the Medical Immunopath. Lab Course Eva · Strongyloidiasis, ascariasis, schistosomiasis) • Nonparasitic infections (eg. EBV, CMV, HIV, M. tuberculosis) • Inflammatory disease

Course 514.020 of the Medical University Vienna

Immunopath. Lab CourseBasic knowledge in allergology

Eva Untersmayr‐ElsenhuberDepartment of Pathophysiology and Allergy Research

Course 514.020 Immunopathological Lab Course

Basic knowledge in allergology

Eva Untersmayr-ElsenhuberDepartment of Pathophysiologyand Allergy ResearchMedical University Vienna




Schedule for Nov. 16 and 17DAY 3: Nov. 16, 2010 Meeting point09:00 – 10:00 Introduction in allergology and

presentation of a case studyPD Dr. Eva Untersmayr‐Elsenhuber

Library at 4Q

10:00‐10:30 Basic techniques in allergology: SDS‐PAGE and ImmunoblotDr. Susanne Diesner & Mrs. Cornelia Schultz

Lab, end of corridor 4Q

10:30 – 10:45 Break10:45 – 12:30 Hands‐on in the lab: SDS‐PAGE

Dr. Diesner & Mrs. SchultzLab, end of corridor 4Q

DAY 4: Nov. 17, 2010 Meeting point09:00 – 09:30 Characteristics of food allergens

PD Dr. Eva Untersmayr‐ElsenhuberLibrary at 4Q

09:30‐10:30 Allergen purification via HPLCMrs. Cornelia Schultz

Christian Doppler Lab

10:30 – 10:45 Break10:45 – 12:30 Hands‐on in the lab: Immunoblot

Dr. Diesner & Mrs. SchultzLab, end of corridor 4Q




detailed case history

• Reactions timely related with exposition

• No symptoms if trigger is avoided

• Family history regarding atopic disorders

• Known allergies

• Diet diary

Diagosis of allergy




serum‐IgE

anti‐IgE

allergen

• Total IgE: PRIST > 100 kU/L• Specific IgE: CAP• Component-resolved diagnosis• Histamin release tests• Skin prick tests with extracts• Prick-to-prick tests with fresh food

Serological and skin testing




Mean serum concentration:IgE: 0.02-0.5 mg/mlIgG: 8-16 mg/ml

Percentage of total Ig:IgE: 0.002 %IgG: 80 %

Serum half life time: 2 days

Peak IgE levels occure 4-6 weeks after peak of pollenseason

Total IgE >1000ng/mLmajor diagnostic criteria forallergic bronchopulmonaryaspergillosis

IgE antibodiesIgE IgG




Diseases with elevated IgE levels

• Atopic diseases• Parasitic infections (eg.

Strongyloidiasis, ascariasis, schistosomiasis)

• Nonparasitic infections (eg. EBV, CMV, HIV, M. tuberculosis)

• Inflammatory disease (eg. Kimura disease, Churg-Stauss vasculitis, Kawasaki disease)

• Malignancies (eg. Hodgkin lymphoma, IgE myeloma)

• Cutaneous diseases (eg. Bullouspemphigoid)

• Cystic fibrosis• Nephrotic syndrome• Primary immunodeficiency

diseases (eg. Hyper-IgEsyndrome, Wiskott-Aldrich syndrome, Omenn syndrome, immune dysregulation, X-linkedinheritance, atypical DiGeorgesyndrome




Ig switch: - transcription through upstream constant switch region- DNA cleavage of ssDNA at transcription site- DNA repair: recombine VDJ domain + new C domain

Immunoglobulin class switch

IgM expressing B-cell IgG1 expressing B-cell

2 signals for IgE switch1) IL4 or IL13 via STAT6:

activates transcriptionat Sε

2) CD40L (T-cells) andCD40 (B-cells):activates DNA switchrecombination




High affinity IgE receptor FcεRITetrameric FcεRIαβγ2

According to: Kraft S, Kinet JP. Nature Immunol 2007

αγ2 β

mast cells, basophils

αβ γ2

Antigen independent

effects

Increased cell survival

Antigen dependenteffects

Mediator release




Trimeric FcεRIαγ2

According to: Kraft S, Kinet JP. Nature Immunol 2007

α

γ2

MHC class II

monocytes, macrophages, dendritic cells, Langerhans cells, eosinophils

Mediator release




ligand:receptor = 1:1 high affinity (1010 M-1) due to low dissociation rate, each binding site has lower intrinsic affinity

1 FcεRIα chain with 2 asymmetric IgE interaction sites:

- 4 solvent-exposed tryptophans large hydrophobic surface- C-C’ loop in the receptor D2 domain

bind dimeric, symmetric IgE Fc (Cε3)

FcεRI and IgE interaction

Garman SC et al. Nature 2000Wan T et al. Nature Immunol 2002

Metzger H. Immunol Rev 1992

Gould HJ et al. Nat Immunol Rev 2008




Glycoprotein, 45 kDa,single chainhomology to C-type(Ca2+-dependent) lectins IgE and CD21 bind tolectin domain

lower affinity for IgE (107 M-1)

