selected topics in clinical Immunology

Allergology

Division Allergology,

Inselspital,

University of Bern,

CH 3010 Bern

Switzerland

Werner J. Pichler MDwerner.pichler@insel.ch

Allergology

• Definition

• Hygiene hypothesis

• Gell & Coombs Classsification

– IgE meditated allergies

– T cell mediated allergies

Allergy

Allergies are immune mediatesd reactions toharmless, non replicating substances, which leadto clinical symptoms;

Symptoms are caused by the immune reaction, not by the „bug“ (virus, bacteria, etc.)

Immune reactions: IgE, T-cells ....

Allergy

Allergies are immune mediatesd reactions toharmless (??), non replicating substances, whichlead to clinical symptoms;

Some allergens appear to have structural featuresable to activate the innate immunity (enzyme likepapain, Der p2, some pollen grains)

Symptoms are caused by the immune reaction, not by the „bug“ (virus, bacteria, etc.)

Immune reactions: IgE, T-cells ....

Allergy

Frequent:

IgE-mediated immune reactions:

hey fever, rhinitis & conjunctivitis, asthma, urtikaria, anaphylaxis ...

T-cell reactions:

contact dermatitis, drug allergies, atopic dermatitisT-cells ....

Definitions of ...

• Sensitization: immune reaction to a foreign

substance (proven in skin tests, serology, cellular

tests...)

• Allergy: immune reaction to a harmless (?), non

replicating substance (protein, chemical, drug,

metal,...), which leads to clinical symptoms

• Atopy: genetic determined readiness to react by

IgE formation to substances taken up via aerogen

or gastro-intestinal routes

Immune response to “non

replicating” substances

(“allergens”)

• No immune response - Ignorance

• (right) immune response - Tolerance

• (false ?) immune response - Allergy

Allergic diseases

IgE:– Hey fever (allergic rhinoconjunktivitis, pollinosis,

pollen induced asthma)

– Insekt venom allergy

– Acute urticaria

– Food allergy

– anaphylaxis: severe generalized allergic reactionsinvolving at least two organ systems and mostlycausing circulatory symptoms

– perennial rhinitis (hous dust mites, professional allergies..)

– asthma bronchiale

– Allergic-pulmonal aspergillosis

– atopische dermatitis

Significance

of IgE

IgE mediated allergies:

• Mainly to proteins (or hapten modified

proteins)

• Exposed to in very small amounts

(<1microg Grassallergen/Saison)

• Mild to very severe reactions

• very frequent diseases

• What is the natural role of IgE ???

„Allergic“ diseases

- non IgE -

• Exogen allergic alveolitis (IgE/IgG/T-cells

• contact dermatitis (T-cells)

• Various, frequent forms of drug allergies

(mostly T-cells)

• „Pseudo - allergies“ (?)

• Some forms of asthma bronchiale

• Some forms of atopic Dermatitis (z.T. T cells)

• Some forms of eosinophilic esophagitis

• chronic urticaria (no antigen/allergen)

Allergen/

Antigen

Epidemiology(IgE-induced Allergies)

Sapaldia/Scarpol Study in Switzerland (1995, 1999)

Ca. 20-25% of population is affected !!

Sensitization Allergy

Insect venom allergy 3,5%

asthma 6,8%

Hey fever 23% 14.2%

Increase of prevalence

1) Well documented, world wide (hey fever, asthma, atopic dermitis, contact eczema....)

Hey fever 1926: < 1%;

1958: 5%

1985: 10%1991: 20%

2050: 100% ??!! (since 2005 constant)

2) Primarily in countries with lwestern life style (east Germany > < west Germany,

Estonia > < Sweden)

Allergy increase – why?

Not definitively clear

Crucial, with high probability : early childhood period(intrauterine ?, surely first year)

• Better Diagnostic/higher readyness to report

– partly correct

• Dirty environment- probably not (SO2, NOx)

• Immunizations – evidence aganst it

• High allergen exposure – controversial, probably not

Allergy increase – why?

Unclear, but

Most evidence speaks for some events in early childhood (1st & 2nd year)

• Improved diagnostic, higher awareness – partly correct

• Air-pollution – probably not (SO2, NOx)

• Immunizations – not proven, analysis did not reveal an asociation

• Higher allergen exposure – controversial, but probably not

Increase of allergic diseases – why?

