Immunologic Research

, Volume 58, Issue 1, pp 40–50 | Cite as

Systemic immunogenicity of para-Phenylenediamine and Diphenylcyclopropenone: two potent contact allergy-inducing haptens

  • Jesper Dyrendom SvalgaardEmail author
  • Carina Særmark
  • Morten Dall
  • Karsten Buschard
  • Jeanne D. Johansen
  • Kåre Engkilde


p-Phenylenediamine (PPD) and Diphenylcyclopropenone (DPCP) are two potent haptens. Both haptens are known to cause delayed-type hypersensitivity, involving a cytokine response and local infiltration of T-cell subpopulations, resulting in contact dermatitis. We investigated the systemic immune effects of PPD and DPCP, two relatively unexplored skin allergens. The dorsal sides of the ears of BALB/c mice were exposed to PPD or DPCP (0.1 % w/v or 0.01 % w/v), or vehicle alone. Mice were treated once daily for 3 days (induction period) and subsequently twice per week for 8 weeks. Local and systemic immune responses in the auricular and pancreatic lymph nodes, spleen, liver, serum, and ears were analyzed with cytokine profiling MSD, flow cytometry, and qPCR. Ear swelling increased significantly in mice treated with 1 % PPD, 0.01 % DPCP or 0.1 % DPCP, compared with vehicle treatment, indicating that the mice were sensitized and that there was a local inflammation. Auricular lymph nodes, pancreatic lymph nodes, spleen, and liver showed changes in regulatory T-cell, B-cell, and NKT-cell frequencies, and increased activation of CD8+ T cells and B cells. Intracellular cytokine profiling revealed an increase in the IFN-γ- and IL-4-positive NKT cells present in the liver following treatment with both haptens. Moreover, we saw a tendency toward a systemic increase in IL-17A. We observed systemic immunological effects of PPD and DPCP. Furthermore, concentrations too low to increase ear thickness and cause clinical symptoms may still prime the immune system. These systemic immunological effects may potentially predispose individuals to certain diseases.


p-Phenylenediamine Diphenylcyclopropenone Contact hypersensitivity Contact allergy Invariant NKT cells Foxp3 



Contact hypersensitivity


Delayed-type hypersensitivity






Auricular lymph nodes


Pancreatic lymph nodes



The authors would like to thank NIH Tetramer Core Facility for providing CD1d tetramers. This study was financed by the Capital Region Research Fund and the Beckett Foundation.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12026_2013_8482_MOESM1_ESM.pdf (364 kb)
Supplementary material 1 (PDF 363 kb)


