Skip Navigation



ToxSci Advance Access published online on April 15, 2003
Toxicological Sciences, doi:10.1093/toxsci/kfg056
Toxicological Sciences © Society of Toxicology 2003; all rights reserved
This Article
Right arrow Advance Access manuscript (PDF) Freely available
Right arrow All Versions of this Article:
73/1/80    most recent
kfg056v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Humphreys, N. E
Right arrow Articles by Kimber, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Humphreys, N. E
Right arrow Articles by Kimber, I.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Received December 9, 2002; accepted January 24, 2003
© 2003 Society of Toxicology

Immunotoxicology

Assessment of Cumulative Allergen-Activated Lymph Node Cell Proliferation Using Flow Cytometry

Neil E Humphreys 1, Rebecca J Dearman 1*, Ian Kimber 1

1 Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, UK

* To whom correspondence should be addressed. E-mail: rebecca.dearman{at}syngenta.com.


  Abstract

The murine local lymph node assay (LLNA) is a method for the prospective identification of chemical contact allergens. The current validated protocol assesses lymphocyte proliferation induced in the draining lymph node as a function of in situ incorporation of radiolabelled thymidine. We have explored the potential utility of an alternative non-radioisotopic marker of cell division, the cytoplasmic dye carboxyfluoresein succinimidyl ester (CFSE). Using this method, the cell phenotype and the number of divisions each cell has undergone can be tracked using flow cytometry. BALB/c strain mice were exposed topically to various concentrations of the contact allergens 2,4-dinitrochlorobenzene (DNCB), oxazolone or hexyl cinnamic aldehyde (HCA), or to the non-sensitizing skin irritant methyl salicylate (MS). Five days later, lymph node cells (LNC) were labelled with CFSE, cultured for 96 h, then incubated with fluorescent labelled anti-CD4 (T helper) and -CD8 (T cytotoxic) cell antibodies, and proliferating CD4+ and CD8+ cells analyzed by flow cytometry. In LNC populations derived from vehicle-treated animals, less than 1% of either cell population had undergone one cell division or more. Topical exposure to MS (2.5% to 20%) did not increase the frequencies of proliferating cells. Exposure to all three allergens, however, resulted in a marked increase in the percentages of both CD4+ and CD8+ cells undergoing division, with up to 5% and 3% of these cells, respectively, proliferating in response to DNCB and oxazolone, with lower levels of proliferation stimulated by HCA. These preliminary data suggest that this method may be applied to provide the basis of a nonradioisotopic end point for the LLNA, particularly for the identification of potent contact allergens.

allergens, irritants, local lymph node assay, nonradioisotopic, flow cytometry .


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.