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. 2011 Nov;90(11):1286–1292. doi: 10.1177/0022034511420431

Expression and Possible Immune-regulatory Function of Ghrelin in Oral Epithelium

K Ohta 1,2,3, NJ Laborde 2, M Kajiya 1,2, J Shin 1, T Zhu 1,2, AK Thondukolam 2, C Min 1, N Kamata 3, NY Karimbux 2, P Stashenko 4, T Kawai 1,2,*
PMCID: PMC3188459  PMID: 21865591

Abstract

Originally found in stomach mucosa, ghrelin is a peptide appetite hormone that has been implicated as an immuno-modulatory factor. Ghrelin has also been found in salivary glands and saliva; however, its expression patterns and biological properties in the oral cavity remain unclear. Therefore, we investigated the expression patterns of ghrelin in saliva, gingival crevicular fluid (GCF), and gingival tissue, as well as its in vitro effects on IL-8 production by TNF-α or LPS-stimulated oral epithelial cells. In the clinical samples obtained from 12 healthy volunteers, the concentration of ghrelin in GCF remarkably exceeded that detected in saliva. The expression of ghrelin mRNAs and growth hormone secretagogue (GHS) receptors could be detected in human oral epithelial cells. Immunohistochemical analysis revealed the expression of ghrelin in gingival epithelium, as well as in fibroblasts in the lamina propria. Ghrelin increased intracellular calcium mobilization and cAMP levels in oral epithelial cells, suggesting that ghrelin acts on epithelial cells to induce cell signaling. Furthermore, synthetic ghrelin inhibited the production of IL-8 from TNF-α or LPS-stimulated oral epithelial cells. These results indicate that ghrelin produced in the oral cavity appears to play a regulatory role in innate immune responses to inflammatory infection.

Keywords: ghrelin, gingival crevicular fluid, saliva, gingival epithelium, IL-8, lipopolysaccharide

Introduction

Ghrelin is a 28-amino-acid acylated peptide hormone mainly secreted by X/A-like cells in the oxyntic mucosa of the stomach, and it is characterized by the presence of an n-octanoyl modification on the serine third position (Kojima et al., 1999). In addition to inducing the production of growth hormone, ghrelin stimulates food intake, and it is involved in the regulation of energy homeostasis (Gualillo et al., 2003). Recent evidence indicated a potential regulatory role of ghrelin in the immune system. For instance, it was reported that ghrelin could down-modulate the proliferation of T-cells, as well as the production of cytokines, from activated T-cells (Xia et al., 2004). Furthermore, analysis of both in vitro and in vivo data showed that ghrelin can suppress pro-inflammatory cytokines produced from monocytes in response to stimulation with lipopolysaccharide (LPS) (Dixit et al., 2004; Waseem et al., 2008). Therefore, ghrelin may function as an important modulator not only for the endocrine and nervous systems, but also for the immune system.

As noted above, ghrelin was initially isolated from gastric mucosa. In addition, ghrelin was found to be widely expressed in different tissues, such as pancreas, kidney, and both small and large intestines (Ghelardoni et al., 2006). In the oral cavity, ghrelin was detected in human saliva, and ghrelin-positive cells were also found in salivary glands and oral squamous cell carcinoma lesions (Gröschl et al., 2005; Alnema et al., 2010). However, it remains unclear if oral epithelial cells, or other cells in the lamina propria, contribute to the production of ghrelin.

Oral epithelial cells are key players in the oral innate immune system. Upon stimulation with the soluble mediators of inflammation, such as TNF-α as well as pathogen-associated molecular patterns (PAMPs), they secrete pro-inflammatory cytokines, including IL-8 (Li et al., 1996; Sugiyama et al., 2002). We know that ghrelin can reduce the LPS-induced pro-inflammatory response in murine macrophages (Waseem et al., 2008). However, its influence on cytokine production by oral epithelial cells is unknown. Therefore, in the present study, we investigated the expressions of ghrelin in saliva, gingival crevicular fluid (GCF), gingival epithelium, and lamina propria. To evaluate the functional role of ghrelin expressed in the oral cavity, we also examined if ghrelin could affect IL-8 production from oral epithelial cell lines in response to LPS or TNF-α.

