Abstract
Recent regulations for cosmetics in Europe prohibit animal testing for evaluating the sensitization potential of chemicals to improve animal welfare. Yet, there is not an acceptable Organization for Economic Co-operation and Development non-animal skin sensitization test method. Several in vitro skin sensitization methods that focus on the activation of Langerhans cells, including human cell lines, are being evaluated as possible alternatives. In our previous study, we optimized our human cell line activation test (h-CLAT) using THP-1 cells (monocytic leukemia cell line) and conducted an inter-laboratory study. We found that measuring CD86/CD54 expression may be useful for predicting skin sensitization. The aim of this study was to confirm the relationship between CD86/CD54 expression and THP-1 cell viability in the h-CLAT. In this study, 21 allergens (e.g., dinitrochlorobenzene, p-phenylenediamine, Ni) and 8 non-allergens (e.g., SLS, lactic acid) were evaluated. For each chemical, more than 10 concentrations that gave a predicted cell viability range of 20–95% were used. The data showed that expression patterns of CD86/CD54 differed depending on chemical. For most allergens, cytotoxicity (65–90% cell viability) was needed for enhancement of CD86/CD54 expression. The criteria of “CD86 ≥ 150 or CD54 ≥ 200” resulted in an accuracy of 93%, which confirms appropriate cut-off criteria for h-CLAT. Furthermore, a good correlation was observed between EC3 of local lymph node assay and EC150(CD86) or EC200(CD54) of h-CLAT (12 or 16 chemicals, respectively), which would provide a useful estimate of allergic potency. These findings suggest that h-CLAT would be a good robust in vitro skin sensitization test.
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Abbreviations
- CV75:
-
cell viability 75% concentration
- DC:
-
dendritic cells
- DMSO:
-
dimethyl sulfoxide
- EC3:
-
estimated concentration that yield a three-fold stimulation
- h-CLAT:
-
human cell line activation Test
- HTD:
-
highest technical dose
- IC50:
-
inhibited cell viability to 50%
- LC:
-
Langerhans cell
- LLNA:
-
local lymph node assay
- MFI:
-
(Geometric) mean fluorescence intensity
- OECD:
-
Organization for Economic Cooperation and Development
- PI:
-
propidium iodide
- RFI:
-
relative fluorescence intensity
- SD:
-
standard deviation
References
Ade N, Martinozzi-Teissier S, Pallardy M, Rousset F. Activation of U937 cells by contact sensitizers: CD86 expression is independent of apoptosis. J Immunotoxicol 2006;3:189–97.
Aeby P, Wyss C, Beck H, Griem P, Scheffler H, Goebel CJ. Characterization of the sensitizing potential of chemicals by in vitro analysis of dendritic cell activation and skin penetration. Invest Dermatol 2004;122:1154–64.
Aiba S, Katz SI. Phenotypic and functional characteristics of in vivo-activated Langerhans cells. J Immunol 1990;145:2791–6.
Aiba S, Terunuma A, Manome H, Tagami H. Dendritic cells differently responded to haptens and irritants by their production of cytokines and expression of co-stimulatory molecules. Eur J Immunol 1997;27:3031–8.
Aiba S, Manome H, Yoshino Y, Tagami H. In vitro treatment of human transforming growth factor-beta1-treated monocyte-derived dendritic cells with haptens can induce the phenotypic and functional changes similar to epidermal Langerhans cells in the initiation phase of allergic contact sensitivity reaction. Immunology 2000;101:68–75.
Aptula AO, Patlewicz G, Roberts DW, Schultz TW. Non-enzymatic glutathione reactivity and in vitro toxicity: a non-animal approach to skin sensitization. Toxicol In Vitro 2006;20:239–47.
Ashikaga T, Hoya M, Itagaki H, Katumura Y, Aiba S. Evaluation of CD86 expression and MHC class II molecule internalization in THP-1 human monocyte cells as predictive endpoints for contact sensitizers. Toxicol In Vitro 2002;16:711–6.
Ashikaga T, Yoshida Y, Hirota M, Yoneyama K, Itagaki H, Sakaguchi H, et al. Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). I. Optimization of the h-CLAT protocol. Toxicol In Vitro 2006;20:767–73.
Azam P, Peiffer JL, Chamousset D, Tissier MH, Bonnet PA, Vian L, et al. The cytokine-dependent MUTZ-3 cell line as an in vitro model for the screening of contact sensitizers. Toxicol Appl Pharmacol 2006;212:14–23.
Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392:245–52.
Basketter DA, Gerberick GF, Kimber I, Loveless SE. The local lymph node assay: a viable alternative to currently accepted skin sensitization test. Food Chem Toxicol 1996;34:986–97.
Basketter DA, Gerberick GF, Kimber I. Strategies for identifying false positive response in predictive skin sensitization tests. Food Chem Toxicol 1998;36:327–33.
Basketter DA, Gilmour N, Dearman RJ, Kimber I, Ryan CA, Gerberick F. Classification of skin sensitisation potency using the Local Lymph Node Assay. Toxicologist 2003;72:101. (Abstract).
Basketter DA, Gilimour N, Ryan CA, Gerberick GF, Dearman RJ, Kimber I. Categorisation of human sensitization potency using local lymph node assay EC3 values. Contact Dermatitis 2004;50:206. (Abstract).
Basketter DA, Casati S, Gerberick GF, Griem P, Philips B, Worth A. Skin sensitisation. Altern Lab Anim 2005;33:83–103.
Basketter DA, Sanders D, Jowsey IR. The skin sensitization potential of resorcinol: experience with the local lymph node assay. Contact Dermatitis 2007;56:196–200.
Becker D, Kolde G, Reske K, Knop J. An in vitro endocytotic activation of murine epidermal Langerhans cells under the influence of contact allergens. J Immunol Methods 1994;169:195–204.