• Hygiene - Hypothesis

1. Insufficient early exposure to gastrointestinal infectious bacteria/viruses/parasites (HP, Hepatitis A,...);

2. Deficient development of immune response (T-reg; Th1 > Th2) because of insufficient contact with(partly harmless, symbiotic) bakteria (LPS) orviruses; farmer`s children have less atopy

3. Unfavorable colonization of GI-tract (because of 2)

4. Tabac-consumption

5. Early craddle, large family size, older siblings: Exposition to bacteria and viruses increased, risk ofallergies reduced

6. Mother milk (?): protects of infections, less fromallergies!

Hygiene-Hypothesis/

Immunology

Western Lifestyle Traditioneal Life style

Low

microbial

load

High

microbial

load*

Th2IL-4, IL-5

TregIL-10, TGF , .....

Allergy Tolerance

No Allergies

*Symbiotic >>> pathogen parasites

Neither cows notr the people living there become

sick by this surely not so clean water.....

Our immune systeme is the result of the evolution,

Which influenced us for the last millions of years via

pathogenic and mainly non pathogenic „bugs“

Since >1

Million

years

Since

hygiene

revolution

(ca. 1880-

1900)

Wolf – no allergy dog (beagle) 30-40%

develop atopic Dermatitis!

Neither cows notr the people living there become

sick by this surely not so clean water.....

Unser Immunsystem ist das Resultat der Evolution,

die wir über mehrere Mill. Jahre zusammen mit den

(kommensalen/symbiotischen) Bakterien durchlebten.

Mazmanian S.K., Cui Hua Liu, Tzianabos A.O., and Kasper D.L.

An Immunomodulatory Molecule of SymbioticBacteria Directs Maturation of the Host Immune System

Cell, 122: 107-118, 2005

Clinical implications of basic research in NEJM

Claudio Fiocchi, M.D

One Commensal Bacterial Molecule — All We Need

for Health?New Engl. J. Medicine 353:2078-2080, 2005

Bacteroides fragilis stimulates immune

system Cell, 122: 107-118, 2005

Without B. fragilis

insufficient

T- cell (CD4)

Development

Conclusion:

Symbiosis of mammal and bacteriae

A bacteriel polysaccharide of bacteroides fragilis ist

sufficient /necessary for a correct

- Systemic CD4+ T cell maturation

- Th1/Th2 differentiation (pathogen free mice have more

Th2 !)

- development of lymphoid organs

?

Hey fever:

symptoms of immediate reaction• Aeyes:

• itching

• conjunctivitis

• NNose:

• sneezingRhinitis

(Fliessschnupfen)

Hey fever:

symptoms of immediate reaction

• Lung:

Broncho-constriction

(Asthma)

• Skin, mucous

membranes: urtikaria,

Angioedema

Symptoms of an IgE-mediated

allergy

acute symptomes

• Pruritus

• swelling (vascular permeability )

• hyper-secretion (sneezing, tears, mucus

production )

• broncho-constriction

Symptoms of the IgE mediated

(immediate) - reaction

acute

(Nose, eyes,

– Red eyes

– Swelling

– pruritus

– sneezing

– Runny nose

(Histamin, PAF, LT, PG)

skin ):

Vascular dilatation

Increased permeabliy

Sensory nerves

Sensory nerves

Hyper secretion

H1

allergic symptoms (IgE)

Acute

• ~ vascular phase of inflammation with dilatation of vesels, permeability , constriktion of smooth muscule and itching

Chronic

• ~ cellular phase of inflammation with infiltration of cells (Eosinophila, Th2-lymphocytee, macrophages,..) and destruction of tissue as consequence of inflammation

Symptoms of IgE induced late reaction

chronic

Swelling

Infiltration by inflammatorycells

Damage to epithelia

Thickening of basal membrane, restructuringof lung tissue

Mucus production

(LT, cytokine, PG, PAF,

ECP, EPO, EDN,... IL-13,

TNFa)

blocked nose

bronchiale

Hyperreactivity

reduced lung function

1 Allergens (= often enzymes)

House dust mite (HDM) Allergens tight

junctionsP

- Pollen

- HDM-etc.

