Sensibilizzazione cutanea: possibilit in vitro. Emanuela Corsini - - PDF document
Sensibilizzazione cutanea: possibilit in vitro. Emanuela Corsini Laboratory of Toxicology, Department of Pharmacological Sciences, Faculty of Pharmacy, Universit degli Studi di Milano, Milan, Italy PRESENTATION LAYOUT Introduction to
Laboratory of Toxicology, Department of Pharmacological Sciences, Faculty of Pharmacy, Università degli Studi di Milano, Milan, Italy
hypersensitivity and respiratory sensitization, both of which can have serious impact on quality of life and represent a common occupational health problem.
increase, although the rate of increase has recently slowed, of allergic diseases like atopic rhinitis, bronchial asthma, urticaria and contact dermatitis.
relation to environmental chemical exposure.
(Poison ivy-type reaction
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.
Induction Sensitization (1st exposure) Elicitation Challenge (subsequent exposure)
STRATUM CORNEUM Chemical EPIDERMIS LYMPH NODE
MIGRATION MATURATION
TNF-α RANTES MCP-1 IL-18 IL-1β GM-CSF
IL-1 IL-6 IL-8 IL-10 IL-12 α MIP-2 IP-10 TNF- IFN-γ
MHC class II
Th0
IL-4, IL-5, IL-6, IL-10
Atopic dermatitis
(Type I hypersensitivity) IL-12, INF-γ, TNF-β
Allergic contact dermatitis
(Type IV hypersensitivity)
CD4+
KC
LC
IL-7 IL-15 IL-18 GM-CSF TGF-α TGF-β Key passages
trauma – proinflammatory cytokine production (danger signals)
DCs maturation and migration
Th cells and the generation
(immunogenicity)
HAPTENS bear both the pro-inflammatory properties (adjuvant) and the antigenic properties through binding to self proteins. STRONG haptens are the one with the most adjuvant properties and are therefore able to sensitize the majority
WEAK haptens have only limited adjuvant effects and can sensitize a minority of people.
Hapten Carrier Langerhans’ / Dendritic cell dermis Lymphatic Vessel Local Lymph Node keratinocytes :
T M T M T M Blood Vessel T M
inflammation T M T M I I
contact, some LDC migrate to local lymph node as before. Other LDC present processed hapten-carrier to memory T cells in skin.
cells secrete cytokines that induce release of inflammatory cytokines from other cell types.
inflammatory cells are recruited to the epidermis from circulation via chemoattractant cytokines and expression
IL-1, IL-6 IFNγ IL-1, Il-6 IL-8, TNFα
Well established methods for contact
Current models and assays as
– Maximization Test – Occlusive Patch Test – Respiratory Challenge – Systemic Anaphylaxix
ID injection w/ and without FCA plus topical application: Days 5-8 Day 20-22 topical challenge Read: 48,72 h after challenge
>30% positive
Guinea Pig Guinea Pig Maximization Maximization Test Test
Topical application - closed patch: Days 0, 6-8, and 13-15 Day 27-28 topical challenge
Read: 21, 24, 48 h after removing patch
> 15% positive
Buehler Buehler Assay Assay
Induction Challenge Endpoint erythema
Criteria
20 animals/ group
Test/vehicle
LOCAL LYMPH NODE ASSAY
Day 0,1,2 Day 5 2 days of rest, 3H TdR 5 hours T T T T T T T T
IMMUNE ACTIVATION Selective clonal expansion of allergen-responsive T lymphocytes
T
The mouse local lymph node assay (LLNA)
Count DPMs
DNCB in A:OO
B B B B B B
0% 0.01% 0.025% 0.05% 0.1% 0.25% 2 4 6 8 10 12 14 16 18 3-fold dpm ± SE x10 -3
B B B B B B
0% 0.0025% 0.005% 0.01% 0.025% 0.05% 2 4 6 8 10 12 14 16 18
OXAZ in A:OO
dpm ± SE x10 -3 3-fold
HCA in A:OO
B B B B B B
0% 2.5% 5% 10% 25% 50% 2 4 6 8 10 12 14 16 18 dpm ± SE x10 -3 3-fold
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.
All adopted 22nd July 2010
STRATUM CORNEUM Chemical EPIDERMIS LYMPH NODE
MIGRATION MATURATION
TNF-α RANTES MCP-1 IL-18 IL-1β GM-CSF
IL-1 IL-6 IL-8 IL-10 IL-12 α MIP-2 IP-10 TNF- IFN-γ
MHC class II
Th0
IL-4, IL-5, IL-6, IL-10
Atopic dermatitis
(Type I hypersensitivity) IL-12, INF-γ, TNF-β
Allergic contact dermatitis
(Type IV hypersensitivity)
CD4+
KC
LC
IL-7 IL-15 IL-18 GM-CSF TGF-α TGF-β
Key events
and local trauma – proinflammatory cytokine production (danger signals)
DCs maturation and migration
Th cells and the generation
(immunogenicity)
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.
to induce/augment cutaneous cytokine production.
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.
Chemical LYMPH NODE
MIGRATION MATURATION
TNF-α
RANTES
MCP-1 IL-1β GM-CSF
MHC class II
Th0
CD4 +
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 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
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)
keratinocyte cell line and IL-18 production (ELISA) as readout.
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 EC50 values were calculated based on the decrease in EE metabolic activity (MTT assay EC50: chemical concentration which results in 50% reduction in cell metabolic activity).
chemical
MTT assay / EC50 calculation
2 4 6 8 25 50 75 100 125
EC50 = 6.778mM DNCB (mM) viable cells (%)
NCTC 2544 chemical IL-18 ELISA
Two tiered cell based assay to distinguish sensitizers from non-sensitizers and to classify sensitizers according to their potency
Gene Set Enrichment Analysis showed upregulation
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.
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.
Chemical LYMPH NODE
MIGRATION MATURATION
TNF-α
RANTES
MCP-1 IL-1β GM-CSF
MHC class II
Th0
CD4 +
ALTERNATIVE USE OF LC:
ALTERNATIVE USE OF DC
monocytic leukemia cell lines).
KEY EVENT IN VITRO OPPORTUNITIES
Reconstituted human epidermis
macro-molecules (i.e., proteins) QSAR/Expert systems; Peptide binding assay
generation of danger signals KeratinosensTM; KC activation; NCTC2544 IL-18 assay; KC gene expression profile
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
presentation to TH cells and memory T-cell generation In vitro T-cell activation
Key events in chemical-induced skin sensitization and in vitro opportunities
allergens are predicted by the up-regulation of CD86 and CD54 expression when cells are exposed to subtoxic concentrations of chemicals.
allergens to bind with nucleophilic amino acids has been shown to correlate to the skin sensitization potential of a chemical.
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.
In vivo toxicity
In vitro model TEST OPTIMIZATION Pre-validation Validation
REGULATORY ACCEPTANCE
VALIDATED TEST
(uninteded stimulation, i.e. contact hypersensitivity)