Principal Investigator
Dr. Rebecca Dearman
Syngenta
Alderley Park
Macclesfield
Cheshire SK10 4TJ
UK
Collaborators
Dr. Brian Flanagan – Department of Immunology – Liverpool University
fla1@liv.ac.uk
Description
Allergic hypersensitivity of the respiratory tract is an important occupational health problem. A major constraint in the development of appropriate methods for the prospective identification of chemical respiratory allergens is the continuing uncertainty regarding the immunobiological mechanisms of this disease and in particular the role of IgE antibody. There is, however, increasing evidence that allergic sensitization of the respiratory tract will be favoured by the induction of a selective type 2 immune response.
The objective of this project was to investigate the potential application of cytokine fingerprinting in predicting which chemicals have the potency to act as chemical respiratory sensitizers. A supplementary aim was to explore, using transcript profiling, whether there exist additional non-cytokine markers that may provide the necessary selectivity and sensitivity for the characterization of respiratory allergens.
The most discriminatory method for the characterization of differential cytokine profiles by respiratory allergens is the measurement of cytokine protein secretion in vitro by culture of draining lymph node cells for 24 and 120 h following chronic (8-13 day) topical exposure of BALB/c strain mice. Several chemicals have been tested including positive and negative control chemicals. Trimellitic anhydride, reactive dye reactive yellow 39, chloramine T and two uronium salts (tetramethyluronium hexafluorophosphate and tetramethyluronium tetrafluoroborate) all of which have been implicated as causing immediate type hypersensitivity reactions have been profiled. Cytokine profiles have been compared with contact allergens 2,4-dinitrochlorobenzene, tetramethylthiuram and eugenol. The nonsensitizing skin irritants methyl salicylate and p-aminobenzoic acid were also tested and failed to stimulate the production of type 1 or type 2 cytokines.
On the contrary, the short listed genes identified by microarray analysis as being up-regulated by the respiratory allergen TMA (IGH-4, CXCL9, CXCL10 and Granzyme B) were analyzed by real time-polymerase change reaction (real time-PCR). Granzyme B up-regulation was the most robust and selective change induced by TMA exposure compared with DNCB treatment. However using a panel of additional chemical allergens and the same exposure conditions Granzyme B up-regulation did not discriminate between contact and respiratory allergens.
Cytokine profiling continues to provide the most promising approach for the identification of chemical respiratory sensitizers, while the selected non-cytokine gene markers do not provide the necessary selectivity and sensitivity for the characterization of respiratory allergens. It is further recommended that local lymph node assay dose response data be used for dose setting, with chemicals tested at doses that provoke relatively vigorous proliferative responses. The standard vehicle acetone:olive oil and DMF are both appropriate for use in cytokine profiling assays and the assay is unaffected by skin irritants.
Related Publications
RJ Dearman, CJ Betts, HT Caddick, BF Flanagen, I Kimber, Cytokine profiling of chemical allergens in mice : impact of mitogen on selectivity of response, in preparation.
RJ Dearman, CJ Betts, HT Caddick, BF Flanagan, I Kimber, Cytokine profiling of chemical allergens in mice: Measurement of message versus protein, Toxicology 2008.
RJ Dearman, DA Basketter, GF Gerberick, I Kimber, Chemical allergy : hazard identification, hazard characterization and risk assessment, in: Immunotoxicology and Immunopharmacology, 3rd edition, eds. R Luebke, R House, I Kimber, CRC Press, New York, 2007, pp 591-606.
RJ Dearman, I Kimber, The use of cytokines in the identification and characterization of chemical allergens, In: Methods in Pharmacology and Toxicology. Cytokines in Human Health. Immunotoxicology, Pathology and Therapeutic Applications, eds. J Descotes, RV House, Humana Press, 2007, pp 67-82.
I Kimber, RJ Dearman, What makes a chemical a respiratory sensitizer?, Current Opinion in Allergy and Clinical Immunology 2005, 5, 119-124.