1

MC

5

IgE ist cytophilic binds to Fc-IgERI

On MC and basophils

IgE

2 LC (Langerhans Zelle)

Uptake & processing

of allergen

migration to LN

costimulation for Th2 primingLC

LN

4 B-cellen mature to IgE-producing

plasma cellen with the help of T cells

(CD40L, IL-4, IL-13.......)

3 In LN „priming“

of Th2-Zellen

(atopic, high

IL-4/IL-5/IL-13,

CD40/CD40L)

3

LC

B4

P IL-5

Eosinophils

P C

IL-4, IL-13

T

TT T

MO

89

T

7

Histamin

LTC4, D4

PG

PAF

8

Vaso-dilatation

Bronchoconstriction

Hypersecretion

Vascular permeability

Symptoms

Cytokine

IL-4, IL-5...

TNF ...

9

cell-acivation

(endothelial cells)

cell- recruting

(eosinophils, basophile

T-cells, monocytes..)

inflammation

10

10 inflammatory

mediators (z.B.

of eosinophils

ECP, EPO, EDN)

damage epithel

ECP

EDN

EPO

10

IL-13 Mucosa-Secretion

P

- Pollen

- HDM etc.

6 Specific IgE will be cross-

7 linked on mast cells (MC)

Freisetzung von Mediatoren

MC

6P

P

- Pollen- HSM-Kot

11

MC

12

13

SO2

O3

NOX

Damage of epithelial barrier

Hyper-reactivity

(= increased sensitivity to

Cold air, NO2, SO2,

ozone....)

12 Anti-dromale reflexes lead to

bronchial obstructionstriktion

13 MC can be stimulated by neuro-

transmitters and MC can

stimulate nervs

MO

T

Histamin

LTC4, D4

PG

PAF

Cytokines

IL-4, IL-5..

TNF ...

cell-activations

(endothial cells)

cell- recruitment

(eosinophils, basophils

T-cells, monocytes...)

inflammation

inflammatory

Mediators (e.g. of

eosinophils

ECP, EPO, EDN)

damage epithelia

ECP

EDN

EPOIL-13 Mucosa-Secretion

P

- Pollen

- HSM-Kot etc.

Specific IgE are cross-linked on

mast cells (MC)

release of mediators

MC

P

1 Allergen avoidance

2 Mast-cell stabilising drugs

(DNCG, etc.)

4 Anti-Histamin

LT-R Antagonists

5 Corticosteroids

5 Corticosteroid

3 Specific immunotherapy

IL-10 , IL-4

LN

1 Allergene (= häufig Enzyme)

HSM-Allergene tight junctionsP

- Pollen

- HSM-Kot etc.

1

MC

5

IgE ist zytophil bindet zu Fc-IgERI

auf MC

IgE

2 LC (Langerhans Zelle)

Allergenaufnahme

Allergenverarbeitung

Wanderung zu LNLC

LN

4 B-Zellen reifen zu IgE-produzierenden

Plasmazellen mit Hilfe von T-Zellen

(CD40L, IL-4, IL-13.......)

3 In LN „priming“

der Th2-Zellen

(Atopiker, hohes

IL-4/IL-5/IL-13)

3

LC

B4

P IL-5

Eosinophilie

P C

IL-4, IL-13

T

TT T

Allergens (mostly „harmless“ (?) molecules, mostly proteins, but

also haptens)

Saisonal: • Baum/Sträucherpollen (Birken, Hasel, Erle, Esche, ...): wichtigstes

Allergen davon: betula verrucosa 1 (bet v1)

• Gräserpollen: Phleum Pratense, Lolium, Roggen (alle sehr ähnlich)

• Kräuterpollen: Beifuss, Spitzwegerich

• Schimmelpilzsporen (Alternaria alternata, Aspergillus fumigatus.....)

Ganzjährlich (perennial): • Milben

• Katzen, Hunde, Pferde, .....

• Küchenschaben

• Nahrungsmittel wie Apfel (mal d1), Milch, Crevetten, Hymenopterengift .....

• Mehle (Bäcker,...)

• ........

IgE - crosslinking by

drugs

Cross-linking of two IgE

molecules on distinct Fc-IgE-

R is necessary;

? How can a small

molecule crosslink two IgE ?

? Difference to protein

antigens ?

