Studies of cell signaling in a reconstructed human epidermis exposed to sensitizers: IL-8 synthesis and release depend on EGFR activation


Models of reconstructed human epidermis (RHE) holding proliferating and fully differentiated cultured keratinocytes allow in vitro investigation of early molecular and cellular epidermal events during the complex response of keratinocytes at the onset of allergic contact dermatitis (ACD) or sensitization. In this study, data collected on RHE exposed to well-characterized sensitizing chemicals, such as dinitrofluorobenzene, oxazolone, cinnamaldehyde and isoeugenol, revealed a transient expression of IL-8 mRNA in association with abundant IL-8 cell release. Investigations of keratinocyte signaling illustrate transient activation by tissue exposure to sensitizing chemicals of the epidermal growth factor receptor (EGFR). This activation of EGFR tyrosine kinase is involved in the expression and release of IL-8. The IL-8 release appears also to be partially dependent on p38 and ERK 1/2 MAPK activation. Moreover, data suggest that heparin-binding EGF-like growth factor (HB-EGF) expression and release induced after exposure of RHE to sensitizing chemicals are also under the control of EGFR tyrosine kinase activity, independently of the IL-8 expression and release. Mechanistic approach of keratinocyte responses in the context of RHE underlying regulation of expression and release of epidermal cytokines and growth factors after topical application of sensitizing chemicals is proposed to identify biomarkers which could then be analysed for in vitro toxicological screening of new or undefined compounds.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9



Atopic dermatitis


Allergic contact dermatitis


Benzalkonium chloride




Dendritic cell




G protein-coupled receptor


Irritant contact dermatitis






Keratinocyte growth factor


Mitogen-activated protein kinase






Reconstructed human epidermis


  1. 1.

    Aiba S, Manome H, Nakagawa S, Mollah ZU, Mizuashi M, Ohtani T, Yoshino Y, Tagami H (2003) p38 Mitogen-activated protein kinase and extracellular signal-regulated kinases play distinct roles in the activation of dendritic cells by two representative haptens, NiCl2 and 2,4-dinitrochlorobenzene. J Invest Dermatol 120:390–399

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Albanesi C, De Pita O, Girolomoni G (2007) Resident skin cells in psoriasis: a special look at the pathogenetic functions of keratinocytes. Clin Dermatol 25:581–588

    PubMed  Article  Google Scholar 

  3. 3.

    Albanesi C, Scarponi C, Giustizieri ML, Girolomoni G (2005) Keratinocytes in inflammatory skin diseases. Curr Drug Targets Inflamm Allergy 4:329–334

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Blaydon DC, Biancheri P, Di WL, Plagnol V, Cabral RM, Brooke MA, van Heel DA, Ruschendorf F, Toynbee M, Walne A, O’Toole EA, Martin JE, Lindley K, Vulliamy T, Abrams DJ, MacDonald TT, Harper JI, Kelsell DP (2011) Inflammatory skin and bowel disease linked to ADAM17 deletion. N Engl J Med 365:1502–1508

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Chiarugi P, Cirri P (2003) Redox regulation of protein tyrosine phosphatases during receptor tyrosine kinase signal transduction. Trends Biochem Sci 28:509–514

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Cook PW, Piepkorn M, Clegg CH, Plowman GD, DeMay JM, Brown JR, Pittelkow MR (1997) Transgenic expression of the human amphiregulin gene induces a psoriasis-like phenotype. J Clin Invest 100:2286–2294

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Coquette A, Berna N, Vandenbosch A, Rosdy M, De Wever B, Poumay Y (2003) Analysis of interleukin-1alpha (IL-1alpha) and interleukin-8 (IL-8) expression and release in in vitro reconstructed human epidermis for the prediction of in vivo skin irritation and/or sensitization. Toxicol In Vitro 17:311–321

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Coquette A, Berna N, Vandenbosch A, Rosdy M, Poumay Y (1999) Differential expression and release of cytokines by an in vitro reconstructed human epidermis following exposure to skin irritant and sensitizing chemicals. Toxicol In Vitro 13:867–877

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    De Luca A, Carotenuto A, Rachiglio A, Gallo M, Maiello MR, Aldinucci D, Pinto A, Normanno N (2008) The role of the EGFR signaling in tumor microenvironment. J Cell Physiol 214:559–567

    PubMed  Article  Google Scholar 

  10. 10.

    Dinkova-Kostova AT, Holtzclaw WD, Cole RN, Itoh K, Wakabayashi N, Katoh Y, Yamamoto M, Talalay P (2002) Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc Natl Acad Sci USA 99:11908–11913

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Divkovic M, Pease CK, Gerberick GF, Basketter DA (2005) Hapten-protein binding: from theory to practical application in the in vitro prediction of skin sensitization. Contact Dermat 53:189–200

    Article  CAS  Google Scholar 

  12. 12.

