September 20, 2013

http://www.gerda2013.com/
Allergologie et Immunologie clinique Lyon-Sud / Gerland
Equipe 8 – INSERM U851
Service Allergologie
et Immunologie Clinique Lyon-Sud
Marc VOCANSON, Inserm U851
Unité de recherche clinique
Lyon-Sud
IMMUNOLOGY OF SKIN ALLERGY / SKIN VACCINATION
Research activities
Pathophysiological research
Skin allergic diseases
Translational research
New immunological assays
Diagnostic
Prediction of allergenicity
Allergic contact dermatitis (ACD)
Atopic dermatitis (AD)
ECZEMAS
MILD - Exanthema
SEVERE – Blistering disease
DRUG ALLERGY
Intradermal vaccination
Fragrance allergy
We know very little!
1.
2.
3.
4.
Fragrances = allergens?
Some fragrances = allergens of concern?
Is it useful to ban allergens of concern?
Is it necessary to reduce the prevalence of ACD to
fragrances?
5. Is it necessary to reduce the prevalence of
fragrance contact allergy?
6. Fragrance contact allergy = positive patch tests
could be irritation or allergy
7. Contact dermatitis to fragrance = eczema
could be irritation or allergy
ALLERGIC CONTACT DERMATITIS
Contact Hypersensitivity
Repeated contact with skin sensitizers
called haptens
Haptens are very diverse chemical
HAPTENS
molecules with 2 properties:
protein chemicals
- Non
pro-inflammatory
(danger signal)
Interact
with
aminoacid
residues
- interaction with amino acids,
modification
of self proteins: new antigenic motifs
DNP et TNP: lysin
Ni: histidin
ACD
is a skin DTH reaction
Hapten-specific T cells
- Strong H: DNP, TNP, oxazolone
Patients with ACD have circulating
ACD in 90% of people
specific T cells which are recruited
in the skin to induce the ACD
inflammation
- Weak H : metals (Ni, Cr, Cu)
ACD in 20% of people
Diagnosis of ACD rely on:
1. Patch-tests
weak H: ACD
in
2.- Very
Immunobiological
tests
< 1%
Professional ACD, drugs
Contact dermatitis/ eczema
= skin inflammation
•
•
•
•
•
Allergic CD
•
Specific T cells
CD8+ T cells/IFNg •
Sensitization &
•
disease
•
Irritant CD
•
No specific T cells
No CD8+ T
cells/IFNg
•
No sensitization
•
•
Pos patch test = Skin inflammation
True +
Contact allergy =
allergy to patch
test
Specific T cells
CD8+ T cells/IFNg
Sensitization
•
•
•
•
•
False +
Contact irritation
No specific T cells
No CD8+ T
cells/IFNg
No sensitization
Take home messages
NO SKIN INFLAMMATION
Immune tolerance
Low dose
SKIN INFLAMMATION
Immune proinflammatory response
Threshold
High dose
Scoring (ICDRG)
IR irritative
Négative
Positive +
+/-
Positive +++
Positive ++
PATCH TEST INTENSITY
+
++
+++
CONTACT ALLERGY
CONTACT IRRITATION
Fragrance Allergy
Contact allergy, allergic contact dermatitis or irritant contact
dermatitis ?
• Pathophysiology of skin irritation and allergy
• How to differentiate allergy from irritation ?
• How to differentiate allergy to patch test from
clinically relevant allergy ?
• Exposure to allergens promotes tolerance
Jean-François NICOLAS, Lyon University Hospital
SKIN INFLAMMATION
ALLERGY OR IRRITATION ?
• Pathophysiology of skin irritation and allergy
• How to differentiate allergy from irritation ?
• How to differentiate allergy to patch test from
clinically relevant allergy ?
• Exposure to allergens promotes tolerance
Jean-François NICOLAS, Lyon University Hospital
Allergic contact dermatitis - Weak Haptens
Sensitization
Fragrance allergens (HCA, EUG, HDCL) 3 sensitizations
Challenge
Ear swelling measue
24-96 h
5 days
Weak hapten
Anti-CD4 mAb depleted
C57BL/6
Les CD4 sont tolérogènes
Œdème de l’oreille (m)
200
150
100
Weak hapten
C57BL/6
Souris tolérante
50
0
0
2
4
6
8
10
Skin irritation conditions the severity of ACD
« L’irritation cutanée fait le lit de l’allergie »
DNFB sur l’oreille
gauche
J0
Mesure de l’œdème
de l’oreille
HO H3 H6 H9 H24
Mesure de l’œdème
de l’oreille
J5/J10
SKIN INFLAMMATION
ALLERGY OR IRRITATION ?
• Pathophysiology of skin irritation and allergy
• How to differentiate allergy from irritation ?
• How to differentiate allergy to patch test from
clinically relevant allergy ?