B-cells, activated T-cells, monocytes, macrophages, eosinophils, Langerhans cells, intestinal epithelial cells, platelets

Low affinity IgE receptor FcεRII/CD23

Mossalayi MD et al. EMBO J 1992




CD23 regulates IgE synthesis

Down-regulation of IgE synthesis

Co-crosslinking of mCD23 and mIgE by allergen-IgEcomplex Inhibition of B-cell proliferation and IgE production

Induction of B-cell apoptosis B-cell populationregulation

B-cell surface

Luo HY et al. J Immunol 1991Sherr E et al. J Immunol 1989

Hibbert RG et al. JEM 2005

From Hibbert RG et al. JEM 2005

Up-regulation of IgE synthesisCo-crosslinking ofmIgE and CD21 bytrimeric sCD23

Aubry JP et al. Nature 1992Hibbert RG et al. JEM 2005

IgE binding tomCD23 protectsmCD23 againstproteolysis andprevents formationof sCD23




high binding affinity and slow dissociation rates of IgE to the allergenIgE: 10-10 bis 10-11 MIgG: 10-7 bis 10-8 M

IgE – allergen interaction

Kim KE et al. Mol Immunol 1996Pierson L et al. J Immunol Meth 1998Jackola DR et al. Mol Immunol 2002Hantusch B et al. Immunol Lett 2005

IgE affinity to allergen among the highest biologicallyrelevant binding strength

IgE epitopes on allergens are mainly conformational

IgE to conformational rather than linear epitopescorrelate with tolerance development in milk and egg allergic children

Foote J et al. PNAS USA 1995

Xia L et al. Mol Immunol 2010Padavattan S et al. J Immunol 2009

Pedraza-Escalona M et al. Mol Immunol 2009

Järvinen KM et al. Allergy 2007Vila L et al. Clin Exp Allergy 2001

repeated epitope presentation on allergen surface required for crosslinking allergens are multimers Tan YW et al. J Biol Chem 2009

Schöll I et al. J Immunol 2005




Sensitisation to allergens

Lymph nodesor mucosa

Galli SJ et al. Nature 2008

Airway Basophils, mastcells, NK T cells,…

Dendritic cell samplingEntrance through disrupted epitheliaProtease activity cleaving of epithelialtight junctions




Early phase of allergy


Allergen-specific IgEcross-linkingFcεRI aggregationmast cell activation

Mediator release

BronchoconstrictionVasodilatationInceased vascularpermeability, increased mucusproduction

Transition to latephase: leukocyte recruitment




Morphology of mast cell

http://www.bu.edu/histology/i/22602ooa.jpg

Tissue based inflammatory cellsRespond to signals of innate and adaptive immunity with immediate and delayed release ofinflammatory mediators

20 μm diameterOvoid or irregularly elongated cells with ovoid nucleusAbundant metachromatic cytoplasmatic granules (metachromatic stainingdue to sulfated proteoglycans)

cKIT (CD117) and FcεRI positiveOther cell-surface receptors depending on location and activation:FcγRIIa (in resting state) FcγRI (CD64) (in presence of IFN-γ)β2-adrenergic receptor, adenosine receptor A2B, prostaglandin E2 receptor,C3a, C5a-receptor, IL-3R, IL-4R, IL-5R, IL-9R, IL-10R, GM-CSFR, IFN- γR,CCR3, CCR5, CXCR2, CXCR4, nerve growth factor R, TLRs, …




Mast cell maturation and tissue distribution

Hematop. Stem cell

Mast cellprogenitor

cKIT (CD 117) – SCF Ligand

Arise from CD34+ progenitors


Mucosalmast cell

Connectivetissuemast cell

Tryptase pos. In respiratoryand GI mucosaincreasedwithinflammation

Tryptase, mast cell-specific chymase pos. In connective tissue (dermis, submucosa of GI tract, heart, conjunctivae, perivascular)Small bowel of end-stage immunodeficiencies

Phenotype of maturemast cells depends on growth factor milieu




• Antigen-crosslinked surface-bound IgE

• Divalent Ab against IgE Fc region

• Anti-idiotypic Ab

• Anti- Fc receptor Ab

• Covalent cross-linked IgE

• Lectins

• Complement (C3a, C5a) throughC3aR, C5aR (CD88)

• Nerve growth factor

• IgG by FcγRI

• TLR ligands (eg. TLR3 dsDNA IFN-γ production in mast cells)

Mechanisms of mast cell activation

C3aC5a7




Mast cell mediators

Granule-ass. preformed mediators• Histamine• Neutral proteases (tryptase in MCT,

tryptase, chymase, cathepsin G, carboxypeptidase in MCTC)

• Proteoglycans (heparin, chondroitinsulfates) neg. charged complexeswith histamine

• Chemotactic and activating factors(ECF-A, NCF)

Newly formed mediators• Lipoxygenase pathway products:

SRS-A (LTC4, LTD4), leukotrienes(LTB4)

