in the effects of estrogen on CCL5 mRNA expressionare observed. These findings indicate that estrogen affects chemokine expression in PDL cells showing a complex pattern involving down-regulation as well as up-regulation of chemokines. Estrogen exerts both anti-inflammatory and pro-inflammatory effectsthrough these mechanisms.In Paper IV, ER expression in human gingival biopsies, and effects of estrogen on cultured gingival epithelial cell (HGEP) proliferation,are investigated. Expression of ERα and ERβ is determined by immunohistochemistry and effects of estrogen on HGEP proliferation monitored by measuring DNA synthesis. Key findings: HGEP cells show strong ERβ immunoreactivity but low ERα immunoreactivity both in vivo and in culture, suggesting that ERβis the predominant ER subtype in HGEP. High, but not low, concentrations of estrogen attenuates proliferation of gingival epithelial cells, indicating a concentration-dependent mechanism.In Paper V, the objective is to investigate the effects of LPS from Escherichia coli and Porphyromonas gingivalis on IL-6 productionin human PDL cells and endothelial cells, and the signaling mechanisms involved. Quantitative real-time PCR is used to determine IL-6 mRNA levels and ELISA to determine IL-6 protein. Key findings: E. coli LPS (but not P. gingivalis LPS) stimulates IL-6 production in PDL cells. Treatment with the non-selective nitric oxide synthase inhibitor L-NAME reduces IL-6 by 30%, while aminoguanidine, an inhibitor of inducible nitric oxide synthase, does not affect IL-6 levels, showing a mechanism probably involving nitric oxide formation via endothelial nitric oxide synthase. Treatment with the glucocorticoid steroid dexamethasone totally prevents E. coli LPS-induced IL-6 in PDL cells.

BACKGROUND AND OBJECTIVE: Estrogen acts via estrogen receptor (ER) α and β. The expression pattern of ERs and their importance in gingival tissues are not fully understood. In this study, we investigate gingival ER expression and effects of estrogen on gingival epithelial cell proliferation.

MATERIAL AND METHODS: Gingival biopsies were obtained from both healthy and diseased sites in three male and three female subjects. Expression of ERα and β was determined by immunohistochemistry. Effects of 17β-estradiol (E(2) ) on cell proliferation, monitored by measuring DNA synthesis, were studied in cultured human gingival epithelial HGEPp.05 cells.

RESULTS: Estrogen receptor β, but not ERα, immunoreactivity was demonstrated in nuclei of epithelial cells in all layers of the gingival epithelium, but also in cells of the lamina propria. No differences were observed between male and female subjects. The same pattern, i.e. high ERβ expression but no ERα expression, was observed in both healthy and diseased sites within each individual. No differences in the intensity of the ERβ immunoreactive signal and the number of ERβ-positive nuclei were observed between healthy and diseased gingiva. Treatment with a physiological concentration of E(2) (10 nm) had no effect on DNA synthesis in ERβ- and ERα-expressing HGEPp.05 cells. In contrast, E(2) at high concentrations (500 nm and 10 μm) reduced DNA synthesis by 60-70%.

CONCLUSION: Human gingival epithelial cells display strong ERβ but low ERα immunoreactivity both in vivo and in culture. Estrogen attenuates gingival epithelial cell DNA synthesis at high but not low concentrations, suggesting a concentration-dependent mechanism.

