Skip to main content
SpringerLink
Log in

A sensitive mouse lymph node assay with two application phases for detection of contact allergens

  • Original Investigations
  • Published:
Archives of Toxicology Aims and scope Submit manuscript

Abstract

A predictive test using mice for the identification of contact sensitizing chemicals was developed. Contact sensitizing activity is measured as a function of draining lymph node activation following application of test chemical. Experimental conditions for assessment of induced lymph node cell (LNC) responses have been optimized. BALB/c mice were initially treated with intradermal injections of test chemical in Freund's complete adjuvant emulsion. Five days after intradermal injection, mice were exposed topically to chemical in vehicle on the ears daily for 3 consecutive days. Next day following the final exposure, changes in lymph node weight, total cell number in the draining lymph nodes and LNC proliferation for 24 h culture were assessed. The performance of the method was evaluated with ten sensitizing chemicals and a non-sensitizing irritant, sodium lauryl sulfate (SLS). The LNC proliferation induced by combination of intradermal injection and topical application of sensitizing chemicals was more clearly increased than that following only topical application. With the single exception of sulfanilic acid, the method developed was able to detect the sensitizing capacity of chemicals that failed to induce sensitization in the local lymph node assay. Under the conditions used, SLS did not induce measurable lymph node responses. These results suggest that the mouse lymph node assay can provide a sensitive screening test for weak to moderate sensitizers. In addition, the assay offers the advantages of objective and quantitative endpoints, and is suitable for the evaluation of colored or irritant chemicals.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

GPMT:

guinea pig maximization test

LLNA:

local lymph node assay

LNC:

lymph node cell

FCA:

Freund's complete adjuvant

TP:

Tinuvin P, 2-(2′-hydroxy-5′-methylphenyl)benzotriazole

SA:

sulfanilic acid, 4-aminobenzenesulfonic acid

CA:

p-chloroaniline

DNCB:

2,4-dinitrochlorobenzene

Benzocaine:

ethyl 4-aminobenzoate

HBA:

4-hydroxybenzoic acid

NiSO4 :

nickel sulfate

SLS:

sodium lauryl sulfate

DPTU:

N,N′-diphenylthiourea

MBT:

2-mercaptobenzothiazole

ZDEC:

zinc diethyldithiocarbamate

DMSO:

dimethylsulfoxide

AOO:

acetone: olive oil (4∶1)

SIp :

stimulation index of lymph node cell proliferation

SIn :

stimulation index of lymph node cell number

SItotal :

stimulation index of total lymph node responses

HEPES:

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

3HTdR:

[methyl-3H]thymidine

MEST:

mouse ear swelling test

PLNA:

popliteal lymph node assay

SD:

standard deviation

References

  • Asherson GL, Zembala M, Perera MACC, Mayhew B, Thomas WR (1977) Production of immunity and unresponsiveness in the mouse by feeding contact sensitizing agents and the role of suppressor cells in the Peyer's patches, mesenteric lymph nodes and other lymphoid tissues. Cell Immunol 33: 145–155

    PubMed  Google Scholar 

  • Basketter DA, Sholes EW (1992) Comparison of the local lymph node assay with the guinea-pig maximization test for the detection of a range of contact allergens. Food Chem Toxicol 30: 65–69

    PubMed  Google Scholar 

  • Basketter DA, Sholes EW, Kimber I, Botham PA, Hilton J, Miller K, Robbins MC, Harrison PTC, Waite SJ (1991) Interlaboratory evaluation of the local lymph node assay with 25 chemicals and comparison with guinea pig test data. Toxicol Methods 1: 30–43

    Google Scholar 

  • Basketter DA, Sholes EW, Cumberbatch M, Evans CD, Kimber I (1992) Sulphanilic acid: divergent results in the guinea pig maximization test and the local lymph node assay. Contact Dermatitis 27: 209–213

    PubMed  Google Scholar 

  • Botham PA, Basketter DA, Maurer T, Mueller D, Potokar M, Bontinck WJ (1991) Skin sensitization — a critical review of predictive test methods in animals and man. Food Chem Toxicol 29: 275–286

    PubMed  Google Scholar 

  • Buehler EV (1965) Delayed contact hypersensitivity in the guinea pig. Arch Dermatol 91: 171–177