• Cyclo-oxygenase products: prostaglandines and thromboxanes

• PAF




Effector function of mediatorsChemo-attractants• NCF• ECF-A• LTB4

Activators• Histamine• PAF• Tryptase• Kininogenase

Spasmogens• Histamine• PGD2• LTC4• LTD4

preformed newly formed

Attractants ofNeutrophils, eosinohils, monocytes, basophils

Vasodil., Vascular permeability

Kinins Vasodil. edemaProteolyt. enzyme, activates C3Microthrombi

• Bronchial smooth muscle contraction

• Mucosal edema• Mucus secretion




Clinical aspects of mast cell activation

Allergen dosageand entranceroute are decisivefor clinical reaction

MCTMCTCintravenoushigh dose

subcutaneouslow dose

inhalationlow dose

uptake withfood

Mast cell activation

Anaphylaxis Wheal andflare reaction

Allerg. rhinitisAsthma

Nausea, pain, vomiting diarreha




Morphology of basophils

http://microanatomy.net/blood/basophil3.jpg

Share many features with mast cells: FcεRIsecretion of Th2 cytokinesmetachromic stainingrelease of histamine after activation

But distinct lineage

5-8 μm diameter, segmented condensed nucleus, little proliferative capacityRapid and potent expression of IL-4 and IL-13Express Cytokine receptors (IL-3R, IL-5R, GM-CSF receptor)

Chemokine receptors (CD11c, CD11c, CD35 and CD88)Ig receptors (FcεRI, FcγRIIb)TLR




http://focosi.altervista.org/blood-cell-development.jpg

Basophil development and activation• CD34+ progenitors• Differentiate and mature in bone marrow• Circulate in periphery, <1% peripheral

leukocytes• Differentiation driven by IL3• Express integrins and chemokine

receptors able to inflitrateinflammed tissue (skin in AD, airway of respiratory allergies)

Activation via IgE crosslinkingC3a, C5a, TLR2 and TLR4 IL-4, IL-13 secretionand potentiation of IgE activationIL-33 through ST2 receptor




Mediators: preformed: Histamine, less heparin, low tryptase levelsnewly synthesized: LTC4, LTD4, LTE4, no PGD2 productioncytokines: IL-4, IL-13, GM-CSF source of early IL-4 for Th2 cell differentiation and amplification of IgE synthesis

Role in health and disease

Karasuyama H et al. Nat Rev Immunol 2009Min B. Nat Immunol 2008

Sullivan BM et al. Immunity 2009

Sullivan BM et al. Immunity 2009

• Physiological function remains unknown (host defense against parasites?)• Innate immunity (TLR2 expression)• Predominant source of IL-4 in allergen and helminth parasite activated

PBMCs• In late-phase allergic responses, found in increased numbers in lungs of

asthma patients dying of asthma




Features of mast cells and basophils

OriginMaturationLifespanLocationSizeNucleusGranulesPeptido-glycansTryptasecontent

Mast cells Basophils

Hematopoietic stem cellConnective tissueMonthsTissue6-12 μmOval or roundSmaller and moreHeparin and chondroitinsulfatesHigh

Hematopoietic stem cellBone marrowDaysIntravascular circulation5-8 μmSegmentedLarger and fewerPredom. chondroitinsulfatesLow

Stone KD et al. JACI 2010




Anaphylaxis= Serious allergic reaction with rapid onset, which can cause death

Anaphylaxis is unpredictable, can occur in anyone, anywhere at any timeUnderrecognized by patients and underdiagnosed by MDs

Incidence: doubled from 21/100.000 person/year (1980s) to 50/100.000 person/year (1990s)

with 70/100.000 person/year younger than19 years

Triggers: Food (peanut, treenut, shellfish, fish, milk, eggs)Medication (β-lactam antibiotics)Insect stings/bitesNatural latex rubberIgG-Ag complexesComplement and coagulation system activation

exercise, cold air or water, medication Simons FER. JACI 2009




http://www.bio.davidson.edu/courses/immunology/Students/spring2006/Witcher/figure%2012-11.jpg

Mechanisms




Nomenclature of food adverse reactions

Food hypersensitivity

Food allergy Non-allergic foodhypersensitivity

IgE-mediatedfood allergy

Non-IgE mediatedfood allergy

immunologicallymediated response

Johansson SG et al. EAACI position paper 2001




IgE-mediated food allergy

Diesner SC. MD thesis MUW 2007




local: Oral allergy syndrome, angioedema, gastrointestinal pain, nausea, vomiting, diarrhea

Systemic: Urticaria, angioedema, eczema, rhinitis, asthma, anaphylaxis

Unclear correlation: Migraine, arthritis, fatigue, behavioral disorders

Symptoms of food allergy




Case study: Patient H. S.35 year old patient with known birch pollenallergy comes to the allergy outpatient clinicreporting increasing severeness of OAS upon food ingestion

Tiggering food: carrot, apple, hazelnut, peanut

CAP class birch: 4CAP class carrot: 2CAP class apple: 3CAP class hazelnut: 2CAP classpeanut: 4

Routes of sensitization for peanut allery?

Documents

the Medical Immunopath. Lab Course Eva · Strongyloidiasis, ascariasis, schistosomiasis) • Nonparasitic infections (eg. EBV, CMV, HIV, M. tuberculosis) • Inflammatory disease