BACKGROUND: Periodontal ligament cells are fibroblast-like cells characterized by collagen production but also possessing some osteoblastic features. In the light of numerous studies presented during recent times, which show that human periodontal ligament cells also produce cytokines and chemokines in response to inflammation promoters, it is reasonable to suggest that periodontal ligament cells play a role as promoters of periodontal inflammation through these mechanisms. MATERIAL AND METHODS: The periodontal ligament, which harbours the periodontal ligament cells, is a part of the attachment apparatus comprised of periodontal ligament cells, extracellular matrix and fibres, attaching the root cement to the alveolar bone. Periodontal ligament cells are in close proximity to bacteria within the plaque and the pocket, and thus these cells are readily accessible to bacterial endotoxins and other promoters of inflammation. RESULTS: Cytokines and chemokines, released by periodontal ligament cells upon stimulation with inflammation promoters, reach the blood vessels easily thanks to rich vascularization of the periodontium stimulating recruitment of white blood cells to the site of inflammation. In addition to classical inflammatory cells, such as leucocytes, macrophages and mast cells, the periodontal ligament cells also contribute to periodontal inflammation via their production and release of cytokines and chemokines. CONCLUSION: Therefore, pharmacological treatment of periodontitis should aim to reduce the release of proinflammatory agents not only from classical inflammatory cells but also from periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Estrogen modulates inflammatory responses, but the mechanisms involved have not yet been identified. Periodontal ligament (PDL) cells produce chemokines (a group of chemoattractant molecules that recruit leukocytes) and it has been suggested that estrogen modulates periodontal inflammation by regulating the expression of chemokines by PDL cells. Therefore, the objectives of this study were to investigate the regulation of chemokine ligand 2 [CCL2/monocyte chemoattractant protein 1 (MCP-1)], chemokine ligand 3 [CCL3/macrophage inflammatory protein-1α (MIP-1α)] and chemokine ligand 5 (CCL5/RANTES) by estrogen in human PDL cells. MATERIAL AND METHODS: PDL cells were obtained from the PDL of premolars, extracted for orthodontic reasons, from two boys and two girls (16 and 17 years of age). PDL cell CCL2, CCL3 and CCL5 mRNA transcripts were determined by quantitative real-time PCR. The concentrations of CCL2, CCL3 and CCL5 proteins were determined by ELISAs. RESULTS: Treatment with 0.5 μg/mL of lipopolysaccharide (LPS, from Escherichia coli) + 100 nm 17β-estradiol (E(2) ) for 24 h reduced the expression of CCL3 mRNA by about 40% compared to PDL cells treated with LPS alone. Attenuation of CCL3 mRNA was not associated with a decrease in CCL3 protein within 48 h, suggesting a slow turnover of the CCL3 protein. Interindividual differences in the effects of E(2) on CCL5 mRNA expression were observed. E(2) (100 nm) increased the expression of CCL5 by 40-60% in PDL cells derived from two subjects but reduced the expression of CCL5 by about 30% in cells from another subject. CCL2 mRNA and CCL2 protein were highly expressed, but not regulated by E(2) . Similar data were observed in cells obtained from both boys and girls. CONCLUSION: Regulation, by estrogen, of chemokine expression in PDL cells shows a complex pattern involving the down-regulation as well as the up-regulation of chemokines, suggesting that estrogen exerts both anti-inflammatory and proinflammatory effects through these mechanisms.

Objective. Non-human primates, dogs, rats, hamsters and ferrets, have frequently been used as laboratory animals in periodontal biology and pathology. In the past, mice have been used less for this purpose, but nowadays attract a lot of interest because gene knockout and transgenic technologies utilize mice primarily. In this study, we investigate the effects of ovariectomy and aging on tooth attachment in female mice. Material and methods. Eight-week-old mice (n=15) were divided into three experimental groups (control, n=5; sham-operated, n=5; ovariectomy, n=5) and ovaries removed bilaterally. Attachment level, assessed by measuring alveolar bone height and apical termination of the junctional epithelium, was determined 6 weeks post-ovariectomy by digital morphometric analysis in sagittal sections of the mandible. The plasma level of the inflammation marker serum amyloid A (SAA) was determined by ELISA. In another series of experiments, tooth attachment was determined in female mice (n=7) at 8–26 weeks of age. Results. Withdrawal of female sex hormone production by ovariectomy had no effect on alveolar bone height and apical termination of the junctional epithelium. The SAA level in plasma was unaffected by removal of the ovaries, suggesting that systemic inflammation is not induced by ovariectomy. Bone height was similar in mice sacrificed at 8–26 weeks of age and apical termination of the junctional epithelium was at the cemento-enamel junction at all ages. Conclusions. Removal of ovarian production of female sex hormones by ovariectomy has no influence on tooth attachment, and further tooth attachment is preserved with age in female mice.

Background: The periodontal ligament cells (PDL cells) play a key role in the formation of the periodontal ligament but these cells have other functions as well. The PDL cells express estrogen receptors but the functional importance is not known. Estrogen modulates inflammation and our hypothesis is that estrogen protects the periodontium via an anti-inflammatory effect on PDL cells. Aim: To identify genes regulated by estrogen in LPS-treated human PDL cells by whole genome arrays. Materials and methods: PDL cells were obtained from human premolars extracted for orthodontic reasons. The cells were divided into two groups. One group was pre-treated with 17β-estradiol (E2, 100 nM) for 2 h and then with Escherichia coli LPS (500ng/ml) for 24 h. The other group was treated with LPS only. Total RNA was extracted and purified by RNeasy Mini Kit (Qiagen®, USA). A whole genome microarray was performed (Affymetrix®, USA) comparing gene expression in the two groups. The cut-off limit was set to a twofold change. Results and Conclusion: Estrogen caused an up-regulation of 38 genes, while 28 genes were down-regulated. Estrogen regulated genes associated with cell-metabolism and cell-signalling but also genes associated with early the inflammatory response. The functional significance of these findings is now determined by e.g. measuring protein levels with ELISA. We conclude that estrogen regulates gene expression in human PDL cells exposed to LPS.