PRESENTATION LAYOUT Introduction to chemical allergy. In vivo models. In vitro models

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1 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

2 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 Cosmetics and Fragrances Dyes Preservatives (formaldehyde) Metals (Ni, Co, e, Cr) Pesticides (Poison ivy-type reaction - delayed type IV)

3 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) Induction Sensitization (1st exposure) Elicitation Challenge (subsequent exposure)

4 MHC class II Chemical IL-1 IL-6 IL-7 IL-8 IL-10 IL-12 IL-15 IL-18 GM-CSF TGF-α TGF-β MIP-2 IP-10 TNF-α IFN-γ KC LC STRATUM CORNEUM EPIDERMIS TNF-α RANTES MCP-1 HAPTENS IL-1β bear both MIGRATION the pro-inflammatory IL-18 properties 2. Protein binding GM-CSF (adjuvant) and the antigenic properties through 3. Antigen binding processing to self proteins. MATURATION 4. Langerhans cells/dermal STRONG haptens are the one with the most adjuvant DCs maturation and migration properties and are therefore able to sensitize the majority of individuals. 5. Antigen presentation to Th cells and the generation WEAK haptens have only limited LYMPH adjuvant NODE effects of memory and can T cells sensitize a minority of people. (immunogenicity) IL-12, INF-γ, TNF-β CD4 + Th0 IL-4, IL-5, IL-6, IL-10 Key passages 1. Absorption and local trauma proinflammatory cytokine production (danger signals) Allergic contact dermatitis (Type IV hypersensitivity) Atopic dermatitis (Type I hypersensitivity) Allergic Contact Dermatitis: Elicitation inflammation keratinocytes: IFNγ IL-1, Il-6 other cytokines IL-8, TNFα T M Hapten Carrier Upon subsequent contact, some LDC migrate to local lymph node as before. Other LDC present processed hapten-carrier to memory T cells in skin. dermis I T M I T M IL-1, IL-6 - T M vascular endothelial cells T M T M Local Lymph Node Langerhans / Dendritic cell lood Vessel Lymphatic Vessel Activated memory T cells secrete cytokines that induce release of inflammatory cytokines from other cell types. Memory T cells and inflammatory cells are recruited to the epidermis from circulation via chemoattractant cytokines and expression of adhesion molecules.

5 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.

6 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 Maximization Test ID injection w/ and without FCA plus topical application: Days animals/ group Induction uehler Assay Topical application - closed patch: Days 0, 6-8, and Day topical challenge Challenge Day topical challenge of the untreated flank for 6 h Read: 48,72 h after challenge >30% positive Endpoint erythema Criteria Read: 21, 24, 48 h after removing patch > 15% positive

7 The mouse local lymph node assay (LLNA) IMMUNE ACTIVATION LOCAL LYMPH NODE ASSAY Test/vehicle T T Day 0,1,2 T T 2 days of rest, 3 H TdR T T T Day 5 T T 5 hours Selective clonal expansion of allergen-responsive T lymphocytes Count DPMs DNC in A:OO OXAZ in A:OO dpm ± SE x dpm ± SE x % 0.01% 0.025% 0.05% 0.1% 0.25% 3-fold 2 0 0% % 0.005% 0.01% 0.025% 0.05% 3-fold HCA in A:OO dpm ± SE x LLNA Dose Response Data % 2.5% 5% 10% 25% 50% 3-fold

8 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 Skin Sensitization: LLNA rdu-elisa All adopted 22 nd July 2010

9 PRESENTATION LAYOUT Introduction to chemical allergy In vivo models In vitro models

10 Choice of experimental model(s) to study hypersensitivity MHC class II Chemical IL-1 KC IL-6 IL-7 IL-8 IL-10 IL-12 IL-15 IL-18 GM-CSF TGF-α TGF-β MIP-2 IP-10 TNF-α IFN-γ LC IL-1β GM-CSF IL-12, INF-γ, TNF-β CD4 + MIGRATION MATURATION Th0 STRATUM CORNEUM EPIDERMIS TNF-α RANTES MCP-1 IL-18 LYMPH NODE IL-4, IL-5, IL-6, IL-10 Key events 1. Absorption (metabolism) and local trauma proinflammatory cytokine production (danger signals) 2. Protein binding 3. Antigen processing 4. Langerhans cells/dermal DCs maturation and migration 5. Antigen presentation to Th cells and the generation of memory T cells (immunogenicity) Allergic contact dermatitis (Type IV hypersensitivity) Atopic dermatitis (Type I hypersensitivity)

