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PATHOGENESIS OF PEMPHIGUS

Prof Pascal JOLY Rouen University Hospital France

CHU_ Hpitaux de Rouen - page 1

PATHOGENESIS OF PEMPHIGUS
Introduction
Pemphigus antibodies Pemphigus antigens Role of genetic background Role of environnemental factors Mechanism of acantholysis

INTRODUCTION

- Autoimmune blistering disease - Ab pathogenic directed against adhesion molecules of desmosomes


1) Antibodies: Identification Pathogenic properties Mechanism of acantholysis

Identification Correspondence with clinical types of pemphigus 3) Mechanisms of the tolerance breakdown

2) Antigens:

PATHOGENESIS OF PEMPHIGUS
Introduction

Pemphigus antibodies
Pemphigus antigens Role of genetic background Role of environnemental factors Mechanism of acantholysis

IN VIVO DEPOSITION OF ANTIBODIES IN PEMPHIGUS


DIF : deposition of IgG and C3 on keratinocyte membrane IgG1 and IgG4 IgA : superficial pemphigus (+IgG) IgA pemphigus PNP dual deposits : IgG : ECS + C3 : BMZ

CIRCULATING ANTIBODIES IN PEMPHIGUS


Circulating Ab are IgG1 and IgG4 Recovery is correlated with the decrease of anti ECS IgG4 Ab Circulating Ab from PV and PF patients in remission are mainly IgG1 Epitopes recognized different EC1-EC2 (active disease) VS EC5 (remission) PNP patients had circulating antibodies that - label both ECS and BMZ human skin - other epithelial and non epithelial tissues (liver, bladder, heart)

PEMPHIGUS ANTIBODIES ARE PATHOGENIC


- Correlation between disease activity and antibody titers.
- Relapse more frequent in patients with persistent circulating antiACS Ab. - Persistent in vivo bound anti ECS Ab: increase risk of relaps? - Neonatal pemphigus

EXPERIMENTAL MODELS OF PEMPHIGUS


Development of in vitro and in vivo models Usefull tools Anhalt GJ et al N Engl J Med 1982 passive transfer of IgG from PV,PF patients to neonatal balb/C mice (intraperitoneal injection) acantholysis dose dependant specific manner

PEMPHIGUS ANTIBODIES ARE PATHOGENIC


Pathogenic properties of IgG4 Ab. Pathogenic Ab are directed against the extracellular part of desmogleins (EC1 EC2 domains) Anti-DSG3 Ab from PV and PNP sera are pathogenic. Anti-DSG1 Ab from PF are pathogenic

OTHER ANIMAL MODELS OF PEMPHIGUS


1) SCID mice grafted with human skin reconstituted with PBL from pemphigus patients 2) mAbes from BALB/c mice upon immunization with recombinant desmogleins 3) reconstitution of Rag-/- mices with splenocytes from desmoglein 3 KO mices immunized with recombinant DSG 3 Usefull models for studying pemphigus pathogenesis and treatments

PATHOGENESIS OF PEMPHIGUS

Introduction Pemphigus antibodies

Pemphigus antigens
Role of genetic background Role of environnemental factors Mechanism of acantholysis

CHARACTERISATION OF PEMPHIGUS ANTIGENS


The 3 steps 1- ultrastructural localization 2- biochemical identification 3 - cloning of genes encoding the different proteins production of recombinant proteins

BIOCHEMICAL IDENTIFICATION OF TARGET ANTIGENS IN PEMPHIGUS


2 mains techniques : - immunoblot analysis - immunoprecipitation Immunoblot analysis - use different Ag sources

bovine tissues (muzzle) human epidermis cultured keratinocytes recombinant proteins

easiest method can detect multiple antigens ONLY detect Ab directed against linear epitopes (Ag are denatured with detergents) (+++)

IMMUNOPRECIPITATION
Use radio labelled cultured keratinocytes extracts Use protein A to precipitate IgG antibodies Complex, time-consuming method Can detect Ab against conformational epitopes

PEMPHIGUS VULGARIS Ag IS DESMOGLEIN 3 A 130-kDa GLYCOPROTEIN OF DESMOSOMES


Pathogenic antibodies are directed against the extracellular region of DSG3. B-cell epitopes targeted by IgG4 antibodies are localized on the EC1-EC2 domains. The whole protein is required to induce pathogenic antibody production by rabbit immunization. Involvement of the intracellular domain in antibody production.

