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Prediction of preservative sensitization potential using surface marker CD86 and/or CD54 expression on human cell line, THP-1

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Abstract

Preservatives are important components in many products, but have a history of purported allergy. Several assays [e.g., guinea pig maximization test (GPMT), local lymph node assay (LLNA)] are used to evaluate allergy potential of preservatives. We recently developed the human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test using human THP-1 cells. This test evaluates the augmentation of CD86 and CD54 expression, which are key events in the sensitization process, as an indicator of allergy following treatment with test chemical. Earlier, we found that a sub-toxic concentration was needed for the up-regulation of surface marker expression. In this study, we further evaluate the capability of h-CLAT to predict allergy potential using eight preservatives. Cytotoxicity was determined using propidium iodide with flow cytometry analysis and five doses that produce a 95, 85, 75, 65, and 50% cell viability were selected. If a material did not have any cytotoxicity at the highest technical dose (HTD), five doses are set using serial 1.3 dilutions of the HTD. The test materials used were six known allergic preservatives (e.g., methylchloroisothiazolinone/methylisothiazolinone, formaldehyde), and two non-allergic preservatives (methylparaben and 4-hydroxybenzoic acid). All allergic preservatives augmented CD86 and/or CD54 expression, indicating h-CLAT correctly identified the allergens. No augmentation was observed with the non-allergic preservatives; also correctly identified by h-CLAT. In addition, we report two threshold concentrations that may be used to categorize skin sensitization potency like the LLNA estimated concentration that yield a three-fold stimulation (EC3) value. These corresponding values are the estimated concentration which gives a relative fluorescence intensity (RFI) = 150 for CD86 and an RFI = 200 for CD54. These data suggest that h-CLAT, using THP-1 cells, may be able to predict the allergy potential of preservatives and possibility classify the potency of an allergen.

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Abbreviations

CV75:

Cell viability 75% concentration

DCs:

Dendritic cells

DMSO:

Dimethyl sulfoxide

DNCB:

2,4-Dinitrochlorobenzene

EC3:

Estimated concentration that yield a three-fold stimulation

FcR:

Fc receptor

GPMT:

Guinea pig maximization test

h-CLAT:

Human Cell Line Activation Test

HPC:

Hematopoietic progenitor cell

HTD:

Highest technical dose

LC:

Langerhans cell

LLNA:

Local lymph node assay

mAb:

Monoclonal antibody

MCI/MI:

Methylchloroisothiazolinone/mthylisothiazolinone

MDBGN:

Methyldibromo gutaronitrile

MFI:

Mean fluorescence intensity

MHC class II:

Class II major histocompatibility complex antigen

MTT:

Methylthiazolydiphenyl-tetrazolium bromide

PBMC:

Peripheral blood mononuclear cell

PI:

Propidium iodide

RFI:

Relative fluorescence intensity

SDS:

Sodium dodecyl sulfate

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Acknowledgment

We thank Dr. Javier Avalos for his critical review of the manuscript and Ms. Satomi Sudo for her excellent technical help and accurate data analysis.

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Authors and Affiliations

  1. Kao Corporation, Safety and Microbial Control Research Center, 2606 Akabane, Ichikai-Machi, Haga-Gun, Tochigi, 321-3497, Japan

    Hitoshi Sakaguchi, Masaaki Miyazawa, Yukiko Yoshida, Yuichi Ito & Hiroyuki Suzuki

Authors
  1. Hitoshi Sakaguchi
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  2. Masaaki Miyazawa
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  3. Yukiko Yoshida
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  4. Yuichi Ito
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  5. Hiroyuki Suzuki
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Correspondence to Hitoshi Sakaguchi.

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Sakaguchi, H., Miyazawa, M., Yoshida, Y. et al. Prediction of preservative sensitization potential using surface marker CD86 and/or CD54 expression on human cell line, THP-1. Arch Dermatol Res 298, 427–437 (2007). https://doi.org/10.1007/s00403-006-0714-9

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  • DOI: https://doi.org/10.1007/s00403-006-0714-9

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