Materials & Methods

Participants

In total, 12 periodontally healthy individuals aged 19-58 yrs participated in this study and provided saliva and GCF specimens (n = 12; two men and 10 women; 34 ± 11.5 yrs, mean age ± SD). We determined periodontal health after establishing the absence of bleeding on probing, probing depths ≤ 3 mm, and attachment loss ≤ 2 mm. For immunohistochemical analysis, cervical gingival tissues were collected at the crown-lengthening procedure from another three individuals (one male, aged 46 yrs, and two females, both aged 35 yrs). Buccal mucosal cells were collected from healthy individuals for RT-PCR analysis (Spivack et al., 2004). The study protocol was reviewed and approved by the Institutional Review Board at the Forsyth Institute, Harvard School of Dental Medicine, and Harvard Medical School, and all participants signed an informed consent statement.

Saliva and GCF Samples

Unstimulated saliva and GCF samples were collected as previously described (Figueredo et al., 2008; Gursoy et al., 2010). Saliva samples were centrifuged at 20,000 x g for 10 min, and the resulting supernatants were kept frozen at -80°C. After the complete removal of dental plaque, GCF was collected from randomly selected quadrants at the mesiobuccal site of every tooth, by means of paperpoints (IDE, Amityville, NY, USA), which were inserted into the pockets and kept in place for 30 sec. The amount of GCF sampled into the paperpoint was measured with a Periotron (OraFlow Inc., Smithtown, NY, USA). The protein levels of IL-8 and ghrelin in saliva and GCF were determined with an IL-8 Duo-ELISA kit (R&D Systems, Minneapolis, MN, USA) and a Ghrelin Enzyme Immuno-Assay kit (Bachem, Torrance, CA, USA), respectively, to detect acylated ghrelin, according to the protocol provided by the manufacturers.

Cell Lines and Synthetic Ghrelin Peptides

Immortalized human oral epithelial cell lines RT7 and OBA9 were cultured in Keratinocytes-SFM (Invitrogen, Carlsbad, CA, USA), as described previously (Hosokawa et al., 2006; Ohta et al., 2008). Immortalized human oral epithelial cell line OKF6/TERT-2 cells were kindly provided by James G. Rheinwald (Brigham and Women’s Hospital, Harvard Institutes of Medicine, Boston, MA, USA) (Dickson et al., 2000). Human gingival fibroblast cell line GT1 cells (Kamata et al., 2004) and human leukemic cell lines HL-60 and THP-1 (De Vriese and Delporte, 2007) were grown in appropriate medium supplemented with 10% fetal calf serum. A full sequence of n-octanoyl ghrelin peptide (28 amino acids) was purchased from Prospec (Rehovot, Israel). Scrambled ghrelin peptide (consisting of the same amino acid sequence as ghrelin but in a different order) was generated by a custom synthesizing service (United Peptide, Bethesda, MD, USA).

RNA Extraction and RT-PCR

Total RNA was prepared from cell lines by means of an RNeasy total RNA isolation Kit (Qiagen, Hilden, Germany). Single-stranded cDNA for a polymerase chain-reaction (PCR) template was synthesized from superscript 3 reverse transcriptase (Invitrogen). cDNA was amplified by PCR with Hot Start Taq DNA polymerase (Qiagen). Specific primers were used: human ghrelin sense 5′-GCCCACCTGTCTGCAACC-3′ and antisense 5′-CTGCT TGACCTCCATCTTCC-3′; human GHS-R1a and GHS-R1b sense 3′-TCTTCCTTCCTGTCTTCTATC-5′; human GHS-R1a antisense 3′-AGTCTGAACACTGCCACC-5′; and human GHS-R1b antisense 3′-TCAGAGAGAAGGGAGAAG G-5′. PCR conditions were 1x (95°C, 15 min), 40x (95°C, 2 min; 55°C, 30 sec; 72°C, 1 min) and 1x (72°C, 7 min). The products were analyzed on 2% agarose gels containing SYBR Green (Invitrogen). β-actin was included as an internal control.

Immunohistochemistry

Frozen sections were fixed with 4% paraformaldehyde followed by incubation with 1.0% H2O2 to neutralize endogenous peroxidase activity. After blocking with 1.5% horse serum in PBS, each section was reacted with polyclonal rabbit IgG specific to n-octanoyl ghrelin (Abcam, Cambridge, MA, USA) overnight at 4°C. Biotin-labeled horse anti-rabbit IgG (Jackson Immuno Research, West Grove, PA, USA) with 1.5% horse serum was reacted for 30 min at room temperature, followed by incubation with avidin/biotin complex with horseradish peroxidase (Vector Laboratories, Burlingame, CA, USA). Specific staining was performed by color development with diaminobenzidine substrate in the presence of H2O2, and counterstaining was done with hematoxylin.