De Smedt AC, Van Den Heuvel RL, Van Tendeloo VF, Berneman ZN, Schoeters GE, Weber E, et al. Phenotypic alterations and IL-1 beta production in CD34(+) progenitor- and monocyte-derived dendritic cells after exposure to allergens: a comparative analysis. Arch Dermatol Res 2002;294:109–16.
Enk AH, Katz SI. Early molecular events in the induction phase of contact sensitivity. Proc Nat Acad Sci U S A 1992;89:1398–402.
Gerberick GF, Robinson MK, Ryan CA, Dearman RJ, Kimber I, Basketter DA, et al. Contact allergenic potency: correlation of human and local lymph node assay data. Am J Contact Dermatitis 2001;12:156–61.
Gerberick GF, Ryan CA, Kern PS, Dearman RJ, Kimber I, Patlewicz GY, et al. A chemical dataset for evaluation of alternative approaches to skin-sensitization testing. Contact Dermatitis 2004;50:274–88.
Hart DNJ. Dendritic cells: unique leukocyte population which control the primary immune response. Blood 1997;90:3245–87.
Hulette BC, Ryan CA, Gerberick GF. Elucidating changes in surface marker expression of dendritic cells following chemical allergen treatment. Toxicol Appl Pharmacol 2002;182:226–33.
Hulette BC, Ryan CA, Gilimour N, Gerberick GF. Relationship of CD86 surface marker expression and cytotoxicity on dendritic cells exposed to chemical allergen. Toxicol Appl Pharmacol 2005;209:159–66.
Jowsey IR, Basketter DA, Westmoreland C, Kimber I. A future approach to measuring relative skin sensitising potency: a proposal. J Appl Toxicol 2006;26:341–350.
Kimber I, Hilton J, Dearman RJ, Gerberick GF, Ryan CA, Basketter DA, et al. Assessment of the skin sensitization potential of topical medicaments using the local lymph node assay: an interlaboratory evaluation. J Toxicol Environ Health 1998;53:563–79.
Miyazawa M, Ito Y, Yoshida Y, Sakaguchi H, Suzuki H. Phenotypic alterations and cytokine production inTHP-1 cells in response to allergens. Toxicol In Vitro 2007;21:428–37.
Miyazawa M, Ito Y, Kosaka N, Sakaguchi H, Suzuki H. Role of MAPK signaling pathway in the activation of dendritic type cell line, THP-1, induced by DNCB and NiSO4. J Toxicol Sci. 2008;33.
Ovigne JM, Verda D, Piroird C, Rousset F. In vitro prediction of the skin sensitization potential of chemicals using the U937/CD86 test. ALTEX 2005;22(Spl):145.
Ozawa H, Nakagawa S, Tagami H, Aiba S. Interleukin-1 beta and granulocyte macrophage colony-stimulating factor mediate Langerhans cell maturation differently. J Invest Dermatol 1996;106:441–5.
Python F, Goebel C, Aeby P. Assessment of the U937 cell line for the detection of contact allergens. Toxicol Appl Pharmacol 2007;220:113–24.
Rougier N, Redziniak G, Mougin D, Schmitt D, Vincent C. In vitro evaluation of the sensitization potential of weak contact allergens using Langerhans-like dendritic cells and autologous T cells. Toxicology 2000;145:73–82.
Rousset F, Verda D, Garrigue JL, Mariani M, Leclaire J. In vitro prediction of contact sensitivity with human cell lines. Contact Dermatitis 2002;46(s4):6. (Abstract).
Ryan CA, Gerberick GF, Cruse LW, Basketter DA, Lea L, Blaikie L, et al. Activity of human contact allergens in the murine local lymph node assay. Contact Dermatitis 2000;43:95–102.
Ryan CA, Hulette BC, Gerberick GF. Approaches for the development of cell-based in vitro methods for contact sensitization. Toxicol In vitro 2001;15:43–5.
Ryan CA, Cruse LW, Skinner RA, Dearman RJ, Kimber I, Gerberick GF. Examination of a vehicle for use with water soluble materials in the murine local lymph node assay. Food Chem Toxicol 2002;40:1719–25.
Sakaguchi H, Ashikaga T, Miyazawa M, Yoshida Y, Ito Y, Yoneyama K, et al. Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT. Toxicol In Vitro 2006;20:774–84.
Sakaguchi H, Miyazawa M, Yoshida Y, Ito Y, Suzuki H. Prediction of preservative sensitization potential using surface marker CD86 and/or CD54 expression on human cell line, THP-1. Arch Dermatol Res 2007;298:427–37.
Smith CK, Hotchkiss SA. Xenobiotics as skin sensitizers: metabolic activation and detoxication, and protein-binding mechanisms. In: Allergic contact dermatitis. London: Taylor and Francis; 2001. p. 119–205.
Staquet MJ, Sportouch M, Jacquet C, Schmitt D, Guesnet J, Peguet-Navarro J. Moderate skin sensitizers can induce phenotypic changes on in vitro generated dendritic cells. Toxicol In Vitro 2004;18:493–500.
Yoshida Y, Sakaguchi H, Ito Y, Okuda M, Suzuki H. Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naïve THP-1 cell line. Toxicol In Vitro 2003;17:221–8.
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We thank Dr. Javier Avalos for his critical review of the manuscript.
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Sakaguchi, H., Ashikaga, T., Miyazawa, M. et al. The relationship between CD86/CD54 expression and THP-1 cell viability in an in vitro skin sensitization test – human cell line activation test (h-CLAT). Cell Biol Toxicol 25, 109–126 (2009). https://doi.org/10.1007/s10565-008-9059-9
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DOI: https://doi.org/10.1007/s10565-008-9059-9