    Esche C, de Benedetto A, Beck LA (2004) Keratinocytes in atopic dermatitis: inflammatory signals. Curr Allergy Asthma Rep 4:276–284

    PubMed  Article  Google Scholar 

  13. 13.

    Funding AT, Johansen C, Gaestel M, Bibby BM, Lilleholt LL, Kragballe K, Iversen L (2009) Reduced oxazolone-induced skin inflammation in MAPKAP kinase 2 knockout mice. J Invest Dermatol 129:891–898

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Giltaire S, Herphelin F, Frankart A, Herin M, Stoppie P, Poumay Y (2009) The CYP26 inhibitor R115866 potentiates the effects of all-trans retinoic acid on cultured human epidermal keratinocytes. Br J Dermatol 160:505–513

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Giltaire S, Lambert S, Poumay Y (2011) HB-EGF synthesis and release induced by cholesterol depletion of human epidermal keratinocytes is controlled by extracellular ATP and involves both p38 and ERK1/2 signaling pathways. J Cell Physiol 226:1651–1659

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Gober MD, Gaspari AA (2008) Allergic contact dermatitis. Curr Dir Autoimmun 10:1–26

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Grone A (2002) Keratinocytes and cytokines. Vet Immunol Immunopathol 88:1–12

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Guinea-Viniegra J, Zenz R, Scheuch H, Hnisz D, Holcmann M, Bakiri L, Schonthaler HB, Sibilia M, Wagner EF (2009) TNFalpha shedding and epidermal inflammation are controlled by Jun proteins. Genes Dev 23:2663–2674

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Itoh Y, Joh T, Tanida S, Sasaki M, Kataoka H, Itoh K, Oshima T, Ogasawara N, Togawa S, Wada T, Kubota H, Mori Y, Ohara H, Nomura T, Higashiyama S, Itoh M (2005) IL-8 promotes cell proliferation and migration through metalloproteinase-cleavage proHB-EGF in human colon carcinoma cells. Cytokine 29:275–282

    PubMed  CAS  Google Scholar 

  20. 20.

    Jans R, Atanasova G, Jadot M, Poumay Y (2004) Cholesterol depletion upregulates involucrin expression in epidermal keratinocytes through activation of p38. J Invest Dermatol 123:564–573

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    Johnston A, Gudjonsson JE, Aphale A, Guzman AM, Stoll SW, Elder JT (2011) EGFR and IL-1 signaling synergistically promote keratinocyte antimicrobial defenses in a differentiation-dependent manner. J Invest Dermatol 131:329–337

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Jost M, Kari C, Rodeck U (2000) The EGF receptor—an essential regulator of multiple epidermal functions. Eur J Dermatol 10:505–510

    PubMed  CAS  Google Scholar 

  23. 23.

    Koeper LM, Schulz A, Ahr HJ, Vohr HW (2007) In vitro differentiation of skin sensitizers by cell signaling pathways. Toxicology 242:144–152

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    Kyriakis JM, Avruch J (2001) Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81:807–869

    PubMed  CAS  Google Scholar 

  25. 25.

    Lambert S, Ameels H, Gniadecki R, Herin M, Poumay Y (2008) Internalization of EGF receptor following lipid rafts disruption in keratinocytes is delayed and dependent on p38 MAPK activation. J Cell Physiol 217:834–845

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Lambert S, Frankart A, Poumay Y (2009) p38 MAPK-regulated EGFR internalization takes place in keratinocyte monolayer during stress conditions. Arch Dermatol Res 302:229–233

    PubMed  Article  Google Scholar 

  27. 27.

    Lambert S, Vind-Kezunovic D, Karvinen S, Gniadecki R (2006) Ligand-independent activation of the EGFR by lipid raft disruption. J Invest Dermatol 126:954–962

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Lee DC, Sunnarborg SW, Hinkle CL, Myers TJ, Stevenson MY, Russell WE, Castner BJ, Gerhart MJ, Paxton RJ, Black RA, Chang A, Jackson LF (2003) TACE/ADAM17 processing of EGFR ligands indicates a role as a physiological convertase. Ann NY Acad Sci 995:22–38

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    Li Y, Yan J, Xu W, Wang H, Xia Y (2011) Lentivirus-mediated ADAM17 RNA interference inhibited interleukin-8 expression via EGFR signaling in lung epithelial cells. Inflammation. doi:10.1007/s10753-01

    Google Scholar 

  30. 30.