  1. 1.
    Macatonia SE, Knight SC, Edwards AJ, Griffiths S, Fryer P. Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate. Functional and morphological studies. J Exp Med. 1987;166:1654–67.CrossRefPubMedGoogle Scholar
  2. 2.
    Saeki H, Moore AM, Brown MJ, Hwang ST. Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes. J Immunol. 1999;162:2472–5.PubMedGoogle Scholar
  3. 3.
    Szczepanik M, Akahira-Azuma M, Bryniarski K, Tsuji RF, Kawikova I, Ptak W, Kiener C, Campos RA, Askenase PW. B-1 B cells mediate required early T cell recruitment to elicit protein-induced delayed-type hypersensitivity. J Immunol. 2003;171:6225–35.CrossRefPubMedGoogle Scholar
  4. 4.
    Akiba H, Kehren J, Ducluzeau MT, Krasteva M, Horand F, Kaiserlian D, Kaneko F, Nicolas JF. Skin inflammation during contact hypersensitivity is mediated by early recruitment of CD8 + T cytotoxic 1 cells inducing keratinocyte apoptosis. J Immunol. 2002;168:3079–87.CrossRefPubMedGoogle Scholar
  5. 5.
    Bour H, Peyron E, Gaucherand M, Garrigue JL, Desvignes C, Kaiserlian D, Revillard JP, Nicolas JF. Major histocompatibility complex class I-restricted CD8 + T cells and class II-restricted CD4 + T cells, respectively, mediate and regulate contact sensitivity to dinitrofluorobenzene. Eur J Immunol. 1995;25:3006–10.CrossRefPubMedGoogle Scholar
  6. 6.
    Kalish RS, Johnson KL. Enrichment and function of urushiol (poison ivy)-specific T lymphocytes in lesions of allergic contact dermatitis to urushiol. J Immunol. 1990;145:3706–13.PubMedGoogle Scholar
  7. 7.
    He D, Wu L, Kim HK, Li H, Elmets CA, Xu H. CD8 + IL-17-producing T cells are important in effector functions for the elicitation of contact hypersensitivity responses. J Immunol. 2006;177:6852–8.PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    He D, Wu L, Kim HK, Li H, Elmets CA, Xu H. IL-17 and IFN-gamma mediate the elicitation of contact hypersensitivity responses by different mechanisms and both are required for optimal responses. J Immunol. 2009;183:1463–70.PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Kish DD, Li X, Fairchild RL. CD8 T cells producing IL-17 and IFN-gamma initiate the innate immune response required for responses to antigen skin challenge. J Immunol. 2009;182:5949–59.PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Kawaguchi M, Adachi M, Oda N, Kokubu F, Huang SK. IL-17 cytokine family. J Allergy Clin Immunol. 2004;114:1265–73.CrossRefPubMedGoogle Scholar
  11. 11.
    Kolls JK, Linden A. Interleukin-17 family members and inflammation. Immunity. 2004;21:467–76.CrossRefPubMedGoogle Scholar
  12. 12.
    Nakae S, Komiyama Y, Narumi S, Sudo K, Horai R, Tagawa Y, Sekikawa K, Matsushima K, Asano M, Iwakura Y. IL-1-induced tumor necrosis factor-alpha elicits inflammatory cell infiltration in the skin by inducing IFN-gamma-inducible protein 10 in the elicitation phase of the contact hypersensitivity response. Int Immunol. 2003;15:251–60.CrossRefPubMedGoogle Scholar
  13. 13.
    Ye P, Rodriguez FH, Kanaly S, Stocking KL, Schurr J, Schwarzenberger P, Oliver P, Huang W, Zhang P, Zhang J, Shellito JE, Bagby GJ, Nelson S, Charrier K, Peschon JJ, Kolls JK. Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense. J Exp Med. 2001;194:519–27.PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Kish DD, Volokh N, Baldwin WM III, Fairchild RL. Hapten application to the skin induces an inflammatory program directing hapten-primed effector CD8 T cell interaction with hapten-presenting endothelial cells. J Immunol. 2011;186:2117–26.PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Brigl M, Brenner MB. CD1: antigen presentation and T cell function. Annu Rev Immunol. 2004;22:817–90.CrossRefPubMedGoogle Scholar
  16. 16.
    Kronenberg M, Gapin L. The unconventional lifestyle of NKT cells. Nat Rev Immunol. 2002;2:557–68.PubMedGoogle Scholar
  17. 17.
    Godfrey DI, Kronenberg M. Going both ways: immune regulation via CD1d-dependent NKT cells. J Clin Invest. 2004;114:1379–88.PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Stetson DB, Mohrs M, Reinhardt RL, Baron JL, Wang ZE, Gapin L, Kronenberg M, Locksley RM. Constitutive cytokine mRNAs mark natural killer (NK) and NK T cells poised for rapid effector function. J Exp Med. 2003;198:1069–76.PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Itakura A, Szczepanik M, Campos RA, Paliwal V, Majewska M, Matsuda H, Takatsu K, Askenase PW. An hour after immunization peritoneal B-1 cells are activated to migrate to lymphoid organs where within 1 day they produce IgM antibodies that initiate elicitation of contact sensitivity. J Immunol. 2005;175:7170–8.CrossRefPubMedGoogle Scholar
  20. 20.