    PubMed  Google Scholar 

  • Cronin E (1980) Contact dermatitis. Churchill Livingston, Edinburgh

    Google Scholar 

  • Fregert S, Traulson L, Zimerson E (1982) Contact allergic reactions to diphenylthiourea and phenylisothiocyanate in PVC adhesive tape. Contact Dermatitis 8: 38–42

    PubMed  Google Scholar 

  • Fregert S, Dahlquist I, Traulson L (1983) Sensitization capacity of diphenylthiourea and phenylisothiocyanate. Contact Dermatitis 9: 87

    PubMed  Google Scholar 

  • Gad SC, Dunn BJ, Dobbs DW, Reilly C, Walsh RD (1986) Development and validation of an alternative dermal sensitization test: the mouse ear swelling test (MEST). Toxicol Appl Pharmacol 84: 93–114

    PubMed  Google Scholar 

  • Gerberick GF, House RV, Fietcher ER, Ryan CA (1992) Examination of the local lymph node assay for use in contact sensitization risk assessment. Fundam Appl Toxicol 19: 438–445

    PubMed  Google Scholar 

  • Gleichmann E, Pals ST, Rolink AG, Radaszkiewicz T, Gleichmann H (1984) Graft-versus-host reactions: clues to the etiopathology of a spectrum of immunological disease. Immunol Today 5: 324–332

    Google Scholar 

  • Gleichmann E, Kimber I, Purchase FH (1989) Immunotoxicity: suppressive and stimulatory effects of drugs and environmental chemicals on the immune system. Arch Toxicol 63: 257–273

    PubMed  Google Scholar 

  • Groot AC, Lien DH (1983) Contact allergy to Tinuvin® P. Contact Dermatitis 9: 324–325

    PubMed  Google Scholar 

  • Ikarashi Y, Tsuchiya T, Nakamura A (1992) Detection of contact sensitivity of metal salts using the murine local lymph node assay. Toxicol Lett 62: 53–61

    PubMed  Google Scholar 

  • Ikarashi Y, Tsuchiya T, Nakamura A (1993 a) Evaluation of contact sensitivity of rubber chemicals using the murine local lymph node assay. Contact Dermatitis 28: 77–80

    PubMed  Google Scholar 

  • Ikarashi Y, Tsukamoto Y, Tsuchiya T, Nakamura A (1993 b) Influence of irritants on lymph-node cell proliferation and the detection of contact sensitivity to metal salts in the murine local lymph node assay. Contact Dermatitis (in press)

  • Kammüller ME, Seinen W (1988) Structural requirements for hydantonis and 2-thiohydantoins to induce lymphoproliferative popliteal lymph node reactions in the mouse. Int J Immunopharmacol 10: 997–1010

    PubMed  Google Scholar 

  • Kammüller ME, Thomas C, De Bakker JM, Bloksma N, Seinen W (1989) The popliteal lymph node assay in mice to screen for the immune dysregulating potential of chemicals. A preliminary study. Int J Immunopharmacol 11: 293–300

    PubMed  Google Scholar 

  • Kaniwa M, Kojima S, Nakamura A, Kantoh H, Itoh M, Ishihara M (1986) Chemical approach to contact dermatitis caused by household products (III). Analysis of dithiocarbamate-type accelerators in commercial rubber gloves and incidence of positive reactions in patch testing. Eisei Kagaku 32: 197–211

    Google Scholar 

  • Kimber I, Weisenberger C (1989) A murine local lymph node assay for the identification of contact allergens: assay development and results of an initial validation study. Arch Toxicol 63: 274–282

    PubMed  Google Scholar 

  • Kimber I, Weisenberger C (1991) Anamnestic responses to contact allergens: application in the murine local lymph node assay. J Appl Toxicol 11: 129–133

    PubMed  Google Scholar 

  • Kimber I, Pierce BB, Mitchell JA, Kinnaird A (1987) Depression of lymph node cell proliferation induced by oxazolone. Int Arch Allergy Appl Immunol 84: 256–262

    PubMed  Google Scholar 

  • Kimber I, Hilton J, Weisenberger C (1989 a) The murine local lymph node assay for identification of contact allergens: a preliminary evaluation of in situ measurement of lymphocyte proliferation. Contact Dermatitis 21: 215–220