IgE-model: size comparison of Bet v1 and amoxicillin

Amoxicillin

Bet v1

IgE

Cross-linking

A simple protein allergen needs two distinct epitopes

to allow crosslinking of two different IgEs

IgE 1 IgE 2

Cross-linking haptens: IgE with the same specificity

might be cross-linked

Epitope 1

Epitope 1

Epitope 2

IgE A

IgE A

IgE B

IgE cross-linking by

protein or drug allergens

• At least two distinct epitopes on

one molecule required;

• Only two distinct IgE molecules

can be cross-linked, but not two

identical IgE molecules

• Can be the same epitope on

different positions of the protein

• IgEs to the same (and other

epitopes) can be cross-linked

A B

B A

αA

Protein

2 epitopes A, B

Only IgE with

distinct

specificities are

crosslinked

A

B

Cross-linking by a single protein: two distinct

Epitopes are needed to allow cross-linking

αA

αA

Hapten modified

protein

IgE with same

or distinct

specificities are

crosslinked

A` A'

Cross-linking

B`

In hapten reactions, the IgE with the same specificity

might be cross-linked! This may facilitate degranulation !

Haptens, specific IgE and cross-

linking

• Haptens may generate many new epitopes

on a single protein

• Even IgE with the same epitope specificity

may be cross-linked, which may explain the

sometimes fulminant reactions

The immune system is highly

specific and needs danger

signals to become activated

The Achill`s heel of the immune system is

a) cross-reactivity

b) the ability of proteins to act immune-

stimulatory

Allergens: their role in innate immunity

Allergens are „harmless“ molecules: not quite so !!

Der p2

• The fact that other members of the MD-2-

like lipid-binding family are allergens, and

that most defined major allergens are

thought to be lipid-binding proteins,

suggests that intrinsic adjuvant activity by

such proteins and their accompanying lipid

cargo may have some generality as a

mechanism underlying the phenomenon of

allergenicity.• Trompette Nature, 457: 585, 2009

The immune system is highly

specific and needs danger

signals to become activated

The Achill`s heel of the immune system is

a) cross-reactivity

b) the ability of proteins to act immune-

stimulatory

cristallographic structures

of Mal d1 and Bet v1

apple

Birch pollen

12

Respiratory and food

allergens: Homologies• Bet v 1-homologe

proteins

• Lipid transfer

proteins

• Taumatin-likeproteins

• Mal d 1, Pru av 1, Pru ar 1,

Pyr c 1, Api g 1, Dau c 1,

Cor a 1, SAM 22

• Mal d 3, Pru p 3, Gly m 1

• Mal d 2, Pru av 2, Cap a 1

Labile food allergen

Stabile food - allergen

Delayed drug hypersensitivity

Subclassification of Type IV into

IVa - IVd

T-cells recognize the drug and exert,

dependent on their function, a

certain pathology

a) How do T-cells recognize drugs ?

– Hapten and p-i mechanism

b) What function do T cells exert ?

– Heterogeneous functions – therefore

hetterogeneous clinical picture

Gell & Coombs Classification (1967!)

Hypersensitivity reactions type I – III & type IV

Type I Type II Type III

Immune

reactantIgE IgG IgG

AntigenSoluble

antigen

Cell- or

matrix-

associated

antigen

Soluble

antigen

EffectorMast-cell

activation

FcR+ cells

(phagocytes,

NK cells)

FcR+ cells

Complement

Example of

hypersen-

sitivity

reaction

Allergic

Rhinitis,

asthma,

systemic

anaphylaxis

Some drug

allergies

(e.g.,

penicillin)

Serum

sickness,

Arthus

reaction

A

g

platelet

s

blood

vesse

l

immune

complexAb - platelet

Type IV

T cell

MHC-presented antigen

T-cells, via cytokines recruitment of

monocytes, eosinophils, neutrophils

Many different diseases: tuberculin skin

test

different forms of exanthema ?, eczema

?, contact dermatitis ? …..