Calcium Flux and cAMP Assays

OBA9 cells were exposed to ghrelin or scrambled ghrelin for 1-2 hrs. Intracellular calcium mobilization and cAMP were measured with the Fluo-8-no-wash calcium assay kit (AAT Bioquest, Sunnyvale, CA, USA) and cAMP assay kit (BioVision, Inc., Mountain View, CA, USA), respectively (SD).

Cytokine Determination

RT7 and OBA9 cells were stimulated with TNF-α (Peprotech, Rocky Hill, NJ, USA), Escherichia coli (E. coli) LPS, Porphyromonas gingivalis (P. gingivalis) LPS (Invivogen, San Diego, CA, USA), and ghrelin for 48 hrs. Supernatants from the cells were collected, and the concentration of IL-8 was measured by IL-8 Duoset ELISA (R&D Systems).

Statistical Analysis

The data were analyzed by Student’s t test or one-way analysis of variance (ANOVA), and the results were presented as the mean ± standard deviation (SD).

Results

Detection of Ghrelin in Saliva and GCF

We examined the concentration of ghrelin in saliva and GCF from 12 healthy volunteers. The concentration of ghrelin measured in GCF was approximately 500-fold greater than in saliva samples (GCF, 19,129 ± 13,280 pg/mL; saliva, 35.7 ± 39.8 pg/mL) (Fig. 1). Like ghrelin, the concentrations of IL-8, as measured in both GCF and saliva samples, showed a trend of prominent elevation in GCF compared with saliva (GCF, 1,349,275 ± 779,733 pg/mL; saliva, 4992 ± 4574 pg/mL). No significant correlation was observed between ghrelin and IL-8 concentrations in either saliva or GCF. The concentration of ghrelin in serum samples from four healthy volunteers in this study was 46.99 ± 9.14 pg/mL, which was lower than that detected in GCF or saliva.

Figure 1.

Figure 1.

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Concentrations of ghrelin and IL-8 in saliva and GCF. (A) Concentrations of ghrelin protein in GCF and saliva. (B) Concentrations of IL-8 protein in GCF and saliva. The protein levels of IL-8 and the levels of ghrelin in saliva and GCF from 12 healthy volunteers were determined by ELISA. *The concentrations of ghrelin in GCF are significantly different from those in saliva (Student’s t test: p < 0.05).

Ghrelin mRNA and GHS Receptors in Oral Epithelial Cells and Gingival Fibroblasts

It is plausible that the secretion of ghrelin by oral epithelial cells may contribute to the elevated levels of ghrelin in GCF. Therefore, we first examined whether oral epithelial cells express ghrelin. RT-PCR assay showed that these oral epithelial cells and fibroblasts constitutively expressed ghrelin mRNA (Fig. 2A). The biological activities of ghrelin are mainly mediated by its ligation to growth hormone secretagogue (GHS) receptors (Howard et al., 1996). As a result of alternative splicing of a single gene, 2 GHS receptor subtypes have been cloned: GHS receptor type 1a (GHS-R1a) and GHS receptor type 1b (GHS-R1b) (Howard et al., 1996). Both of them were detected in oral epithelial cells and fibroblasts (Fig. 2A). The mRNA of ghrelin and these 2 GHS receptors were detected in human buccal epithelial cells from three healthy volunteers (Fig. 2B).

Figure 2.

Figure 2.

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Expression of ghrelin in oral epithelium. (A) Ghrelin mRNA expression in RT7, OBA9, and OKF6/TERT-2 epithelial cell lines. Total RNA was isolated from each cell line at a confluent culture, after which RT-PCR was performed for ghrelin and β-actin. Human leukemic cell lines HL-60 and THP-1, which were reported to express ghrelin mRNA, were used as positive controls (De Vriese and Delporte, 2007). (B) Ghrelin mRNA expression in human buccal mucosal cells from three healthy volunteers (one man, aged 40 yrs, and two women, ages 35 and 31 yrs). The samples were obtained from healthy individuals after they provided informed consent (Spivack et al., 2004). Total RNA was isolated from each participant, after which RT-PCR was performed for ghrelin and β-actin. (C) Ghrelin immunostaining in gingival epithelium. Periodontal tissues collected from healthy human participants were stained with polyclonal rabbit IgG anti-ghrelin antibodies (a) and control non-immunized rabbit IgG antibodies (b). The gingival epithelium shown in (C) was isolated from the cervical epithelium that faces the inner side of gingival crevice. Original magnification, 200x.

