TECHNIQUES IN EPIDERMAL BIOLOGY: AN INTEGRATED APPROACH TO AUTOIMMUNE SKIN DISEASE

TECHNIQUES IN EPIDERMAL BIOLOGY: AN INTEGRATED APPROACH TO AUTOIMMUNE SKIN DISEASE

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Contents

Preface

Chapter 1: Pathophysiology of the Epidermis: Lesson from Pemphigus

Introduction

Pemphigus Vulgaris

Immunopathology of PV

Plasminogen Activation Hypothesis

Direct Steric Hindrance of Cell-Cell Adhesion and Desmoglein Compensation Theory

Acetylcholine Receptors and the “Multiple Hit” Hypothesis

Intracellular Signaling in Acantholysis and Related Theories on Pemphigus Blistering

The Keratinocyte Death Hypothesis and Apoptolysis

The Basal Cell Shrinkage Hypothesis

Novel Concepts and Future Directions in Pemphigus Research

Chapter 2: Patients, Sera, and Experimental Models

Introduction

Materials and Methods

Patients

Purification of Total IgG Fractions and Anti-Dsg3-L IgG

In Vitro Model of PV Using Keratinocyte Monolayers

In Vitro Models with Mouse Skin Organ Cultures

In Vivo Passive Transfer of Sera Using Neonatal Mice

Standard Immunofluorescence Microscopy and Histology

Evaluatiotion of Acantholysis in Cell Monolayers

Morphology

Morphometric Analysis

Results

Selection of Patients and Characterization of Sera

PV Serum Induces Specific Changes in KIF Arrangement

Detection of Acantholysis Induced by Concentrated PV Sera through Dsg3 Staining

In Vitro Model of PV with Mouse Skin Cultures

In Vivo Model of PV with Passive Transfer of Sera in Neonatal Mice

Discussion

Chapter 3: Optimization of the Experimental Procedures: Semi-Quantitative LCIF Microscopy, Cirillo’s HSU Buffer, KAD Medium

Introduction

Materials and Methods

Culture Conditions and In Vitro Models

Western Blot Analysis and Immunoprecipitation

In Vitro Model of PV Using Cultured Keratinocytes

Semiquantitative Living Cell Immunofluorescence (LCIF) Microscopy

Results

Optimization of Extraction Rate and Expression Levels of Dsgs

Cell-Cell Detachment and KIF Collapse Can Be Revealed by PV Igg Staining

Time-Course of PV-Induced Acantholytic Dysmorphisms on Keratinocytes

Discussion

Chapter 4: Study of Anti-Desmoglein Autoimmunity: Pemphigus Vulgaris as a Desmoglein- Remodeling Disease

Introduction

Materials and Methods

Antibodies and Reagents

Sera, Total IgG Fractions, and Purified Anti-Dsg3-L IgG

Cell Cultures and Treatments

Protein Extraction and Western Blot Analysis

Immunoprecipitation

Standard and Semiquantitative Living-CellImmunofluorescence (LCIF) Microscopy

Results

Pemphigus Serum Induces Reduction of Dsg3 Half-Life

PV IgG do Not Induce an Immediate Disruption of AssembledDesmosomes

PV IgG-Bound Dsg3 is Not Early Internalized but Does NotForm Desmosomes

Anti-Dsg1 IgG-Containing Sera Induce Internalization of Dsg1but Not Its Early Depletion from Desmosomes

PF and PF-Like Autoantibodies Mobilize PrevalentlyNon-Clustered Dsg1

Changes in Dsg1 Synthesis in Response toAnti-Dsg1 Antibodies

Cleavage of Dsg3 in Keratinocytes Exposed to PV Serum

Depletion of Dsg3 and Its Localization during Acantholysisby Semiquantitative LCIF

