Clinical Oral Investigations

, Volume 19, Issue 2, pp 385–399

Sterile-filtered saliva is a strong inducer of IL-6 and IL-8 in oral fibroblasts

  • Barbara Cvikl
  • Adrian Lussi
  • Andreas Moritz
  • Anton Sculean
  • Reinhard Gruber
Original Article



Saliva has been implicated to support oral wound healing, a process that requires a transient inflammatory reaction. However, definitive proof that saliva can provoke an inflammatory response remained elusive.

Materials and methods

We investigated the ability of freshly harvested and sterile-filtered saliva to cause an inflammatory response of oral fibroblasts and epithelial cells. The expression of cytokines and chemokines was assessed by microarray, RT-PCR, immunoassays, and Luminex technology. The involvement of signaling pathways was determined by Western blot analysis and pharmacologic inhibitors.


We report that sterile-filtered whole saliva was a potent inducer of IL-6 and IL-8 in fibroblasts from the gingiva, the palate, and the periodontal ligament, but not of oral epithelial cells. This strong inflammatory response requires nuclear factor-kappa B and mitogen-activated protein kinase signaling. The pro-inflammatory capacity is heat stable and has a molecular weight of <40 kDa. Genome-wide microarrays and Luminex technology further revealed that saliva substantially increased expression of other inflammatory genes and various chemokines. To preclude that the observed pro-inflammatory activity is the result of oral bacteria, sterile-filtered parotid saliva, collected under almost aseptic conditions, was used and also increased IL-6 and IL-8 expression in gingiva fibroblasts. The inflammatory response was, furthermore, independent of MYD88, an adapter protein of the Toll-like receptor signaling pathway.


We conclude that saliva can provoke a robust inflammatory response in oral fibroblasts involving the classical nuclear factor-kappa B and mitogen-activated protein kinase signaling pathway.

Clinical relevance

Since fibroblasts but not epithelial cells show a strong inflammatory response, saliva may support the innate immunity of defect sites exposing the oral connective tissue.