    Mascia F, Cataisson C, Lee TC, Threadgill D, Mariani V, Amerio P, Chandrasekhara C, Souto Adeva G, Girolomoni G, Yuspa SH, Pastore S (2010) EGFR regulates the expression of keratinocyte-derived granulocyte/macrophage colony-stimulating factor in vitro and in vivo. J Invest Dermatol 130:682–693

    PubMed  Article  CAS  Google Scholar 

  31. 31.

    Mascia F, Mariani V, Girolomoni G, Pastore S (2003) Blockade of the EGF receptor induces a deranged chemokine expression in keratinocytes leading to enhanced skin inflammation. Am J Pathol 163:303–312

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    Mathay C, Giltaire S, Minner F, Bera E, Herin M, Poumay Y (2008) Heparin-binding EGF-like growth factor is induced by disruption of lipid rafts and oxidative stress in keratinocytes and participates in the epidermal response to cutaneous wounds. J Invest Dermatol 128:717–727

    PubMed  Article  CAS  Google Scholar 

  33. 33.

    Mathay C, Pierre M, Pittelkow MR, Depiereux E, Nikkels AF, Colige A, Poumay Y (2011) Transcriptional profiling after lipid raft disruption in keratinocytes identifies critical mediators of atopic dermatitis pathways. J Invest Dermatol 131:46–58

    PubMed  Article  CAS  Google Scholar 

  34. 34.

    McGovern T, Risse PA, Tsuchiya K, Hassan M, Frigola G, Martin JG (2010) LTD induces HB-EGF-dependent CXCL8 release through EGFR activation in human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 299:808–815

    Article  Google Scholar 

  35. 35.

    Migdal C, Foggia L, Tailhardat M, Courtellemont P, Haftek M, Serres M (2010) Sensitization effect of thimerosal is mediated in vitro via reactive oxygen species and calcium signaling. Toxicology 274:1–9

    PubMed  Article  CAS  Google Scholar 

  36. 36.

    Migdal C, Tailhardat M, Courtellemont P, Haftek M, Serres M (2010) Responsiveness of human monocyte-derived dendritic cells to thimerosal and mercury derivatives. Toxicol Appl Pharmacol 246:66–73

    PubMed  Article  CAS  Google Scholar 

  37. 37.

    Miller LS, Sorensen OE, Liu PT, Jalian HR, Eshtiaghpour D, Behmanesh BE, Chung W, Starner TD, Kim J, Sieling PA, Ganz T, Modlin RL (2005) TGF-alpha regulates TLR expression and function on epidermal keratinocytes. J Immunol 174:6137–6143

    PubMed  CAS  Google Scholar 

  38. 38.

    Minner F, Poumay Y (2009) Candidate housekeeping genes require evaluation before their selection for studies of human epidermal keratinocytes. J Invest Dermatol 129:770–773

    PubMed  Article  CAS  Google Scholar 

  39. 39.

    Mitamura T, Higashiyama S, Taniguchi N, Klagsbrun M, Mekada E (1995) Diphtheria toxin binds to the epidermal growth factor (EGF)-like domain of human heparin-binding EGF-like growth factor/diphtheria toxin receptor and inhibits specifically its mitogenic activity. J Biol Chem 270:1015–1019

    PubMed  Article  CAS  Google Scholar 

  40. 40.

    Mitjans M, Galbiati V, Lucchi L, Viviani B, Marinovich M, Galli CL, Corsini E (2010) Use of IL-8 release and p38 MAPK activation in THP-1 cells to identify allergens and to assess their potency in vitro. Toxicol In Vitro 24:1803–1809

    PubMed  Article  CAS  Google Scholar 

  41. 41.

    Pastore S, Mascia F, Girolomoni G (2006) The contribution of keratinocytes to the pathogenesis of atopic dermatitis. Eur J Dermatol 16:125–131

    PubMed  CAS  Google Scholar 

  42. 42.

    Pastore S, Mascia F, Mariani V, Girolomoni G (2006) Keratinocytes in skin inflammation. Expert Rev Dermatol 1:279–291

    Article  CAS  Google Scholar 

  43. 43.

    Pastore S, Mascia F, Mariani V, Girolomoni G (2008) The epidermal growth factor receptor system in skin repair and inflammation. J Invest Dermatol 128:1365–1374

    PubMed  Article  CAS  Google Scholar 

  44. 44.

    Pastore S, Mascia F, Mariotti F, Dattilo C, Mariani V, Girolomoni G (2005) ERK1/2 regulates epidermal chemokine expression and skin inflammation. J Immunol 174:5047–5056

    PubMed  CAS  Google Scholar 

  45. 45.