• Exposure to allergens promotes tolerance
Jean-François NICOLAS, Lyon University Hospital
Les haptènes forts activent fortement les LT CD4+ reg
qui limitent l’inflammation de l’EAC
Sensitization (Day 0)
DNFB (haptène fort)
following days
104
Naive
8.4
4,5%
1.46
103
draining
lymph nodes
(dLNs)
FACS analysis
104
DNFB – T reg
7.2
16%
3.66
103
3.66%
1.46%
102
102
101
101
10
0
10
2.96%
87.2
0
100
2.96
10
1
10
2
10
3
10
13.1%
4
76.1
100
13.1
101
102
103
104
Les haptènes faibles (parfums) activent fortement les
LT CD4+ reg/tol qui préviennent l’immunisation et donc qui
empêchent la survenue de l’EAC
Sensitization (Day 0)
Parfum (HCA)
(haptène faible)
following days
10
7.95
10
0.46
10 3
10
1
CD25
10
0.46%
2.56%
89
0
10
0
HCA – T tol
4
8.15
ICOS
1
10
.
2
10
3
10
4
2.01
10
2
10
1
10
0
2.01%
7.59%
2.56
10
10%
10 3
.
2
.
10
FACS analysis
3%
Isopropanol
4
draining
lymph nodes
(dLNs)
82.2
10
0
7.59
10
1
10
.
2
10
3
10
4
SKIN INFLAMMATION
ALLERGY OR IRRITATION ?
• Pathophysiology of skin irritation and allergy
• How to differentiate allergy from irritation ?
• How to differentiate allergy to patch test from
clinically relevant allergy ?
• Exposure to allergens promotes tolerance
Jean-François NICOLAS, Lyon University Hospital
Irritant versus Allergic Contact Dermatitis
Irritant versus Allergic Contact Dermatitis ?
SLS
How to differentiate irritation from allergy?
•
•
•
10 positive patch tests
Are there irritant or allergic ?
Are true positive tests relevant ?
By demonstrating the existence of an
allergic reaction
1. Modify the patch testing protocol
– Patch-test applied for 24 hrs (12 hrs)
– Reading at 48/72 hrs
2. Characterize the skin DTH reaction: biopsy
– T cell infiltration
– T cell activation
3. Characterize circulating specific T cells
– LTT
– Elispot
Strong and weak haptens
Nom
Source
Pouvoir sensibilisant
2,4-Dinitrochlorobenzène
Chimie
Fort
2,4-Dinitrofluorobenzène
Chimie
Strong/Fort
2,4-Dinitroiodobenzène
Fort
Disperses Blue 124 ou 106
Chimie
Textile (Colorants)
Dichromate de potassium
Bâtiment (ciment)
Modéré
Sulfate de nickel
Bâtiment, Bijoux fantaisie
Modéré
Formaldéhyde
Cosmétique, Textiles, Désinfectant
Faible
Glutaraldéhyde
Conservateur, Antiseptique
Faible
p- Phénylenédiamine
Cosmétique (colorant capillaire)
Eugénol
Cosmétique, Antiseptique
Faible
Weak/Faible
Hexylcinnamique aldéhyde
Cosmétique (parfum)
Weak/Faible
Hydroxycitronellal
Cosmétique (parfum)
Weak/Faible
Amoxicilline, Cyanamide,
Cetrimide...
Médicament
Weak/Faible
Faible
Testing of skin sensitization, Basketter, 2002
DRUG ALLERGY - INTRODUCTION
1
2
3
4
5
6
7
Mortality
Prevalence
- TEN: Toxic Epidermal Necrolysis
- DRESS: Drug Rash with Eosinophilia and Systemic
symptoms
- AGEP: Acute Generalized Exanthematous Pustulosis
- FDE: Fixed Drug Eruption
- Erythema multiform
- Others severe reactions
Drug allergy – DTH reactions or not ?
What is the offending drug ?
Maculo-papular exanthema Clamoxyl (peni A)
MPE after clamoxyl + ibuprofene
replaced by sulfamethoxazole +
tenoxicam
MPE to peni M (Bristopen) + Oropivalone
+ ketoprofene + paracetamol
FIRST PATCH TESTING – Allergic and irritant positive patch tests
Mr V.