Saliva Inflammation Fibroblasts Cytokines NFκB Microarray Luminex technology 


  1. 1.
    du Toit DF, Nortje C (2004) Salivary glands: applied anatomy and clinical correlates. SADJ 59:65–66, 69-71, 73-4PubMedGoogle Scholar
  2. 2.
    Cheaib Z, Lussi A (2011) Impact of acquired enamel pellicle modification on initial dental erosion. Caries Res 45:107–112. doi:10.1159/000324803 CrossRefPubMedGoogle Scholar
  3. 3.
    Jakubovics NS, Kolenbrander PE (2010) The road to ruin: the formation of disease-associated oral biofilms. Oral Dis 16:729–739. doi:10.1111/j.1601-0825.2010.01701.x CrossRefPubMedGoogle Scholar
  4. 4.
    Yao Y, Berg EA, Costello CE, Troxler RF, Oppenheim FG (2003) Identification of protein components in human acquired enamel pellicle and whole saliva using novel proteomics approaches. J Biol Chem 278:5300–5308. doi:10.1074/jbc.M206333200 CrossRefPubMedGoogle Scholar
  5. 5.
    Nobbs AH, Jenkinson HF, Jakubovics NS (2011) Stick to your gums: mechanisms of oral microbial adherence. J Dent Res 90:1271–1278. doi:10.1177/0022034511399096 CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Bodner L, Knyszynski A, Adler-Kunin S, Danon D (1991) The effect of selective desalivation on wound healing in mice. Exp Gerontol 26:357–363CrossRefPubMedGoogle Scholar
  7. 7.
    Bodner L, Dayan D, Rothchild D, Hammel I (1991) Extraction wound healing in desalivated rats. J Oral Pathol Med 20:176–178CrossRefPubMedGoogle Scholar
  8. 8.
    Grossman N, Binyamin LA, Bodner L (2004) Effect of rat salivary glands extracts on the proliferation of cultured skin cells—a wound healing model. Cell Tissue Bank 5:205–212. doi:10.1007/s10561-004-4367-7 PubMedGoogle Scholar
  9. 9.
    Proksch S, Steinberg T, Keller C, Wolkewitz M, Wiedmann-Al-Ahmad M, Finkenzeller G, Hannig C, Hellwig E, Al-Ahmad A (2012) Human saliva exposure modulates bone cell performance in vitro. Clin Oral Investig 16:69–77. doi:10.1007/s00784-010-0506-7 CrossRefPubMedGoogle Scholar
  10. 10.
    Komine K, Kuroishi T, Ozawa A, Komine Y, Minami T, Shimauchi H, Sugawara S (2007) Cleaved inflammatory lactoferrin peptides in parotid saliva of periodontitis patients. Mol Immunol 44:1498–1508. doi:10.1016/j.molimm.2006.09.003 CrossRefPubMedGoogle Scholar
  11. 11.
    Ohshima M, Sato M, Ishikawa M, Maeno M, Otsuka K (2002) Physiologic levels of epidermal growth factor in saliva stimulate cell migration of an oral epithelial cell line, HO-1-N-1. Eur J Oral Sci 110:130–136CrossRefPubMedGoogle Scholar
  12. 12.
    Humphreys-Beher MG, Macauley SP, Chegini N, van Setten G, Purushotham K, Stewart C, Wheeler TT, Schultz GS (1994) Characterization of the synthesis and secretion of transforming growth factor-alpha from salivary glands and saliva. Endocrinology 134:963–970PubMedGoogle Scholar
  13. 13.
    Oudhoff MJ, Bolscher JG, Nazmi K, Kalay H, van ’t Hof W, Amerongen AV, Veerman EC (2008) Histatins are the major wound-closure stimulating factors in human saliva as identified in a cell culture assay. FASEB J 22:3805–3812. doi:10.1096/fj.08-112003 CrossRefPubMedGoogle Scholar
  14. 14.
    Kato T, Imatani T, Minaguchi K, Saitoh E, Okuda K (2002) Salivary cystatins induce interleukin-6 expression via cell surface molecules in human gingival fibroblasts. Mol Immunol 39:423–430CrossRefPubMedGoogle Scholar
  15. 15.
    Pradhan L, Cai X, Wu S, Andersen ND, Martin M, Malek J, Guthrie P, Veves A, Logerfo FW (2011) Gene expression of pro-inflammatory cytokines and neuropeptides in diabetic wound healing. J Surg Res 167:336–342. doi:10.1016/j.jss.2009.09.012 CrossRefPubMedGoogle Scholar
  16. 16.
    Papanicolaou DA, Wilder RL, Manolagas SC, Chrousos GP (1998) The pathophysiologic roles of interleukin-6 in human disease. Ann Intern Med 128:127–137CrossRefPubMedGoogle Scholar
  17. 17.
    Baggiolini M, Walz A, Kunkel SL (1989) Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils. J Clin Invest 84:1045–1049. doi:10.1172/JCI114265 CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Scheres N, Laine ML, de Vries TJ, Everts V, van Winkelhoff AJ (2010) Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis. J Periodontal Res 45:262–270. doi:10.1111/j.1600-0765.2009.01229.x CrossRefPubMedGoogle Scholar
  19. 19.
    Uehara A, Takada H (2007) Functional TLRs and NODs in human gingival fibroblasts. J Dent Res 86:249–254CrossRefPubMedGoogle Scholar
  20. 20.
    Gornowicz A, Bielawska A, Bielawski K, Grabowska SZ, Wojcicka A, Zalewska M, Maciorkowska E (2012) Pro-inflammatory cytokines in saliva of adolescents with dental caries disease. Ann Agric Environ Med AAEM 19:711–716Google Scholar
  21. 21.
    Ertugrul AS, Sahin H, Dikilitas A, Alpaslan N, Bozoglan A (2013) Comparison of CCL28, interleukin-8, interleukin-1beta and tumor necrosis factor-alpha in subjects with gingivitis, chronic periodontitis and generalized aggressive periodontitis. J Periodontal Res 48:44–51. doi:10.1111/j.1600-0765.2012.01500.x CrossRefPubMedGoogle Scholar
  22. 22.
    Scott AE, Milward M, Linden GJ, Matthews JB, Carlile MJ, Lundy FT, Naeeni MA, Lorraine Martin S, Walker B, Kinane D, Brock GR, Chapple IL (2012) Mapping biological to clinical phenotypes during the development (21 days) and resolution (21 days) of experimental gingivitis. J Clin Periodontol 39:123–131. doi:10.1111/j.1600-051X.2011.01825.x CrossRefPubMedGoogle Scholar