PEMPHIGUS FOLIACEUS ANTIGEN IS DESMOGLEIN 1 = 160-kDa


DSG1 belongs with DSG3 to the cadherin family. DSG3 gene has high sequence homology and identity with DSG3 gene. Pathogenic Ab are directed against the extracellular region of DSG1. B Cell epitopes are localized on the EC2, EC3 and EC4 domain of the extracellular region. Only 50 % PF sera recongnized DSG1 by immunoblot analysis.

PARANEOPLASTIC PEMPHIGUS ANTIBODIES RECOGNIZE SEVERAL PROTEINS OF THE DESMOSOMAL PLAQUE

- PNP Ab are deposited both on the extracellular area and the intrecellular plaques of desmosomes - And on the hemidesmosomes of the basement membrane zone

PARANEOPLASTIC PEMPHIGUS ANTIBODIES RECOGNIZE SEVERAL PROTEINS OF THE DESMOSOMAL PLAQUE

- PNP sera recognize an antigenic complex composed of proteins that belong to the plakin family
-Desmoplakin I : 250-kD -BPAG1 : 230-kD -Desmoplakin 2 and Envoplakin : 210-kD -Periplakin : 190-kD -Plectin : 600-kD -Unidentified : 170-kD

Genes encoding these proteins have high sequence homology

THERE IS AN OVERLAP OF AUTOANTIBODY RESPONSE BETWEEN THE DIFFERENT TYPES OF PEMPHIGUS


- Half PV sera have anti-DSG1 antibodies in addition to anti-DSG2 Ab. - Some PV patients may relapse with clinical and histological features of PF. - Anti-DSG1 antibodies present in some PV sera are pathogenic in animal models.

THERE IS AN OVERLAP OF AUTOANTIBODY RESPONSE BETWEEN THE DIFFERENT TYPES OF PEMPHIGUS


PNP sera react with DSG3 and DSG1 in immunoblotting experiments Confirmed by ELISA
(anti-DSG3 Ab in 100 % PNPsera) (anti-DSG1 Ab in 65 % PNP sera)

Anti-DSG3 Ab from PNP sera are pathogenic.


(their immunoabsorption with recombinant DSG3 abolish the pathogenic effect of PNP sera)

These findings explain the clinical and histological similarities between PV and PNP patients.

THIRD STEP : CLONING AND SEQUENCING GENES ENCODING FOR PEMPHIGUS ANTIGENS
Desmoglein 1 and 3 belong to cadherins a family of proteins involved in cell adhesion.

PNP antigens belong to plakins a family of desmosomal plaque proteins.

PEMPHIGUS ANTIBODIES SPECIFICALLY INHIBIT ADHESIVE FUNCTION OT THE TARGET ANTIGEN


DSG3 is mostly responsible for cell-cell adhesion in the oral mucosa. DSG3 and DSG1 are both involved in cell-cell adhesion of the basilar layer of epidermis. DSG1 : only responsible for adhesion in the superficial layers.

PEMPHIGUS ANTIBODIES SPECIFICALLY INHIBIT ADHESIVE FUNCTION OF THE TARGET ANTIGEN


- PV patients with only anti-DSG3 Ab have mucosal lesions. - PV patients with both anti-DSG3 and anti-DSG1 Ab have both mucosal and skin lesions with a supra basilar acantholysis - PF patients who only have anti-DSG1 antibodies only have skin lesions characterized by a superficial cleavage

PATHOGENESIS OF PEMPHIGUS

Introduction Pemphigus antibodies Pemphigus antigens

Role of genetic background


Role of environnemental factors Mechanism of acantholysis

AUTO ANTIBODY PRODUCTION IN PEMPHIGUS Role of Genetic background


Genetic factors play a role in the disease susceptibility - Familial cases - Detection of anti-ECS Ab in patients relatives - Increased frequency in some ethnic groups (Ashkenary Jews, Japanese, regions of central America)

AUTO ANTIBODY PRODUCTION IN PEMPHIGUS Role of Genetic background

AUTO ANTIBODY PRODUCTION IN PEMPHIGUS Role of Genetic background


- All the alleles involved in PV share conserved residues at key positions (26, 67, 70, 71) - These conserved residues are responsible for the productions of a negative charge of the P4 pocket of the HLA molecule, which is necessary for selective presentation of DSG3 peptides. The association between HLA and pemphigus is probably the direct consequence of the ability of the susceptibility alleles to present peptides of DSG3 to auto reactive T-cells.