    Piepkorn M, Predd H, Underwood R, Cook P (2003) Proliferation-differentiation relationships in the expression of heparin-binding epidermal growth factor-related factors and erbB receptors by normal and psoriatic human keratinocytes. Arch Dermatol Res 295:93–101

    PubMed  Article  CAS  Google Scholar 

  46. 46.

    Poumay Y, Coquette A (2007) Modelling the human epidermis in vitro: tools for basic and applied research. Arch Dermatol Res 298:361–369

    PubMed  Article  Google Scholar 

  47. 47.

    Poumay Y, Dupont F, Marcoux S, Leclercq-Smekens M, Herin M, Coquette A (2004) A simple reconstructed human epidermis: preparation of the culture model and utilization in in vitro studies. Arch Dermatol Res 296:203–211

    PubMed  Article  CAS  Google Scholar 

  48. 48.

    Reich K, Garbe C, Blaschke V, Maurer C, Middel P, Westphal G, Lippert U, Neumann C (2001) Response of psoriasis to interleukin-10 is associated with suppression of cutaneous type 1 inflammation, downregulation of the epidermal interleukin-8/CXCR2 pathway and normalization of keratinocyte maturation. J Invest Dermatol 116:319–329

    PubMed  Article  CAS  Google Scholar 

  49. 49.

    Roupe KM, Nybo M, Sjobring U, Alberius P, Schmidtchen A, Sorensen OE (2010) Injury is a major inducer of epidermal innate immune responses during wound healing. J Invest Dermatol 130:1167–1177

    PubMed  Article  CAS  Google Scholar 

  50. 50.

    Samet JM, Tal TL (2010) Toxicological disruption of signaling homeostasis: tyrosine phosphatases as targets. Annu Rev Pharmacol Toxicol 50:215–235

    PubMed  Article  CAS  Google Scholar 

  51. 51.

    Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387:569–572

    PubMed  Article  CAS  Google Scholar 

  52. 52.

    Terao M, Murota H, Kitaba S, Katayama I (2010) Tumor necrosis factor-alpha processing inhibitor-1 inhibits skin fibrosis in a bleomycin-induced murine model of scleroderma. Exp Dermatol 19:38–43

    PubMed  Article  CAS  Google Scholar 

  53. 53.

    Trompezinski S, Migdal C, Tailhardat M, Le Varlet B, Courtellemont P, Haftek M, Serres M (2008) Characterization of early events involved in human dendritic cell maturation induced by sensitizers: cross talk between MAPK signalling pathways. Toxicol Appl Pharmacol 230:397–406

    PubMed  Article  CAS  Google Scholar 

  54. 54.

    White KJ, Maffei VJ, Newton-West M, Swerlick RA (2011) Irritant activation of epithelial cells is mediated via protease-dependent EGFR activation. J Invest Dermatol 13:435–442

    Article  Google Scholar 

  55. 55.

    Xu Y, Voorhees JJ, Fisher GJ (2006) Epidermal growth factor receptor is a critical mediator of ultraviolet B irradiation-induced signal transduction in immortalized human keratinocyte HaCaT cells. Am J Pathol 169:823–830

    PubMed  Article  CAS  Google Scholar 

  56. 56.

    Zhang Z, Oliver P, Lancaster JR Jr, Schwarzenberger PO, Joshi MS, Cork J, Kolls JK (2001) Reactive oxygen species mediate tumor necrosis factor alpha-converting, enzyme-dependent ectodomain shedding induced by phorbol myristate acetate. FASEB J 15:303–305

    PubMed  Article  CAS  Google Scholar 

  57. 57.

    Zhu YM, Woll PJ (2005) Mitogenic effects of interleukin-8/CXCL8 on cancer cells. Future Oncol 1:699–704

    PubMed  Article  CAS  Google Scholar 

Download references


Technical help from D. Van Vlaender, F. Herphelin and V. De Glas is gratefully acknowledged. Contract grant sponsor was Henkel AG & CoKGaA, FNRS and FRFC 2.4.522.10F.

Conflict of interest

The authors have no conflict of interest to declare.

Author information



Corresponding author

Correspondence to Yves Poumay.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 189 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Frankart, A., Coquette, A., Schroeder, KR. et al. Studies of cell signaling in a reconstructed human epidermis exposed to sensitizers: IL-8 synthesis and release depend on EGFR activation. Arch Dermatol Res 304, 289–303 (2012).

Download citation


  • Keratinocytes
  • RHE (reconstructed human epidermis)
  • Sensitizers
  • IL-8
  • EGFR signaling