Cytokines

cytotoxicity

bullous exanthem (bullous E.)

maculopapular

exanthem (MPE)

Drug hypersensitivity:

a generalized immune reaction to drugs

Acute generalized

exanthematous

pustulosis (AGEP)

Drug specific T-cell clones (TCC)from blood and tissue

cell culturewith drug

proliferation

Expansion of a

single cell

T-cells

T-cells

phenotype

function: cytokines,

cytotoxicity

interaction with

APCs, tissue cells

drug recognition

cross-reactivity

TCC

Perforin - CD4

red - brown

Immunohisto-

chemistry

TCC

cytotoxicity

cytokin-production

(IFN , IL-5, IL-8, ....)

specificity,

cross-reactivity

phenotyp

(CD4/CD8, TCR-V

cell-interactions

Characterisation of drug-specific

T-cell clones (TCC) in vitroCD4--MPE

T-cells recognize the drug and exert, dependent on

their function, a certain pathology

Amoxicillin

bullous E.

MHC-I (+ MHC-II)

CD8+ > CD4+

cytotoxicity (CD8+)

IFN ; IL-5

MPEMHC-II

CD4+cytotoxicity (CD4+)

IL-5; IFN

AGEP

MHC-II + I

CD4+ & CD8+

cytotoxicity

IL-8; IL-5 (IL-17?)

Delayed hypersensitivity reactions Type IV a, IV b, IV c, IV

d

Type IV a Type IV b Type IV c Type IV d

Immune

reactantTH1 cells TH2 cells CTL PMN

AntigenSoluble antigen

cells

Soluble antigen

or direct T cell

stimulation

Cell-associated

antigen or direct

T cell stimulation

Soluble antigen

or direct T cell

stimulation

Effector Macrophage Eosinophils T cells PMN

Example of

hypersen-

sitivity

reaction

Contact

dermatitis,

tuberculin

reaction

Chronic asthma,

chronic allergic

rhinitis, maculo-

papular

exanthema with

eosinophilia,

atopic dermatitis

Contact

dermatitis

Macolopapula

r and bullous

exanthema

AGEP

Behçet diseae

IFN- TH

1

chemokines,

cytokines,

cytotoxins

cytokines,

inflammatory

mediators

TH2

IL-4

IL-5eotaxin

CTL

PM

N

CXCL8

GM-

CSF

Cytokines, Inflammatory

mediators

Antibody mediated hypersensitivity reactions (I-III) and delayed type hypersensitivity reactions (IV a-d)

Type I Type II Type IIIType IV a Type IV b Type IV

cType IV d

Immune

reactantIgE IgG IgG

IIFN , TNF

(TH1 cells)

IL-5, IL-4/IL-13

(TH2 cells)

Perforin/

GranzymeB

(CTL)

CXCL-8, IL-17 (?).

GM-CSF

(T-cells)

Antigen Soluble antigenCell- or matrix-

associated antigenSoluble antigen

Antigen presented

by cells or direct T

cell stimulation

Antigen presented

by cells or direct T

cell stimulation

Cell-associated

antigen or direct T

cell stimulation

Soluble antigen

presented by cells

or direct T cell

stimulation

EffectorMast-cell activation

FcR+ cells

(phagocytes, NK

cells)

FcR+ cells

Complement

Macrophage

activationEosinophils T cells Neutrophils

Example

of

hypersen-

sitivity

reaction

Allergic rhinitis,

asthma, systemic

anaphylaxis

Some drug

allergies (e.g.,

penicillin)

Serum

sickness,

Arthus reaction

Tuberculin

reaction

,contact

dermatitis (with

IVc)

Chronic

asthma,

chronic allergic

rhinitis

Maculopapular

exanthema

with

eosinophilia

Contact

dermatitis

Maculopapular

and bullous

exanthema

hepatitis

AGEP

Behçet

disease

Ag

platelets

blood

vessel

immune complex

IFN- TH1

chemokines, cytokines,

cytotoxinscytokines, inflammatory

mediators

TH2

IL-4

IL-5eotaxin

CTL

PMN

CXCL8

GM-CSF

cytokines, inflammatory

mediators

Subclassification of drug-hypersensitivity

- type IVa-d reactions -

TYPE IV a TYPE IV b TYPE IV c TYPE IV d

Th1 Th2 Cytotoxic T cells T cells

IFN-γ, TNF-α IL-5, IL-4, IL-13,

eotaxin

Perforin, granzyme B,

FasL

CXCL-8, GM-CFS

IL-17 ?