Drug-induced maculo-papular exanthema
January 2004 – 5 days after a
prostatectomy + GA
1st testing (patch removal 72h):
Tracrium +
Ampicillin (IV solution + petrolatum)
Gentamycin +
SLS 0,5 et 0,25+
• Several positive patch tests
• Irritation control (SLS) positive
• No conclusion
SECOND PATCH TESTING – Early removal of patch tests decreases the irritation
properties of chamicals
FIRST PATCH TESTING
Patch removal at 12
hours
-Ampicillin solution ++
-Ampicillin petrolatum ++
- Tacrium patch: - Gentamycin patch: -SLS 0.25:-
SKIN BIOPSIES – Presence of T cells infiltrating the patch tests
Results: T cell infiltration/activation found only
in ampicillin positive patch test
SLS 0,25 / Ampi
Actine
CD4
3,5
3
CD8
2,5
2
1,5
1
0,5
0
8
7
6
IFNg
5
4
3
2
1
0
SLS
CD8
IFN-
Ampi
9
Ratio (DO CD4 / DO Actine )x100
FIRST PATCH TESTING
4
SLS
Ratio (DO IFNg / DO Actine )x100
Skin Biopsies (4mm diameter). Deep frozen
RNA extraction
Probes for CD4, CD8 and IFNg (cDNA)
Semi-quantitative RT-PCR
Ratio (DO CD8 / DO Actine )x100
RT-PCR analysis of T cell infiltration and T cell activation4,5
20
18
16
14
12
10
8
6
4
2
0
Ampi
CD4
SLS
Ampi
BLOOD - Presence of hapten-specific T cells
Detection and enumeration of hapten-specific T cells in blood
CFSE
3
2,5
2
1,5
1
0,5
0
Nbre de spots / 1.106
PBMC
Totapen
1mg/ml
Totapen 0,6 Totapen
mg/ml
0,3mg/ml
Gentalline
1mg/ml
Gentalline
0,6mg/Ml
% CD8+CFSE Low
Index de stimulation
LTT:
6
5
4
3
2
1
0
Totapen 1mg/ml
Gentalline 1mg/ml
-LTT positive ampicillin SI=2,8 /
CD8+CFSE low ampicillin 5,1%
-ELISPOT positive ampicillin 52
spots / 1.106 PBMC
-Tests negative for gentamycin
ELISPOT IFN-
60
50
40
30
20
10
0
Totapen 1mg/ml
Gentalline 1mg/ml
CONCLUSION: presence of
ampicillin-specific T cells in
blood
BLOOD – Characterization of T cell cross-reactivities to different but related
chemicals
3
70
IFN- SFC / 1.106 PBMC
Index de stimulation
2,5
2
1,5
1
0,5
0
LTT
60
50
40
30
20
10
0
ELISPOT IFN
 Cross-reactivity between ticarcillin, peni A, peni M, peni G:
These antibiotics are contra-indicated
 No cross-reactivity with ceftriaxon (Rocephine®)
SKIN INFLAMMATION
ALLERGY OR IRRITATION ?
• Pathophysiology of skin irritation and allergy
• How to differentiate allergy from irritation ?
• How to differentiate allergy to patch test from
clinically relevant allergy ?
• Exposure to allergens promotes tolerance
Jean-François NICOLAS, Lyon University Hospital
• False + patch-tests
• Sensitization versus allergy
• Clinical relevance of skin tests
• Open tests et ROAT
12/14 patchpos, roatneg ?
- irritants
- patchpos non pertinents
Contact Allergy to fragrances
• Prevalence of fragrance contact allergy: 7%
• Positive patch-test = Fragrance-induced skin inflammation
– Innate immunity: contact irritation (inflammasome; IL-1 & other primary
cytokines)
– Adaptive immunity: contact allergy
(T cell- mediated, CD8+ T cells, IFNg)
• What is the real prevalence of fragrance contact allergy ?
– Which proportion of positive tests are true positive and represent
allergic tests?
– Which proportion of positive tests are false positive and represent
irritative tests?
– Open tests and repeated open tests (ROAT) are needed for diagnosis
+/-
PATCH TEST INTENSITY
+
++
+++
CONTACT ALLERGY
CONTACT IRRITATION
Contact Dermatitis to fragrances
• Clinical lesion of eczema
• Skin inflammation
– Innate immunity: irritant contact dermatitis (inflammasome;
IL-1 & other primary cytokines)
– Adaptive immunity: allergic contact dermatitis
(T cell- mediated, CD8+ T cells, IFNg)
• Prevalence of CD to fragrances unknown
– No strong epidemiological data
– No cosmetovigilance data
• Prevalence of ACD to fragrances unknown
Misinterpretation:
– Irritant contact dermatitis
– Atopic dermatitis (facial eczema, eyelid eczema)
• Prevalence of ACD to fragrances probably very low
(example though the coumarine studies)
• Studies needed on ACD to fragrances and not on
contact allergy to fragrances: epidemiology,
cosmetovigilance, immunology etc..
ALLERGIC CONTACT DERMATITIS
Contact Hypersensitivity
Question 1: IS THIS AN
ECZEMA/DERMATITIS ?
Question 2: IS THIS A
CONTACT DERMATITIS ?
Question 2: IS THIS AN
ALLERGIC CD ?
ALLERGIC CONTACT DERMATITIS
Contact Hypersensitivity
Question 1: irritation (false +)
or allergy (real +) ?
- irritation: non-specific inflammation
- allergy: specific, T cell-mediated,
inflammation
Question 2: Is the real + test
clinically relevant ?
- sensitization, DTH, chemical- specific
T cells
- allergy to patch test
- patch testing is a maximization test
and real+ patch means the existence
of specific T cells
- real + patch test does not mean that
these T cells could be activated in
normal conditions of exposure to the
chemical