    Campos RA, Szczepanik M, Itakura A, Akahira-Azuma M, Sidobre S, Kronenberg M, Askenase PW. Cutaneous immunization rapidly activates liver invariant Valpha14 NKT cells stimulating B-1 B cells to initiate T cell recruitment for elicitation of contact sensitivity. J Exp Med. 2003;198:1785–96.PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Gerberick GF, Cruse LW, Miller CM, Ridder GM. Selective modulation of B-cell activation markers CD86 and I-Ak on murine draining lymph node cells following allergen or irritant treatment. Toxicol Appl Pharmacol. 1999;159:142–51.CrossRefPubMedGoogle Scholar
  22. 22.
    Krasteva M, Bons B, Ryan C, Gerberick GF. Consumer allergy to oxidative hair coloring products: epidemiologic data in the literature. Dermatitis. 2009;20:123–41.PubMedGoogle Scholar
  23. 23.
    Thyssen JP, Carlsen BC, Sosted H, Menne T, Johansen JD. Frequency of p-phenylenediamine sensitization among Danish eczema patients tested between 1985 and 2007. Contact Dermat. 2008;59:184–5.CrossRefGoogle Scholar
  24. 24.
    Damian DL, Shannon KF, Saw RP, Thompson JF. Topical diphencyprone immunotherapy for cutaneous metastatic melanoma. Australas J Dermatol. 2009;50:266–71.CrossRefPubMedGoogle Scholar
  25. 25.
    Upitis JA, Krol A. The use of diphenylcyclopropenone in the treatment of recalcitrant warts. J Cutan Med Surg. 2002;6:214–7.CrossRefPubMedGoogle Scholar
  26. 26.
    Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000;132:365–86.PubMedGoogle Scholar
  27. 27.
    Watanabe H, Unger M, Tuvel B, Wang B, Sauder DN. Contact hypersensitivity: the mechanism of immune responses and T cell balance. J Interferon Cytokine Res. 2002;22:407–12.CrossRefPubMedGoogle Scholar
  28. 28.
    Engkilde K, Menne T, Johansen JD. Inverse relationship between allergic contact dermatitis and type 1 diabetes mellitus: a retrospective clinic-based study. Diabetologia. 2006;49:644–7.CrossRefPubMedGoogle Scholar
  29. 29.
    Engkilde K, Menne T, Johansen JD. Inflammatory bowel disease in relation to contact allergy: a patient-based study. Scand J Gastroenterol. 2007;42:572–6.CrossRefPubMedGoogle Scholar
  30. 30.
    Engkilde K, Thyssen JP, Menne T, Johansen JD. Association between cancer and contact allergy: a linkage study. BMJ Open. 2011;1:e000084.PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Engkilde K, Thyssen JP, Bangsgaard N, Menne T, Johansen JD. Inverse association between rheumatoid arthritis and contact allergy. Acta Derm Venereol. 2012;92:175–6.CrossRefPubMedGoogle Scholar
  32. 32.
    Mayer CT, Floess S, Baru AM, Lahl K, Huehn J, Sparwasser T. CD8 + Foxp3 + T cells share developmental and phenotypic features with classical CD4 + Foxp3 + regulatory T cells but lack potent suppressive activity. Eur J Immunol. 2011;41:716–25.CrossRefPubMedGoogle Scholar
  33. 33.
    Goubier A, Vocanson M, Macari C, Poyet G, Herbelin A, Nicolas JF, Dubois B, Kaiserlian D. Invariant NKT Cells Suppress CD8(+) T-Cell-Mediated Allergic Contact Dermatitis Independently of Regulatory CD4(+) T Cells. J Invest Dermatol. 2013;133:980–7.CrossRefPubMedGoogle Scholar
  34. 34.
    Long SA, Rieck M, Tatum M, Bollyky PL, Wu RP, Muller I, Ho JC, Shilling HG, Buckner JH. Low-dose antigen promotes induction of FOXP3 in human CD4 + T cells. J Immunol. 2011;187:3511–20.PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Engkilde K, Buschard K, Hansen AK, Menne T, Johansen JD. Prevention of diabetes in NOD mice by repeated exposures to a contact allergen inducing a sub-clinical dermatitis. PLoS ONE. 2010;5:e10591.PubMedCentralCrossRefPubMedGoogle Scholar
  36. 36.
    Crowe NY, Coquet JM, Berzins SP, Kyparissoudis K, Keating R, Pellicci DG, Hayakawa Y, Godfrey DI, Smyth MJ. Differential antitumor immunity mediated by NKT cell subsets in vivo. J Exp Med. 2005;202:1279–88.PubMedCentralCrossRefPubMedGoogle Scholar
  37. 37.
    Askenase PW, Majewska-Szczepanik M, Kerfoot S, Szczepanik M. Participation of iNKT cells in the early and late components of Tc1-mediated DNFB contact sensitivity: cooperative role of gammadelta-T cells. Scand J Immunol. 2011;73:465–77.CrossRefPubMedGoogle Scholar
  38. 38.
    Dey N, Szczepanik M, Lau K, Majewska-Szczepanik M, Askenase PW. Stimulatory lipids accumulate in the mouse liver within 30 min of contact sensitization to facilitate the activation of Naive iNKT cells in a CD1d-dependent fashion. Scand J Immunol. 2011;74:52–61.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Jesper Dyrendom Svalgaard
    • 1
    • 2
    Email author
  • Carina Særmark
    • 2
  • Morten Dall
    • 2
  • Karsten Buschard
    • 2
  • Jeanne D. Johansen
    • 2
  • Kåre Engkilde
    • 1
    • 2
  1. 1.Department of Dermato-Allergology, National Allergy Research Centre, Gentofte HospitalUniversity of CopenhagenHellerupDenmark
  2. 2.The Bartholin Institute, RigshospitaletCopenhagen BiocenterCopenhagenDenmark

Personalised recommendations