Qualitative and quantitative evaluation of a local lymph node assay based on ex vivo interleukin-2 production.
The local lymph node assay (LLNA) is a regular method for the detection of sensitizing chemicals in mice which measures the incorporation of tritiated thymidine in lymph node cells. We have evaluated an alternative to this method based on the interleukin-2 (IL-2) production of lymph node cells. At the mRNA level, no change in the IL-2 gene expression level was detected by real-time PCR analysis. At the protein level, various experimental conditions were checked in order to improve the irritant versus sensitizer discrimination with a restricted set of prototypic compounds. In particular, the use of phytohemagglutinin A (PHA) in an ex vivo cell culture step showed an improvement of both signal and discrimination. In these optimised conditions, a panel of irritants and potency-graded sensitizers was used to assess the performance of the modified method. IFN-gamma production was used as a positive control. For each compound, a dose-response was performed and stimulation indexes (SI) were determined. Effective concentrations (EC) for each sensitizers were then extracted and compared to the literature data of the regular LLNA. The IL-2-based LLNA showed similar performances at both qualitative and quantitative levels compared to regular LLNA.
Azam
Philippe
P
AFSSAPS, Unité BCM, 635 Rue de la Garenne, 34740 Vendargues, France.
Peiffer
Jean-Luc
JL
Ourlin
Jean-Claude
JC
Bonnet
Pierre-Antoine
PA
Tissier
Marie-Hélène
MH
Vian
Laurence
L
Fabre
Isabelle
I
D000826:Animal Testing Alternatives; D000818:Animals; D017449:Dermatitis, Allergic Contact / Q000209:etiology / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007371:Interferon-gamma / Q000235:genetics / Q000276:immunology; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000235:genetics; D007509:Irritants / Q000633:toxicity*; D023441:Local Lymph Node Assay; D008198:Lymph Nodes / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007376:Interleukin-2; D007509:Irritants; D012333:RNA, Messenger; D007371:Interferon-gamma