  23. 23.
    Teles RP, Likhari V, Socransky SS, Haffajee AD (2009) Salivary cytokine levels in subjects with chronic periodontitis and in periodontally healthy individuals: a cross-sectional study. J Periodontal Res 44:411–417. doi:10.1111/j.1600-0765.2008.01119.x CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    Severino VO, Napimoga MH, de Lima Pereira SA (2011) Expression of IL-6, IL-10, IL-17 and IL-8 in the peri-implant crevicular fluid of patients with peri-implantitis. Arch Oral Biol 56:823–828. doi:10.1016/j.archoralbio.2011.01.006 CrossRefPubMedGoogle Scholar
  25. 25.
    Groschl M (2009) The physiological role of hormones in saliva. Bioessays 31:843–852. doi:10.1002/bies.200900013 CrossRefPubMedGoogle Scholar
  26. 26.
    Services USDoHaH (2004) Guidance for industry, sterile drug products produced by aseptic processing—current good manufacturing practice. FDA, RockvilleGoogle Scholar
  27. 27.
    Jin J, Sundararaj KP, Samuvel DJ, Zhang X, Li Y, Lu Z, Lopes-Virella MF, Huang Y (2012) Different signaling mechanisms regulating IL-6 expression by LPS between gingival fibroblasts and mononuclear cells: seeking the common target. Clin Immunol 143:188–199. doi:10.1016/j.clim.2012.01.019 CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Hwang YS, Lee SK, Park KK, Chung WY (2012) Secretion of IL-6 and IL-8 from lysophosphatidic acid-stimulated oral squamous cell carcinoma promotes osteoclastogenesis and bone resorption. Oral Oncol 48:40–48. doi:10.1016/j.oraloncology.2011.08.022 CrossRefPubMedGoogle Scholar
  29. 29.
    Tang CH, Hsu CJ, Yang WH, Fong YC (2010) Lipoteichoic acid enhances IL-6 production in human synovial fibroblasts via TLR2 receptor, PKCdelta and c-Src dependent pathways. Biochem Pharmacol 79:1648–1657. doi:10.1016/j.bcp.2010.01.025 CrossRefPubMedGoogle Scholar
  30. 30.
    Thomadaki K, Helmerhorst EJ, Tian N, Sun X, Siqueira WL, Walt DR, Oppenheim FG (2011) Whole-saliva proteolysis and its impact on salivary diagnostics. J Dent Res 90:1325–1330. doi:10.1177/0022034511420721 CrossRefPubMedCentralPubMedGoogle Scholar
  31. 31.
    Miyazono K, Hellman U, Wernstedt C, Heldin CH (1988) Latent high molecular weight complex of transforming growth factor beta 1. Purification from human platelets and structural characterization. J Biol Chem 263:6407–6415PubMedGoogle Scholar
  32. 32.
    Takanashi N, Tomosada Y, Villena J, Murata K, Takahashi T, Chiba E, Tohno M, Shimazu T, Aso H, Suda Y, Ikegami S, Itoh H, Kawai Y, Saito T, Alvarez S, Kitazawa H (2013) Advanced application of bovine intestinal epithelial cell line for evaluating regulatory effect of lactobacilli against heat-killed enterotoxigenic Escherichia coli-mediated inflammation. BMC Microbiol 13:54. doi:10.1186/1471-2180-13-54 CrossRefPubMedCentralPubMedGoogle Scholar
  33. 33.
    Yan W, Apweiler R, Balgley BM, Boontheung P, Bundy JL, Cargile BJ, Cole S, Fang X, Gonzalez-Begne M, Griffin TJ, Hagen F, Hu S, Wolinsky LE, Lee CS, Malamud D, Melvin JE, Menon R, Mueller M, Qiao R, Rhodus NL, Sevinsky JR, States D, Stephenson JL, Than S, Yates JR, Yu W, Xie H, Xie Y, Omenn GS, Loo JA, Wong DT (2009) Systematic comparison of the human saliva and plasma proteomes. Proteomics Clin Appl 3:116–134. doi:10.1002/prca.200800140 CrossRefPubMedCentralPubMedGoogle Scholar
  34. 34.
    Loo JA, Yan W, Ramachandran P, Wong DT (2010) Comparative human salivary and plasma proteomes. J Dent Res 89:1016–1023. doi:10.1177/0022034510380414 CrossRefPubMedCentralPubMedGoogle Scholar
  35. 35.
    Odqvist L, Sanchez-Beato M, Montes-Moreno S, Martin-Sanchez E, Pajares R, Sanchez-Verde L, Ortiz-Romero PL, Rodriguez J, Rodriguez-Pinilla SM, Iniesta-Martinez F, Solera-Arroyo JC, Ramos-Asensio R, Flores T, Palanca JM, Bragado FG, Franjo PD, Piris MA (2013) NIK controls classical and alternative NF-kappaB activation and is necessary for the survival of human T-cell lymphoma cells. Clin Cancer Res Off J Am Assoc Cancer Res 19:2319–2330. doi:10.1158/1078-0432.CCR-12-3151 CrossRefGoogle Scholar
  36. 36.
    Abend JR, Uldrick T, Ziegelbauer JM (2010) Regulation of tumor necrosis factor-like weak inducer of apoptosis receptor protein (TWEAKR) expression by Kaposi’s sarcoma-associated herpesvirus microRNA prevents TWEAK-induced apoptosis and inflammatory cytokine expression. J Virol 84:12139–12151. doi:10.1128/JVI.00884-10 CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Barbara Cvikl
    • 1
    • 2
    • 3
  • Adrian Lussi
    • 1
  • Andreas Moritz
    • 3
  • Anton Sculean
    • 4
  • Reinhard Gruber
    • 1
    • 2
    • 4
  1. 1.Department of Preventive, Restorative and Pediatric Dentistry, School of Dental MedicineUniversity of BernBernSwitzerland
  2. 2.Laboratory of Oral Cell Biology, School of Dental MedicineUniversity of BernBernSwitzerland
  3. 3.Department of Conservative Dentistry and PeriodontologyMedical University of ViennaViennaAustria
  4. 4.Department of Periodontology, School of Dental MedicineUniversity of BernBernSwitzerland

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