Monocyte,

Macrophage

Eosinophilic

inflammationCytotoxic T cells Neutrophils

Tuberkulin skin test,

Contact dermatitis

Maculopapular

exanthem with

eosinophilia

Contact dermatitis

bullous & maculo-

papular exanthema

Pustular exanthema

Acute generalised exanthematous Pustulosis

(AGEP)

clinic

–generalised, sterile pustels

–Non follikular, intraepidermal

–fever (>38°C)

–Leukocytosis

DD

Psoriasis (pustular)

Frequency

ca. 5-10 cases per 1x106 inhabitants

(CH)

AGEP – skin test with relevant drug

can imitate pustulosis

48 hrsvesicles,

not yet filled with PMN

96 hrspustules,

filled with PMN

Skin tests with amoxicillin

T cells infiltrate the skin before neutrophils

Britschgi et al., J Clin Invest, 2001

CD

8

NEUTROP

HIL

ELASTASE

CD4

acute

phase

patch-test

after 48 h

Perforin/granzyme B and Fas/FasL expression in

the diseased skin of AGEP

granzyme B

Schmid S. et al., Am J Pathol, 2003

acute

phase

patch-test

after 48 h

perforin Fas FasL

Immunohistochemical staining (ABC/AP method) of patch test lesions

of patient JS (a–d) with anti-FasL-antibody.

Anti FasL antibody (ABC/AP)

S. Schmid et al, Am J Pathol, 2003

0

10

20

30

40

1 : 1 3 : 1 10 : 1 30 : 1

Drug-specific T cells from AGEP kill via

perforin/granzyme and Fas/FasL in vitro

Perforin/granzyme-

medated killing

autologous target cells

(B-LCL) as targets

Schmid et al., Am J Pathol, 2002

0

20

40

60

80

100

0.3 : 1 1 : 1 3 : 1 10 : 1 30 : 1

spec

ific

ly

sis

(%)

E : T ratio

CD

4+

CD

8+

E : T ratio

0

10

20

30

40

0.3 : 1 1 : 1 3 : 1 10 : 1 30 : 1

E : T ratio

CD

8+

Fas/FasL-

mediated killing

Fas-transfected

cell line as targetspec

ific

ly

sis

(%)

Drug-specific T cells from AGEP

produce IL-8 (CXCL-8)

0,001

0,01

0,1

1

10

stimulation with

anti-CD3/anti-CD28

non-AGEP T cellscontact dermatitis

bullous exanthema

tetanus toxoid-specific

AGEP T cell clones

33

980

Pro

du

ctio

n o

f IL

-8 (

ng/m

l)

P 0.0001

Britschgi et al., J Clin Invest, 2001

Britschgi and Pichler, Curr Opin Allergy Clin Immunol, 2002

b) expansion

venule

dermis

AGEP: a human model system for T cell-mediated

orchestration of neutrophilic disorders (1)

c) migration

to skin

IFN- TNF-IL-4, IL-5, RANTES

cytotoxicity

IL-8

GM-CSF

CD4+ and CD8+

d) functional activity of T cells in the skin

inflammation

neutrophil

activation/attraction

tissue

destruction

drug-specific

T cell

professional

APC

a) activation

drug

Phase 1

lym

ph

no

des

Britschgi and Pichler, Curr Opin Allergy Clin Immunol, 2002

AGEP: a human model system for T cell-mediated orchestration

of neutrophilic disorders (2)

b) cytotoxic killing and massive production of

IL-8 by T cells and keratinocytes

Phase 2

subcorneal vesicle formation

a) drug () presentation by LCs and

keratinocytes; further activation of T cells

Langerhans’ cell (LC) keratinocyte

IL-8

IL-8IL-8

Britschgi and Pichler, Curr Opin Allergy Clin Immunol, 2002

Phase 3

IL-8

gra

dien

t

venulePMN

Recruitment of neutrophils

(PMN) to the skin

subcorneal sterile

pustule

venule

Phase 3

PMN

AGEP: a human model system for T cell-mediated orchestration

of a neutrophilic inflammation (3)

Recruitment of neutrophils

(PMN) to the skin

CXCL8

gradient

subcorneal sterile

pustule

NEUTROPHIL ELASTASE

CD8

CD4

Immunhistochemistry of skin

biopsy from acute AGEP