title: Development of a flow cytometry assay for the identification and differentiation of chemicals with the potential to elicit irritation, IgE-mediated, or T cell-mediated hypersensitivity responses.
abstract: These studies were conducted to investigate the potential use of a flow cytometric analysis method for the identification and differentiation of chemicals with the capacity to induce irritation, IgE- or T cell-mediated hypersensitivity responses. An initial study investigated the ability of equally sensitizing concentrations (determined by local lymph node assay) of IgE-mediated (Toluene Diisocyanate-TDI) and T cell-mediated (Dinitrofluorobenzene-DNFB) allergens to differentially modulate the IgE+B220+ population in the lymph nodes draining the dermal exposure site. Sodium lauryl sulfate (SLS) was also tested as a nonsensitizing irritant control. Female B6C3F1 mice were dermally exposed once daily for 4 consecutive days, with the optimum time point for analysis determined by examining the IgE+B220+ population 8, 10, and 12 days post-initial chemical exposure. At the peak time point, day 10, the IgE+B220+ population was significantly elevated in TDI (41%), while moderately elevated in DNFB (18%) exposed animals when compared to the vehicle (0.8%), and remained unchanged in SLS (2.2%) exposed animals when compared to the ethanol control (2.5%). Experiments in our laboratory and others have demonstrated that the draining lymph node B220+ population becomes significantly elevated following exposure to allergens (IgE- and T cell-mediated), not irritants, allowing for their differentiation. An existing mouse ear swelling assay was used to identify chemical irritants. Therefore, using the endpoints of percent ear swelling, percent B220+ cells, and percent IgE+B220+ cells, a combined irritancy/phenotypic analysis assay was developed and tested with tetradecane (irritant), toluene diisocyanate, trimellitic anhydride (IgE-mediated allergens), benzalkonium chloride, dinitrofluorobenzene, oxazolone, and dinitrochlorobenzene (T cell-mediated allergens) over a range of concentrations. Based upon the pattern of response observed, a paradigm was developed for continued evaluation: Irritant exposure will result in significant ear swelling without altering the B220+ or IgE+B220+ populations. Exposure to sensitizers (IgE-mediated or T cell-mediated) will increase the B220+ population and the percent ear swelling will remain unchanged or will significantly increase, depending on the irritancy capacity of the chemical. Both the IgE+B220+ and B220+ populations will become elevated at the same test concentration following exposure to IgE-mediated, hypersensitivity inducing allergens. At its peak, the percent of IgE+B220+ cells will be equal to the percent of B220+ cells. The B220+ population will increase at a lower test concentration than the IgE+B220+ population, following exposure to T cell-mediated, hypersensitivity inducing allergens. At its peak, the percent of IgE+B220+ cells will reach less than half that of the percent of B220+ cells. The irritancy/phenotypic analysis method may represent a single murine assay able to identify and differentiate chemicals with the capacity to induce irritation, or IgE-mediated or T cell-mediated responses.
authors.affiliation: Department of Pharmacology and Toxicology, Medical College of Virginia/VCU, Richmond, USA.
authors.forename: T S
authors.initials: TS
authors.lastname: Manetz
authors.forename: B J
authors.initials: BJ
authors.lastname: Meade
mesh_terms: D000279:Administration, Cutaneous; D000818:Animals; D003877:Dermatitis, Contact / Q000209:etiology; D004139:Dinitrofluorobenzene / Q000009:adverse effects*; D004305:Dose-Response Relationship, Drug; D004487:Edema / Q000139:chemically induced; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006967:Hypersensitivity / Q000209:etiology*; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification; D007509:Irritants / Q000302:isolation & purification*; D008198:Lymph Nodes / Q000166:cytology; D051379:Mice; D010641:Phenotype; D012967:Sodium Dodecyl Sulfate / Q000009:adverse effects; D013047:Specific Pathogen-Free Organisms; D013601:T-Lymphocytes / Q000276:immunology; D014051:Toluene 2,4-Diisocyanate / Q000009:adverse effects*
chemical_list: D007074:Immunoglobulin G; D007509:Irritants; D014051:Toluene 2,4-Diisocyanate; D012967:Sodium Dodecyl Sulfate; D004139:Dinitrofluorobenzene