title: Cytokine profiling of chemical allergens in mice: impact of mitogen on selectivity of response.
abstract: A major constraint in the development of methods for the identification of chemical respiratory allergens is the continuing uncertainty regarding the mechanisms of this disease and in particular the role of IgE antibody. There is, however, increasing evidence that respiratory sensitization is favoured by the induction of a selective type 2 cytokine response. The current investigations focus on the potential application of cytokine profiling to the identification of chemical respiratory sensitizers. The objective is to determine the optimal configuration of the method for discrimination between chemical contact and respiratory sensitizers. The reference contact sensitizer 2,4-dinitrochlorobenzene (DNCB) and reference respiratory sensitizer trimellitic anhydride (TMA), which have been shown to induce type 1 and type 2 cytokine profiles, respectively, were utilized. Variables investigated included cell concentration, time in culture, dosing regimens (a 13 day and a truncated 8 day protocol) and the impact of restimulation in vitro with T cell mitogens. Cell culture conditions were critical for the selectivity of the response, with the addition of mitogen resulting in a less discriminatory pattern of cytokine expression, particularly for the type 1 cytokine interferon gamma (IFN-gamma). Furthermore, a 13 day exposure period was required for vigorous expression of IFN-gamma by DNCB-activated cells, whereas type 2 cytokine expression by TMA-stimulated cells was recorded after 8 days. These data demonstrate that the most optimal method for cytokine profiling is a chronic (13 day) exposure regime followed by culture of lymph node cells at 10(7 )cells ml(-1) for 120 h in the absence of mitogen.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M139PT, UK. rebecca.dearman@manchester.ac.uk
authors.forename: Rebecca Jane
authors.initials: RJ
authors.lastname: Dearman
authors.forename: Catherine Jean
authors.initials: CJ
authors.lastname: Betts
authors.forename: Helen Theresa
authors.initials: HT
authors.lastname: Caddick
authors.forename: Ian
authors.initials: I
authors.lastname: Kimber
mesh_terms: D000485:Allergens / Q000276:immunology* / Q000494:pharmacology*; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004396:Coloring Agents / Q000378:metabolism; D016207:Cytokines / Q000032:analysis* / Q000378:metabolism; D004137:Dinitrochlorobenzene / Q000494:pharmacology; D005260:Female; D008198:Lymph Nodes / Q000033:anatomy & histology / Q000166:cytology / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008934:Mitogens / Q000494:pharmacology*; D010796:Phthalic Anhydrides / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013997:Time Factors; D023382:Toxicity Tests, Acute; D036401:Toxicity Tests, Chronic; D014343:Trypan Blue / Q000378:metabolism
chemical_list: D000485:Allergens; D004396:Coloring Agents; D016207:Cytokines; D004137:Dinitrochlorobenzene; D008934:Mitogens; D010796:Phthalic Anhydrides; C015559:trimellitic anhydride; D014343:Trypan Blue