The NCTC 2544 IL‐18 Assay for the In Vitro Identification of Contact Allergens

Valentina Galbiati1, Emanuela Corsini1

1 Department of Pharmacological Sciences, Università degli Studi di Milano, Milan, Italy
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 20.8
DOI:  10.1002/0471140856.tx2008s54
Online Posting Date:  November, 2012
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Abstract

Assessment of allergenic potency of low‐molecular‐weight compounds is generally performed using animal models, such as the murine Local Lymph Node Assay (LLNA) and the Guinea Pig Maximization Test (GMT). Progress in understanding the mechanism of skin sensitization, including effects on the production of cytokines by different cell types within the skin, provides the opportunity to develop in vitro tests as an alternative to in vivo sensitization testing. This unit will describe a method for differentiating contact allergens from low‐molecular‐weight respiratory allergens and irritants, based on the selective induction of intracellular interleukin 18 (IL‐18) in the human keratinocyte cell line NCTC 2544. Similar results could also be obtained using primary human keratinocytes or other human keratinocyte cell lines, e.g., HaCaT, HPKII, etc. IL‐18 was chosen because this cytokine has been demonstrated to favor Th‐1‐type immune responses by enhancing the secretion of pro‐inflammatory mediators such as TNF‐α, IL‐8, and IFN‐γ, and to play a key proximal role in the induction of allergic contact dermatitis. Curr. Protoc. Toxicol. 54:20.8.1‐20.8.18. © 2012 by John Wiley & Sons, Inc.

Keywords: contact sensitizers; contact dermatitis; keratinocytes; cytokines; in vitro toxicology; alternative method

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Selective Up‐Regulation of Intracellular IL‐18 by Contact Allergens in the Human Keratinocyte Cell Line NCTC 2544
  • Support Protocol 1: Guidelines for Making Dilutions of Test Substances
  • Basic Protocol 2: MTT Assay for Assessment of Cell Viability (Range Finding Study)
  • Basic Protocol 3: Cell Treatment for Intracellular IL‐18 Production
  • Basic Protocol 4: Determining Intracellular IL‐18 Content by ELISA
  • Support Protocol 2: Total Protein Determination
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Selective Up‐Regulation of Intracellular IL‐18 by Contact Allergens in the Human Keratinocyte Cell Line NCTC 2544

  Materials
  • Master cell and working cell bank: NCTC 2544 cells can be obtained from Istituto Zooprofilattico di Brescia, Cat. n. BS CL 143 (Via Bianchi, 9 ‐ 25124 Brescia: Tel. +3903022901; Fax: +39030 2425251; e‐mail: )
  • RPMI 1640 culture medium
  • Glutamine
  • Penicillin/streptomycin (pen/strep)
  • Gentamicin
  • Fetal bovine serum (FBS)
  • Dulbecco's phosphate‐buffered saline (D‐PBS)
  • Trypsin/EDTA
  • 0.4% Trypan blue
  • 75‐cm2 flasks
  • Hemacytometer (e.g., Burker, Neubauer) or equivalent cell counter
  • Incubator, 37°C, 5% CO 2, 95% humidity
  • 24‐well plates or 96‐well plates
  • Additional reagents and equipment for counting cells using a hemacytometer and trypan blue staining (Phelan, )
NOTE: The handling of this cell line is not very different from any other cell lines. No specific recommendations apply.

Support Protocol 1: Guidelines for Making Dilutions of Test Substances

  Materials
  • Solid and liquid compounds
  • Dulbecco's phosphate‐buffered saline (D‐PBS)
  • Dimethyl sulfoxide (DMSO)
  • p‐Phenylediamine (PPD)
  • Complete culture medium (RPMI medium supplemented with 2 mM glutamine, 100 U/ml penicillin/100 µg/ml streptomycin, and 10% heat‐inactivated FBS)
  • Laboratory balance (accuracy 0.1 mg)

Basic Protocol 2: MTT Assay for Assessment of Cell Viability (Range Finding Study)

  Materials
  • NCTC 2544 cells
  • Complete culture medium (RPMI medium supplemented with 2 mM glutamine, 100 U/ml penicillin/100 µg/ml streptomycin, and 10% heat‐inactivated FBS)
  • Dulbecco's phosphate‐buffered saline (D‐PBS)
  • Dimethyl sulfoxide (DMSO)
  • Test chemicals (see protocol 2)
  • MTT [3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] solution (see recipe)
  • Acidic isopropanol (see recipe)
  • Adjustable micropipets: 2‐20 µl, 20‐200 µl, 200‐1000 µl
  • Adjustable multistep pipet
  • Incubator, 37°C, 5% CO 2, 95% humidity
  • Paper towels
  • ELISA plate reader equipped with 595‐nm and 405‐ to 450‐nm filters

