Sensibilizzazione cutanea: possibilità in vitro. Emanuela Corsini Laboratory of Toxicology, Department of Pharmacological Sciences, Faculty of Pharmacy, Università degli Studi di Milano, Milan, Italy PRESENTATION LAYOUT • Introduction to chemical allergy • In vivo models • In vitro models
CHEMICAL ALLERGY • The two most frequent manifestation of chemical-induced allergy are contact hypersensitivity and respiratory sensitization , both of which can have serious impact on quality of life and represent a common occupational health problem. • Chemical agents cause approximately 40% of cases of occupational asthma. • Over the past few decades industrialized countries have faced a significant increase, although the rate of increase has recently slowed, of allergic diseases like atopic rhinitis, bronchial asthma, urticaria and contact dermatitis. • Hypersensitivity reactions are often considered a major health problem in relation to environmental chemical exposure. Potential Contact Sensitizers Potential Contact Sensitizers • Cosmetics and Fragrances • Dyes • Preservatives (formaldehyde) • Metals (Ni, Co, Be, Cr) • Pesticides (Poison ivy-type reaction - delayed type IV)
Hypersensitivity Definition excessive humoral or cellular response to an antigen which can lead to tissue damage. Hypersensitivity reactions are the result of normally beneficial immune responses acting inappropriately. Two Stages (Distinguishes from irritation) Elicitation Induction Challenge Sensitization (subsequent (1st exposure) exposure)
MHC class II Chemical STRATUM CORNEUM Key passages IL-1 KC EPIDERMIS IL-6 1. Absorption and local IL-7 trauma – proinflammatory IL-8 LC cytokine production TNF- α IL-10 (danger signals) RANTES IL-12 MCP-1 HAPTENS bear both the pro-inflammatory properties MIGRATION 2. Protein binding IL-1 β IL-18 IL-15 GM-CSF (adjuvant) and the antigenic properties through binding to 3. Antigen processing IL-18 self proteins. MATURATION GM-CSF 4. Langerhans cells/dermal STRONG haptens are the one with the most adjuvant TGF- α DCs maturation and migration properties and are therefore able to sensitize the majority TGF- β MIP-2 of individuals. 5. Antigen presentation to IP-10 Th cells and the generation WEAK haptens have only limited adjuvant effects and can α LYMPH NODE TNF- of memory T cells sensitize a minority of people. IFN- γ (immunogenicity) CD4+ Th0 IL-4, IL-5, IL-6, IL-10 IL-12, INF- γ , TNF- β Allergic contact dermatitis Atopic dermatitis (Type IV hypersensitivity) (Type I hypersensitivity) Allergic Contact Dermatitis : : Elicitation Elicitation Allergic Contact Dermatitis • Upon subsequent Hapten contact, some LDC inflammation migrate to local lymph node as before. Other Carrier LDC present processed keratinocytes : IFN γ T M IL-1, Il-6 hapten-carrier to other cytokines IL-8, TNF α memory T cells in skin. T • Activated memory T M Langerhans’ / dermis cells secrete cytokines Dendritic cell that induce release of T inflammatory cytokines M from other cell types. vascular endothelial cells I I Blood Vessel T IL-1, IL-6 M • Memory T cells and T - M inflammatory cells are Lymphatic Vessel recruited to the epidermis from circulation via Local T chemoattractant M Lymph cytokines and expression Node of adhesion molecules.
PRESENTATION LAYOUT • Introduction to chemical allergy • In vivo models • In vitro models Toxicological Approaches to Skin Sensitisation � Well established methods for contact hypersensitivity. � Current models and assays as inadequate predictors for system hypersensitivity reaction.