    PubMed  Google Scholar 

  • Kimber I, Shepherd CJ, Mitchell JA, Turk JL, Baker D (1989 b) Regulation of lymphocyte proliferation in contact sensitivity: homeostatic mechanisms and a possible explanation of antigenic competition. Immunology 66: 577–582

    PubMed  Google Scholar 

  • Kimber I, Bentley AN, Hilton J (1990 a) Contact sensitization of mice to nickel sulphate and potassium dichromate. Contact Dermatitis 23: 325–330

    PubMed  Google Scholar 

  • Kimber I, Hilton J, Botham PA (1990 b) Identification of contact allergens using the murine local lymph node assay: comparisons with the Buehler occluded patch test in guinea pigs. J Appl Toxicol 10: 173–180

    PubMed  Google Scholar 

  • Kimber I, Hilton J, Botham PA, Basketter DA, Sholes EW, Miller K, Robbins MC, Harrison PTC, Gray TJB, Waite SJ (1991) The murine local lymph node assay: results of an inter-laboratory trial. Toxicol Lett 55: 203–213

    PubMed  Google Scholar 

  • Kligman AM, Epstein WL (1959) Some factors affecting contact sensitization in man. In: Shaffer JH, Logrippo GA, Chase MW (eds) Mechanisms of hypersensitivity. Little Brown, Boston, pp 713–722

    Google Scholar 

  • Magnusson B, Kligman AM (1969) The identification of contact allergens by animal assay. The guinea pig maximization test method. J Invest Dermatol 52: 268–276

    PubMed  Google Scholar 

  • Magnusson B, Kligman AM (1970) Allergic contact dermatitis in the guinea pig. Charles C. Thomas, Springfield, Ill.

    Google Scholar 

  • Maguire HC Jr (1973) The bioassay of contact allergy in the guinea pig. J Soc Cosmet Chem 24: 151–162

    Google Scholar 

  • Maurer T, Weirich EG, Hess R (1980) The optimization test in the guinea pig in relation to other predictive sensitization methods. Toxicology 15: 163–171

    PubMed  Google Scholar 

  • Miller SD, Claman HN (1976) The induction of hapten-specific T cell tolerance using hapten-modified lymphoid cells. 1. Characteristics of tolerance induction. J Immunol 117: 1519–1526

    PubMed  Google Scholar 

  • Moorhead JW (1976) Tolerance and contact sensitivity of DNFB in mice. VI. Inhibition of afferent sensitivity by suppressor T cells in adoptive tolerance. J Immunol 117: 802–806

    PubMed  Google Scholar 

  • Niklasson B, Bjorkner B (1989) Contact allergy to the UV-absorber Tinuvin P in plastics. Contact Dermatitis 21: 330–334

    PubMed  Google Scholar 

  • Robinson MK, Sneller DL (1990) Use of an optimized in vitro lymphocyte blastogenesis assay to detect contact sensitivity to nickel sulfate in mice. Toxicol Appl Pharmacol 104: 106–116

    PubMed  Google Scholar 

  • Sholes EW, Basketter DA, Sarll AE, Kimber I, Evans CD, Miller K, Robbins MC, Harrison PTC, Waite SJ (1992) The local lymph node assay: results of a final inter-laboratory validation under field conditions. J Appl Toxicol 12: 217–222

    PubMed  Google Scholar 

  • Thomas WR, Watkins MC, Asherson GL (1979) Suppressor cells for the afferent phase of contact sensitivity of picryl chloride: inhibition of DNA synthesis induced by T cells from mice injected with picryl sulfonic acid. J Immunol 122: 2300–2303

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Medical Devices, National Institute of Hygienic Sciences, 18-1, Kamiyoga 1-Chome, Setagaya-ku, 158, Tokyo, Japan

    Yoshiaki Ikarashi, Toshie Tsuchiya & Akitada Nakamura

Authors
  1. Yoshiaki Ikarashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Toshie Tsuchiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Akitada Nakamura
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ikarashi, Y., Tsuchiya, T. & Nakamura, A. A sensitive mouse lymph node assay with two application phases for detection of contact allergens. Arch Toxicol 67, 629–636 (1993). https://doi.org/10.1007/BF01974070

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01974070

Key words

Search

Navigation