Ghrelin Expression in Oral Epithelium

To examine if ghrelin is expressed in situ, we carried out immunohistochemical evaluation on healthy human epithelial tissues. The results displayed ghrelin-positive staining in gingival epithelium (Fig. 2C). However, fibroblasts present in the lamina propria showed more prominent expression of ghrelin than those in gingival epithelium (Fig. 2C), indicating that fibroblasts, as well as gingival epithelium, albeit to a lesser extent, may contribute to the higher level of ghrelin found in GCF compared with saliva.

Effects of Ghrelin on Intracellular Calcium and cAMP Levels

GHS-R1a binds to ghrelin and leads to intracellular calcium concentration (Kojima et al., 1999). Further, GHS-R1a increases intracellular cAMP, which is a second messenger that activates the subsequent signaling cascade (Kojima and Kangawa, 2005). Therefore, we examined intracellular calcium mobilization and cAMP levels in ghrelin-stimulated oral epithelial cells. A full sequence of active ghrelin, but not control scrambled ghrelin, increased the intracellular calcium and cAMP levels in OBA9 (Fig. 3). These results indicate that oral epithelial cells can elicit intracellular signals in response to ghrelin, possibly via ligation to GHS-R1a.

Figure 3.

Figure 3.

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The effects of ghrelin on intracellular calcium and cAMP levels. (A) Confluent OBA9 cells in 96-well plates were exposed to various concentrations of ghrelin or scrambled ghrelin for 1 hr. Intracellular calcium mobilization was measured by means of a Fluo-8 no-wash calcium assay kit. Relative fluorescence intensity ratios are presented as the mean ± SD of 3 independent experiments relative to that of buffer alone. The ratios among the various concentrations of ghrelin showed statistical differences (ANOVA, p < 0.01). *Significantly different from non-treated cells (Student’s t test: p < 0.05). N.D.: no significant difference. (B) Confluent OBA9 in 6-well plates were exposed to various concentrations of ghrelin or scrambled ghrelin for 2 hrs. Medium was removed, and cells underwent lysis in 0.1 M HCl. Intracellular cAMP levels were determined by means of a cAMP assay kit. The cAMP levels among the various concentrations of ghrelin showed statistical differences (ANOVA, p < 0.01). Data are shown as the mean ± standard deviation of 3 independent experiments. *Significantly different from non-treated cells (Student’s t test: p < 0.05). N.D.: no significant difference.

Effect of Ghrelin on IL-8 Production by Oral Epithelial Cell Line Stimulated with TNF-α or LPS

Finally, because an increase of cAMP in cytoplasm is reported to elicit intracellular signals that down-modulate cytokine production in immune responses (Zidek, 1999), the finding that ghrelin increased cAMP in OBA9 cells (Fig. 3) led us to examine ghrelin’s possible immune-regulatory effect on IL-8 production from oral epithelial cells. The stimulation of oral epithelial cell lines with TNF-α up-regulated the production of IL-8. However, the addition of ghrelin into the culture of the oral epithelial cell line significantly suppressed the production of IL-8 from TNF-α-stimulated oral epithelial cells (Fig. 4). Furthermore, ghrelin also suppressed IL-8 production induced by E. coli LPS or P. gingivalis LPS (Fig. 4). It is noteworthy that control scrambled ghrelin did not affect the production of IL-8 from TNF-α- or LPS-stimulated OBA9 cells.

Figure 4.

Figure 4.