METHODS IN IMMUNOTOXICOLOGY Hypersensitivity Testing • Guinea Pig Tests (OECD 406): – Maximization Test – Occlusive Patch Test – Respiratory Challenge – Systemic Anaphylaxix • Mouse Tests: – Local lymph node assay (OECD 429) – Mouse Ear Swelling Test GUINEA PIG MODELS GUINEA PIG MODELS Guinea Pig Guinea Pig Buehler Buehler Maximization Maximization Assay Assay Test Test Topical application - closed ID injection w/ and without 20 animals/ group patch: FCA plus topical application: Induction Days 0, 6-8, and 13-15 Days 5-8 Day 27-28 topical challenge Day 20-22 topical challenge Challenge of the untreated flank for 6 h Read: 21, 24, 48 h after Endpoint erythema Read: 48,72 h after challenge removing patch >30% positive Criteria > 15% positive
The mouse local lymph node assay (LLNA) IMMUNE ACTIVATION LOCAL LYMPH NODE ASSAY Test/vehicle Day 0,1,2 T T T 2 days of rest, 3 H TdR T T T Day 5 T T 5 hours T Selective clonal expansion of allergen-responsive T lymphocytes Count DPMs 18 18 DNCB in A:OO OXAZ in A:OO 16 16 B 14 14 12 12 B B dpm ± SE 10 dpm ± SE 10 x10 -3 x10 -3 8 8 6 6 B 4 4 B B 2 2 B 3-fold 3-fold B B B B 0 B 0 0% 0.01% 0.025% 0.05% 0.1% 0.25% 0% 0.0025% 0.005% 0.01% 0.025% 0.05% 18 HCA in A:OO 16 14 LLNA Dose 12 dpm ± SE 10 Response Data x10 -3 8 B 6 B 4 2 3-fold B B B B 0 0% 2.5% 5% 10% 25% 50%
NEW OECD GUIDELINES - Update OECD 429 – Skin sensitization: reduced LLNA It also includes the Performance Standards that can be used to evaluate the validation status of new and /or modified test methods that are functionall and mechanistically similar to the LLNA. - OECD 442A - Skin Sensitization: LLNA DA - OECD 442B - Skin Sensitization: LLNA BrdU-ELISA All adopted 22 nd July 2010
PRESENTATION LAYOUT • Introduction to chemical allergy • In vivo models • In vitro models
Choice of experimental model(s) to study hypersensitivity MHC class II Chemical STRATUM CORNEUM Key events IL-1 KC EPIDERMIS IL-6 1. Absorption (metabolism) IL-7 and local trauma – IL-8 LC TNF- α proinflammatory cytokine IL-10 RANTES production (danger signals) IL-12 MCP-1 MIGRATION IL-1 β IL-18 2. Protein binding IL-15 GM-CSF IL-18 3. Antigen processing MATURATION GM-CSF 4. Langerhans cells/dermal TGF- α DCs maturation and TGF- β migration MIP-2 5. Antigen presentation to IP-10 Th cells and the generation α TNF- LYMPH NODE of memory T cells IFN- γ (immunogenicity) CD4+ Th0 IL-4, IL-5, IL-6, IL-10 IL-12, INF- γ , TNF- β Allergic contact dermatitis Atopic dermatitis (Type IV hypersensitivity) (Type I hypersensitivity)
KERATINOCYTES • In principle, a test system comprised of KC alone may not be useful in establishing MHC class II allergenic potency as these cells lack antigen Chemical presenting capacity. � However, in addition to chemical processing, LC activation requires the binding of cytokines produced by KC as a TNF- α result of initial chemical exposure. RANTES IL-1 β MIGRATION MCP-1 • Chemical must cause sufficient local trauma GM-CSF MATURATION to induce/augment cutaneous cytokine production. • The irritant capacity of allergens might present an additional risk factor so that irritant + CD4 LYMPH NODE allergens may be stronger allergens than Th0 non-irritant ones (Grabbe et al ., 1996). � In this case, the potency of chemicals to induce cutaneous sensitization may be assessed as a function of KC cytokine expression.
Exposure of NCTC 2544 cells to contact allergens results in a dose-related induction of intracellular IL-18, whereas exposure to respiratory allergens and irritants fails to induce IL-18 production Similar results were also obtained using primary human KC and other keratinocyte cell lines, confirming the relevance of the proposed model and the possibility to use different source of KC 33
IL-18 SECRETION AS A MARKER FOR IDENTIFICATION OF CONTACT SENSITIZERS IN THE EPIDERM IN VITRO HUMAN SKIN MODEL Deng, W., Oldach, J., Armento, A., Ayehunie, S., Kandarova, H., Letasiova, S., Klausner, M., and Hayden, P. MatTek Corporation, Ashland, MA, USA. Presented at SOT 2011, Abstract #2571 Chemical Tested: 2,4-Dinitrochlorobenzene (DNCB), 2-Mercaptobenzothiazole (2- MBT), 4-Nitrobenzylbromide (4-NBB), Cinnamaldehyde, Cinnamyl Alcohol, Eugenol, Glycerol, Glyoxal, Isoeugenol, Lactic Acid, Phenol, p-Phenylenediamine (ppd), Resorcinol, Salicylic Acid, Tetramethylthiurame disulfide (TMTD) Two tiered cell based assay to distinguish sensitizers from non-sensitizers and to classify sensitizers according to their potency • Tier 1 Distinguishes sensitizers from non-sensitizers using NCTC 2544 keratinocyte cell line and IL-18 production (ELISA) as readout. chemical IL-18 ELISA NCTC 2544 • Tier 2 Determines sensitizer potency. Sensitizers selected in tier 1 were used in tier 2. Epidermal equivalents (EE) were topically exposed for 24 hours to sensitizers selected from tier 1 in a dose response manner and EC 50 values were calculated based on the decrease in EE metabolic activity (MTT assay EC 50 : chemical concentration which results in 50% reduction in cell metabolic activity). 125 viable cells (%) chemical 100 75 MTT assay / EC 50 calculation 50 25 0 0 2 4 6 8 DNCB (mM) EC 50 = 6.778mM
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