11 IL-1β GM-CSF MHC class II Chemical MIGRATION MATURATION CD4+ Th0 TNF-α RANTES MCP-1 LYMPH NODE KERATINOCYTES In principle, a test system comprised of KC alone may not be useful in establishing allergenic potency as these cells lack antigen presenting capacity. However, in addition to chemical processing, LC activation requires the binding of cytokines produced by KC as a result of initial chemical exposure. Chemical must cause sufficient local trauma to induce/augment cutaneous cytokine production. The irritant capacity of allergens might present an additional risk factor so that irritant allergens may be stronger allergens than 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.

12 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

13 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 (DNC), 2-Mercaptobenzothiazole (2- MT), 4-Nitrobenzylbromide (4-N), 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 NCTC 2544 IL-18 ELISA 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). chemical MTT assay / EC 50 calculation viable cells (%) DNC (mm) EC 50 = 6.778mM

14 Gene Set Enrichment Analysis showed upregulation of Keap1 dependent and oxidative stress gene lists. KC expression profiling can identify contact sensitizers. Moreover, data suggest that contact sensitizers induce the oxidative stress pathway in KC. LANGERHANS CELLS IL-1β GM-CSF MHC class II Chemical TNF-α RANTES MIGRATION MCP-1 MATURATION CD4+ Th0 LYMPH NODE LC form a sentinel network able to detect, capture, and process antigens such as invading bacteria, viruses, products of tissue damage and haptens. Upon antigen capture, the LC undergo a maturation process leading to the upregulation of co-stimulatory molecules (CD54, CD86, CD80, CD40), MHC class II molecules and the CD83 protein. Thereafter, LC migrate to the T-cell areas of lymphoid organs where they lose antigen-processing activity and become potent immunostimulatory cells.

15 Langerhans cells: restrictions Difficult to isolate from human skin. Low viability. Shortage of available human skin ALTERNATIVE USE OF LC: Human peripheral blood mononuclear cells CD34+ hematopoietic progenitor cells from cord blood ALTERNATIVE USE OF DC Use of cell lines such as THP-1, KG-1, MUTZ-3 (human monocytic leukemia cell lines).

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17 Key events in chemical-induced skin sensitization and in vitro opportunities KEY EVENT IN VITRO OPPORTUNITIES 1. Skin penetration Human skin biopsis, pig skin; Reconstituted human epidermis 2. inding to macro-molecules (i.e., proteins) QSAR/Expert systems; Peptide binding assay 3. Local trauma and generation of danger signals Keratinosens TM ; KC activation; NCTC2544 IL-18 assay; KC gene expression profile 4. Langerhans cells maturation and migration DC-like up-regulation of class II antigens and costimulatory molecules, i.e. CD54, CD86; Cytokine release, i.e. IL-8; LC-like MUTZ-3 cells migration assay; DC-like gene expression profile 5. Antigen presentation to T H cells and memory T-cell generation In vit ro T-cell activation Methods under validation at ECVAM THP-1: human Cell Line Activation Test (h-clat), chemical allergens are predicted by the up-regulation of CD86 and CD54 expression when cells are exposed to subtoxic concentrations of chemicals. U937: CD86 upregulation Peptide binding assay: the ability of known chemical allergens to bind with nucleophilic amino acids has been shown to correlate to the skin sensitization potential of a chemical.

18 Methods under validation at JaCVAM THP-1: IL-8 Luc assay Takahashi T, Kimura Y, Saito R, Nakajima Y, Ohmiya Y, Yamasaki K, Aiba S. An in vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THP-G8. Toxicol Sci. 124(2):359-69, 2011 Sep 13. Development of alternative in vitro test In vivo toxicity In vitro model TEST (uninteded stimulation, i.e. contact hypersensitivity) Validation Pre-validation OPTIMIZATION VALIDATED TEST REGULATORY ACCEPTANCE

19 FINAL CONCLUSIONS In vitro methods to assess unintended contact hypersensitivity are available, although none have been formally validated. These methods can be used in-house for pre-screening, prioritization, and hazard identification of direct immunotoxicants.