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Effects of ghrelin on IL-8 expression from TNF-α- or LPS-stimulated oral epithelial cells. RT7 and OBA9 cells were cultured for 48 hrs in the presence or absence of (A) TNF-α (10 ng/mL), (B) E. coli LPS (50 µg/mL), or (C) P. gingivalis LPS (50 µg/mL) with different concentrations of ghrelin, after which the level of IL-8 in the culture supernatant was measured by ELISA. Data are shown as the mean ± SD of 3 independent experiments. *Significantly different from TNF-α or LPS alone (Student’s t test: p < 0.05). (D) OBA9 cells were stimulated with TNF-α (10 ng/mL), E. coli LPS (50 µg/mL), or P. gingivalis LPS (50 µg/mL) for 48 hrs in the presence or absence of scrambled ghrelin (10 µg/mL). The level of IL-8 in culture supernatant was measured by ELISA. Data are shown as the mean ± SD of 3 independent experiments. N.D.: no significant difference.

Discussion

The present study demonstrated that the oral epithelium and fibroblasts produce ghrelin, and that a much higher concentration of ghrelin is present in GCF compared with saliva. Furthermore, ghrelin demonstrated its ability to suppress IL-8 production by TNF-α- or LPS-stimulated oral epithelial cells, suggesting the modulatory role of ghrelin in the innate immune response mediated by IL-8 in the context of the oral cavity.

Ghrelin plays a major role in the regulation of gastrointestinal tract function, stimulating gastric contractility and acid secretion (Date et al., 2001). Ghrelin is also responsible for a metabolic response to starvation by modulating insulin and glucose levels via its ligation to the receptor GHS-R1a expressed in the pituitary gland (Papotti et al., 2000). In addition, ghrelin was recently found to function as a negative regulator of lymphocyte proliferation (Xia et al., 2004). In the oral cavity, it has been reported that ghrelin is produced by salivary glands and released into saliva (Gröschl et al., 2005). However, the present study revealed that the concentration of ghrelin found in GCF was approximately 500-fold higher than that detected in saliva. Furthermore, ghrelin measured in serum was lower than the concentration found in either GCF or saliva. This result is supported by a study reporting higher levels of ghrelin in the saliva, as compared with the plasma, of healthy individuals (Aydin et al., 2005). Although ghrelin-positive staining was detected in the outer layer of epithelium, little or no ghrelin staining was found in the basal layer. Therefore, autocrine signaling through GHS receptors may occur prominently in the outer layer of epithelial cells. The high level of ghrelin found in GCF is thought to have a greater impact on the regulation of IL-8 production from gingival and oral epithelium than epithelial autocrine ghrelin. Therefore, we hypothesize that the high level of ghrelin detected in GCF produced prominently by fibroblasts in lamina propria and, to a lesser extent, by gingival epithelial cells, but not from the serum exudates, plays a role in IL-8 regulation.

Multiple species of bacteria colonize tooth and root surfaces and are present in the gingival crevice, and some of them are considered to be opportunistic pathogens for gingivitis as well as periodontitis. GCF plays a pivotal role in inhibiting the overgrowth of these bacteria by providing a mechanical washout flow as well as by delivering innate immune leukocytes (Delima and Van Dyke, 2003). However, it has been reported that IL-8-activated neutrophils produce superoxide, which might, in turn, injure oral epithelial cells (Altman et al., 1992). Furthermore, analysis of clinical data indicates that the concentration of pro-inflammatory cytokines, including IL-8, in GCF is associated with periodontal status (Gamonal et al., 2000). These findings imply that overactivation of neutrophils can cause periodontal tissue injury and resulting inflammation based on the production of reactive oxygen species produced from neutrophils in the context of chronic periodontal disease (Kantarci et al., 2003; Iwata et al., 2009). However, since we have established that ghrelin can suppress the production of neutrophil chemoattractant factor IL-8 from oral epithelial cells, it is plausible that ghrelin secreted in GCF may play an important role in regulating neutrophil-mediated innate immune responses, which may, in turn, protect periodontal tissue from the oxidative damage caused by the overaccumulation of neutrophils.

The concentration of ghrelin showing in vitro inhibitory effects on IL-8 produced from oral epithelial cells was higher than that measured in the GCF. However, in the physiological condition, the constant flow of GCF reaches a cumulative level of ghrelin that is sufficient to suppress the overexpression of IL-8. A previous study suggested that ghrelin secreted from saliva may promote the proliferation of oral keratinocytes (Gröschl et al., 2005). Furthermore, treatment of human microvascular endothelial cells with ghrelin increased their proliferation, migration, and angiogenesis (Li et al., 2007). Based on the findings in this study, along with previous reports, as noted above, it is suggested that ghrelin may play a role in the maintenance of gingival mucosal tissue turnover, as well as down-regulating the overreaction of innate immune response that involves IL-8-mediated neutrophil recruitment.

