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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)

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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

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Acknowledgment

We thank Dr. Javier Avalos for his critical review of the manuscript.

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Authors and Affiliations

  1. Safety Science Research Laboratories, Kao Corporation, 2606 Akabane, Ichikai-Machi, Haga-Gun, Tochigi, 321-3497, Japan

    Hitoshi Sakaguchi, Masaaki Miyazawa, Nanae Kosaka, Yuichi Ito & Hiroyuki Suzuki

  2. Safety Quality Assurance Center, Shiseido Co., Ltd, Yokohama, Kanagawa, 236-8643, Japan

    Takao Ashikaga, Katsurako Yoneyama, Sakiko Sono, Hiroshi Itagaki & Hidekazu Toyoda

Authors
  1. Hitoshi Sakaguchi
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  2. Takao Ashikaga
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  3. Masaaki Miyazawa
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  4. Nanae Kosaka
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  5. Yuichi Ito
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  6. Katsurako Yoneyama
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  8. Hiroshi Itagaki
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  9. Hidekazu Toyoda
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  10. Hiroyuki Suzuki
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Correspondence to Hitoshi Sakaguchi.

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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

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