Basic Protocol 3: Cell Treatment for Intracellular IL‐18 Production

  Materials
  • NCTC 2544 cells
  • Dimethyl sulfoxide (DMSO)
  • Complete culture medium (RPMI medium supplemented with 2 mM glutamine, 100 U/ml penicillin/100 µg/ml streptomycin, and 10% heat‐inactivated FBS)
  • p‐Phenylediamine (PPD)
  • Dulbecco's phosphate‐buffered saline (D‐PBS)
  • Triton X‐100
  • Ice
  • 24‐well plate
  • Adjustable micropipets: 2‐20 µl, 20‐200 µl, 200‐1000 µl
  • Adjustable multi‐step pipet
  • Incubator, 37°C, 5% CO 2, 95% humidity
  • −80°C freezer

Basic Protocol 4: Determining Intracellular IL‐18 Content by ELISA

  Materials
  • Anti‐human IL‐18 monoclonal antibody, 1 mg/ml
  • Coating buffer: 100 mM carbonate/bicarbonate buffer in water, pH 9.6
  • Ice
  • Wash buffer: D‐PBS + 0.01% Tween 20
  • Blocking and dilution buffer: D‐PBS + 1% BSA
  • Concentrated and working standards (see recipes)
  • Anti‐human IL‐18 biotinylated monoclonal Ab, 0.5 mg/ml
  • Streptavidin‐conjugated polyhorseradish peroxidase (HRP)
  • 3, 3′,5 ,5′‐Tetramethylbenzidine (TMB) liquid substrate for ELISA
  • Stop solution: 1 M H 2SO 4 in distilled H 2O
  • Adjustable micropipets: 2‐20 µl, 20‐200 µl, 200‐1000 µl
  • Adjustable multi‐step pipet
  • 96‐well flat‐bottom, ELISA plates
  • Plate sealer
  • 4°C incubator
  • Paper towels
  • ELISA plate reader equipped with 595‐nm and 405‐ to 450‐nm filters
  • Additional reagents and equipment for measuring total cell protein (see protocol 6)

Support Protocol 2: Total Protein Determination

  Materials
  • Protein standard curve (1.0 mg/ml → 0.04 mg/ml)
  • BSA for standard curve
  • Lysis buffer (0.5% Triton X‐100)
  • Bicinchoninic acid solution (BSA; Sigma)
  • Copper(II) Sulfate solution (Sigma)
  • 96‐well plate
  • Plate reader with 540‐ to 560‐nm absorbance filter
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Figures

Videos

Literature Cited

Literature Cited
   Andersen, K.E., Frankild, S., Wahlberg, J.E., and Boman, A. 1996. Non‐specific hyperreactivity related to the use of Freund's complete adjuvant. Contact Dermatitis 35:127.
   Antonopoulos, C., Cumberbatch, M., Mee, J.B., Dearman, R.J., Wei, X.Q., Liew, F.Y., Kimber, I., and Groves, R.W. 2008. IL‐18 is a key proximal mediator of contact hypersensitivity and allergen‐induced Langerhans cell migration in murine epidermis. J. Leukoc. Biol. 83:361‐367.
   Berghard, A., Gradin, K., and Toftgard, R. 1990. Serum and extracellular calcium modulate induction of cytochrome P‐450IA1 in human keratinocytes. J. Biol. Chem. 265:21086‐21090.
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   Corsini, E., Mitjans, M., Galbiati, V., Lucchi, L., Galli, C.L., and Marinovich, M. 2009. Use of IL‐18 production in a human keratinocyte cell line to discriminate contact sensitizers from irritants and low molecular weight respiratory allergens. Toxicol. In Vitro 23:789‐796.
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   Picardo, M., Zompetta, C., De Luca, C., Cristaudo, A., Cannistraci, C., Faggioni, A., and Santucci, B. 1990. Nickel‐keratinocyte interaction: A possible role in sensitization. Br. J. Dermatol. 122:729‐735.
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   Teunis, M., Corsini, E., Smits, M., Madsen, C.B., Eltze, T., Ezendam, J., Galbiati, V., Gremmer, E., Krul, C., Landin, A., Landsiedel, R., Pieters, R., Rasmussen, T.F., Reinders, J., Roggen, E., Spiekstra, S., and Gibbs, S. 2012. Transfer of a two‐tiered keratinocyte assay: IL‐18 production by NCTC2544 to determine the skin sensitizing capacity and epidermal equivalent assay to determine sensitizer potency. Toxicol. In Vitro In press.
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