In conclusion, this is the first demonstration that ghrelin (1) is produced from oral epithelium, (2) is present in GCF, and (3) can inhibit TNF-α- or LPS-induced IL-8 production in oral epithelial cells, suggesting that ghrelin may function as an immune-modulation factor in the context of oral mucosa.

Footnotes

This study was supported by NIH grants DE-018310 and DE-018499 from the NIDCR and by a Pilot Grant from Harvard Catalyst. Jane Shin was the recipient of a 2010 Student Research Fellowship from the AADR.

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

References

  1. Alnema MM, Aydin S, Ozkan Y, Dagli AF, Ozercan HI, Yildirim N, et al. (2010). Ghrelin and obestatin expression in oral squamous cell carcinoma: an immunohistochemical and biochemical study. Mol Cell Biochem 339:173-179 [DOI] [PubMed] [Google Scholar]
  2. Altman LC, Baker C, Fleckman P, Luchtel D, Oda D. (1992). Neutrophil-mediated damage to human gingival epithelial cells. J Periodontal Res 27:70-79 [DOI] [PubMed] [Google Scholar]
  3. Aydin S, Halifeoglu I, Ozercan IH, Erman F, Kilic N, Aydin S, et al. (2005). A comparison of leptin and ghrelin levels in plasma and saliva of young healthy subjects. Peptides 26:647-652 [DOI] [PubMed] [Google Scholar]
  4. Date Y, Nakazato M, Murakami N, Kojima M, Kangawa K, Matsukura S. (2001). Ghrelin acts in the central nervous system to stimulate gastric acid secretion. Biochem Biophys Res Commun 280:904-907 [DOI] [PubMed] [Google Scholar]
  5. De Vriese C, Delporte C. (2007). Autocrine proliferative effect of ghrelin on leukemic HL-60 and THP-1 cells. J Endocrinol 192:199-205 [DOI] [PubMed] [Google Scholar]
  6. Delima AJ, Van Dyke TE. (2003). Origin and function of the cellular components in gingival crevice fluid. Periodontol 2000 31:55-76 [DOI] [PubMed] [Google Scholar]
  7. Dickson MA, Hahn WC, Ino Y, Ronfard V, Wu JY, Weinberg RA, et al. (2000). Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol 20:1436-1447 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dixit VD, Schaffer EM, Pyle RS, Collins GD, Sakthivel SK, Palaniappan R, et al. (2004). Ghrelin inhibits leptin- and activation-induced proinflammatory cytokine expression by human monocytes and T cells. J Clin Invest 114:57-66 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Figueredo CM, Rescala B, Teles RP, Teles FP, Fischer RG, Haffajee AD, et al. (2008). Increased interleukin-18 in gingival crevicular fluid from periodontitis patients. Oral Microbiol Immunol 23:173-176 [DOI] [PubMed] [Google Scholar]
  10. Gamonal J, Acevedo A, Bascones A, Jorge O, Silva A. (2000). Levels of interleukin-1 beta, -8, and -10 and RANTES in gingival crevicular fluid and cell populations in adult periodontitis patients and the effect of periodontal treatment. J Periodontol 71:1535-1545 [DOI] [PubMed] [Google Scholar]
  11. Ghelardoni S, Carnicelli V, Frascarelli S, Ronca-Testoni S, Zucchi R. (2006). Ghrelin tissue distribution: comparison between gene and protein expression. J Endocrinol Invest 29:115-121 [DOI] [PubMed] [Google Scholar]
  12. Gröschl M, Topf HG, Bohlender J, Zenk J, Klussmann S, Dötsch J, et al. (2005). Identification of ghrelin in human saliva: production by the salivary glands and potential role in proliferation of oral keratinocytes. Clin Chem 51:997-1006 [DOI] [PubMed] [Google Scholar]
  13. Gualillo O, Lago F, Gómez-Reino J, Casanueva FF, Dieguez C. (2003). Ghrelin, a widespread hormone: insights into molecular and cellular regulation of its expression and mechanism of action. FEBS Lett 552:105-109 [DOI] [PubMed] [Google Scholar]
  14. Gursoy UK, Könönen E, Pradhan-Palikhe P, Tervahartiala T, Pussinen PJ, Suominen-Taipale L, et al. (2010). Salivary MMP-8, TIMP-1, and ICTP as markers of advanced periodontitis. Clin Periodontol 37:487-493 [DOI] [PubMed] [Google Scholar]