AUTO REACTIVE T-CELLS ARE NECESSARY FOR PEMPHIGUS ANTIBODY PRODUCTION


T cell clones directed against multiples epitopes of DSG3 and DSG1 can be detected in PBL from PV and PF patients respectively. Most of them are CD4+ memory T-cells with a Th2 cytokine profile (IL4, IL6 and IL10) (cytokines involved in the IgG4 switch). CD4+ T cell clones with T 1 profile can also be detected. The role of the cellular immune response is probably crucial in the production of antibodies by B cells.

PATHOGENESIS OF PEMPHIGUS

Introduction Pemphigus antibodies Pemphigus antigens Role of genetic background

Role of environnemental factors


Mechanism of acantholysis

ROLE OF ENVIRONNEMENTAL FACTORS


Exemple of Fogo Selvagem - endemic form of PF in some areas in Brazil - very high incidence rate : 3 % population - numerous epidemiological data suggesting the auto immune response in PF may be linked to exposure to hematophagous insects

PRECLINICAL PHASE TO DISEASE


Anti DSG1 Ab : - 98 % PF patients - 2 % healthy individuals from USA/europe - 55 % normal subjects from Limaoverde - 19 % people living in surounding areas

PRECLINICAL PHASE TO DISEASE


Normal subjects living in endemic areas - Ig1 (+++) - low titers of IgG4 Mainly directed against EC5

Patients : exclusive IgG4 Abx70 fold higher response than healthy individuals mainly directed against EC1 EC2

PRECLINICAL PHASE TO DISEASE

Evidence for a switch (IgG1 IgG4) of auto antibody response

Evidence for an epitope spreading phenomenom EC5 EC1 EC2


heathly individuals patients

PRECLINICAL PHASE TO DISEASE


Anti DSG1 antibodies in sera from patients with parasitic infections (blood transmitted) - onchocerchiasis : 83 % - Leishmaniasis : 43 % - Chagas disease : 58 % Hypothesis : component of insect vector saliva (rather than the parasite itself) may trigger the Ab response to EC5

ENDEMIC PEMPHIGUS
Environnemental factors Insect-bite transmitted Infectious diseases ? Component of insect vector saliva insect bite Production of Anti DSG1 Ab (IgG1-2-3) EC5 Healthy individuals genetic factors HLA DR3-DR4-DR14

switch

pathogenic IgG4 anti DSG1 Ab EC1 EC2 patients

PATHOGENESIS OF PEMPHIGUS

Introduction Pemphigus antibodies Pemphigus antigens Role of genetic background Role of environnemental factors

Mechanism of acantholysis

THE WAY PEMPHIGUS ANTIBODIES INDUCE ACANTHOLYSIS REMAINS CONTROVERSIAL


- Desmogleins are adhesion molecules: DSG3 KO mice have mucosal lesions similar to PV - DSG1 and DSG3 interact in an homotypic manner - Ab directed against desmogleins inhibit their adhesive function

MECHANISM OF ACANTHOLYSIS
Exact mechanism IC Ab causes loss of cell adhesion : unclear. Hypothesis : 1) IgG could block adhesion sites of DSG. 2) The paratope of some anti-DSG Ab may contain adhesive sequences and interact with the adhesive function of DSG by mechanism of molecular mimicry.

MECHANISM OF ACANTHOLYSIS

Hypothesis : 3) IgG could stimulate the release of proteolystic enzymes. (SSSS is caused by a toxin that binds specifically DSG1 and cleaves it)

MECHANISM OF ACANTHOLYSIS
Hypothesis : 4) IC IG could trigger a signalling event via the intracellular signalling pathway (phosphokinase C) that : interfere with DSG function or structure inducing dissociation from plakoglobin, leading to disruption of desmosomes. reorganisation of the cytoskeleton of keratinocytes

COMPLEMENT OS NOT NECESSARY FOR INDUCTION OF ACANTHOLYSIS


Arguments for a role of complement 1) C3 and the membrane attack complex: C5b9 are detected on ECS of PV patients by DIF Local activation of the complement system. 2) Depletion of complement by cobra venom pretreatment induces a delay in experimental acantholysis

CONCLUSION
Significant advances in the understanding of pemphigus pathogenesis New techniques facilitate the diagnosis and the evaluation of patients prognosis Use of recombinant Ag for targeting auto-reactive Bcells open a way for specific immunotherapy Understanding of genetic susceptibility and triggering factors of pemphigus will possibly provide opportunity to prevent and/or to use causative treatments in the future

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