Depletion of Dsg3 by PV Serum and IgG Fractions

High-Dose Anti-Dsg3-L Can Induce Acantholysis andReduction of Dsg3 Levels

Discussion

Chapter 5: Role of Non-Desmoglein andNon-IgG Autoimmunity in PV

Introduction

Materials and Methods

Antibodies and Reagents

Preparation of IgG-Free Sera and Igg Purification

Cell Cultures, Treatments and siRNA transfections

Protein Extraction, Western Blotting, and Immunoprecipitation

Gel Purification of 130 kDa Bands

Immunofluorescence Microscopy

Metabolic activity Assay Using MTT

Cell Dissociation Assay

Results

PV IgG Immunoprecipitate a PBMC 130 kDa Protein Other than Dsg3

The Novel 130 kDa Antigen Is Expressed on PBMC Surface

PV IgG-depleted Sera Induce Dramatic Morphological Changeson Keratinocytes

Effects of IgG-free PV Sera on Keratinocyte Viability and Cell-Cell Adhesion

PERK expression is altered in pemphigus vulgaris and isdependent on non-IgG factors

Discussion

Chapter 6: Downstream Signaling Involved in Acantholysis: Part I: Changes in Cell Cycle and Protein Phosphorylation

Introduction

Materials and Methods

Antibodies and Reagents

Keratinocyte Culture Experiments

Cell Cycle Analysis

Western Blot Analysis

Protein Phosphorylation Assay

siRNA Treatment

Immunofluorescence and Immunohistochemistry

Morphometric Analysis of Cell-Cell Detachment

Results

PV Sera Affect Cell Phosphorylation Status

PV serum promotes activation of a number protein kinases in kerotinocytes

Cell Cycle Analysis

PV Sera Modulate Dose-Dependently cdk2 Expression butModestly Other Kinases

Silencing of cdk2 Prevents Cell-Cell Detachment Inducedby PV Sera

Discussion

Chapter 7: Downstream Signaling Involved in Acantholysis: Part II: Secretion of Proteinases and the “Specific Proteolysis Hypothesis” of Pemphigus

Introduction

Materials and Methods

Keratinocyte Culture Experiments

Quantitation of Apoptosis

Western Blot Analysis and Immunofluorescence

Zymography

Results

MMP-9 Is over-Expressed in Keratinocyte Mololayers Exposedto PV Serum

MMP-9 Overexpression in the Organ-Culture Model of PV

Secretion of MMP9 in Keratinocyte Intercellular SpacesPrecedes Cell-Cell Detachment

Secretion of MMP-9 in Culture Supernatants

Processing of Dsg3 by Gelatinases

Discussion

The Specific Proteolysis Hypothesis

Beyond Anti-Desmoglein Autoimmunity

Chapter 8: Microarray Analysis of Diseased Tissues: Cell Adhesion Is Down- Regulated on the Gene Level in Pemphigus

Introduction

Materials and Methods

Passive Transfer of Sera in Neonatal Mice

Cell Cultures and Treatments

RNA Extraction, Microarrays Analysis, and RT-PCR

Ontology Assessment

Results

Modulation of Gene Expression by PV Sera in KeratinocytesDetected by DNA Microarray

Evidence that cdk2 Inhibition through Roscovitine Prevents Acantholysis in the Neonatal Mouse Model of PV

Modulation of Gene Expression by PV Sera in Mouse Skin Lesions Detected by DNA Microarray

Shared Microarray Results

Discussion

Chapter 9: Pharmacological Block of Acantholysis: Towards Novel Therapies for the Treatment of Pemphigus

Introduction

Materials and Methods

Passive Transfer of Sera in Neonatal Mice

Cell Cultures and Treatments

RNA Extraction, Microarrays Analysis, RT-PCR,and Ontology Assessment

Standard Immunofluorescence Microscopyand Histology

Results

Inhibition of Phosphorylation Events PreventsPemphigus Acantholysis

Evidence that cdk2 Inhibition through Roscovitine PreventsAcantholysis in the Neonatal Mouse Model of PV

Cdk2 Partially Modulates Gene Expression by PV Sera inMouse Skin by DNA Microarray

Discussion

Concluding Remarks

Acknowledgments

References

Index