  15. Hosokawa I, Hosokawa Y, Komatsuzawa H, Gonçalves RB, Karimbux N, Napimoga MH, et al. (2006). Innate immune peptide LL-37 displays distinct expression pattern from beta-defensins in inflamed gingival tissue. Clin Exp Immunol 146:218-225 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, et al. (1996). A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 273:974-976 [DOI] [PubMed] [Google Scholar]
  17. Iwata T, Kantarci A, Yagi M, Jackson T, Hasturk H, Kurihara H, et al. (2009). Ceruloplasmin induces polymorphonuclear leukocyte priming in localized aggressive periodontitis. J Periodontol 80:1300-1306 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kamata N, Fujimoto R, Tomonari M, Taki M, Nagayama M, Yasumoto S. (2004). Immortalization of human dental papilla, dental pulp, periodontal ligament cells and gingival fibroblasts by telomerase reverse transcriptase. J Oral Pathol Med 33:417-423 [DOI] [PubMed] [Google Scholar]
  19. Kantarci A, Oyaizu K, Van Dyke TE. (2003). Neutrophil-mediated tissue injury in periodontal disease pathogenesis: findings from localized aggressive periodontitis. J Periodontol 74:66-75 [DOI] [PubMed] [Google Scholar]
  20. Kojima M, Kangawa K. (2005). Ghrelin: structure and function. Physiol Rev 85:495-522 [DOI] [PubMed] [Google Scholar]
  21. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. (1999). Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656-660 [DOI] [PubMed] [Google Scholar]
  22. Li A, Cheng G, Zhu GH, Tarnawski AS. (2007). Ghrelin stimulates angiogenesis in human microvascular endothelial cells: implications beyond GH release. Biochem Biophys Res Commun 353:238-43 [DOI] [PubMed] [Google Scholar]
  23. Li J, Ireland GW, Farthing PM, Thornhill MH. (1996). Epidermal and oral keratinocytes are induced to produce RANTES and IL-8 by cytokine stimulation. J Invest Dermatol 106:661-666 [DOI] [PubMed] [Google Scholar]
  24. Ohta K, Shigeishi H, Taki M, Nishi H, Higashikawa K, Takechi M, et al. (2008). Different regulation of CXCL9, CXCL10, and CXCL11 expression induced by IFN-γ, TNF-α and IL-4 in human oral keratinocytes and fibroblasts. J Dent Res 87:1161-1165 [DOI] [PubMed] [Google Scholar]
  25. Papotti M, Ghè C, Cassoni P, Catapano F, Deghenghi R, Ghigo E, et al. (2000). Growth hormone secretagogue binding sites in peripheral human tissues. J Clin Endocrinol Metab 85:3803-3807 [DOI] [PubMed] [Google Scholar]
  26. Spivack SD, Hurteau GJ, Jain R, Kumar SV, Aldous KM, Gierthy JF, et al. (2004). Gene-environment interaction signatures by quantitative mRNA profiling in exfoliated buccal mucosal cells. Cancer Res 64:6805-6813 [DOI] [PubMed] [Google Scholar]
  27. Sugiyama A, Uehara A, Iki K, Matsushita K, Nakamura R, Ogawa T, et al. (2002). Activation of human gingival epithelial cells by cell-surface components of black-pigmented bacteria: augmentation of production of interleukin-8, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor and expression of intercellular adhesion molecule 1. J Med Microbiol 51:27-33 [DOI] [PubMed] [Google Scholar]
  28. Waseem T, Duxbury M, Ito H, Ashley SW, Robinson MK. (2008). Exogenous ghrelin modulates release of pro-inflammatory and anti-inflammatory cytokines in LPS-stimulated macrophages through distinct signaling pathways. Surgery 143:334-342 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Xia Q, Pang W, Pan H, Zheng Y, Kang JS, Zhu SG. (2004). Effects of ghrelin on the proliferation and secretion of splenic T lymphocytes in mice. Regul Pept 122:173-178 [DOI] [PubMed] [Google Scholar]
  30. Zidek Z. (1999). Adenosine-cyclic AMP pathways and cytokine expression. Eur Cytokine Netw 10:319-328 [PubMed] [Google Scholar]

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