Eosinophil localization to the basement membrane zone is autoantibody- and complement-dependent in a human cryosection model of bullous pemphigoid

Kelly N Messingham; Jeffrey W Wang; Heather M Holahan; Rupasree Srikantha; Samantha C Aust; Janet A Fairley

doi:10.1111/exd.12883

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Eosinophil localization to the basement membrane zone is autoantibody- and complement-dependent in a human cryosection model of bullous pemphigoid

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Eosinophil localization to the basement membrane zone is autoantibody- and complement-dependent in a human cryosection model of bullous pemphigoid

Kelly N Messingham, Jeffrey W Wang, Heather M Holahan, Rupasree Srikantha, Samantha C Aust and Janet A Fairley

Experimental dermatology, Vol.25(1), pp.50-55

01/2016

DOI: 10.1111/exd.12883

PMCID: PMC4730885

PMID: 26475989

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Abstract

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by antibodies (IgG and IgE) targeting cell-substrate adhesion proteins. A variety of BP models suggest that autoantibody-dependent neutrophil degranulation is essential for blister formation. However, lesional biopsies reveal a predominance of eosinophils and few neutrophils. Our goal was to evaluate the role of antibodies and complement in eosinophil localization, degranulation and split formation at the dermo-epidermal junction (DEJ) utilizing a human skin cryosection model of BP paired with a human eosinophilic cell line, 15HL-60. Expression of receptors for IgG (FcγRII), IgE (FcεRI) and complement (CR1 and CR3) was confirmed on 15HL-60 cells using flow cytometry. 15HL-60 expression of granule protein [eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO)] mRNA and their degranulation in vitro was confirmed using RT-PCR and ELISA, respectively. For cryosection experiments, BP or control sera or IgG and IgE antibodies purified from BP sera were utilized in combination with 15HL-60 cells ± fresh complement. Both BP serum and fresh complement were required for localization of 15-HL60 cells to the DEJ. Interestingly, eosinophil localization to the DEJ was dependent on IgG, but not IgE, and complement. However, no subepidermal split was observed. Additionally, the 15HL-60 cells did not degranulate under any experimental conditions and direct application of cell lysate to cryosections did not result in a split. Our observation that eosinophil localization to the DEJ is dependent on IgG mediated complement fixation provides additional insight into the sequence of events during the development of BP lesions.

Cell Line

Enzyme-Linked Immunosorbent Assay

Immunoglobulin G - blood

Eosinophil Peroxidase - metabolism

Autoantibodies - blood

Humans

Neutrophils - immunology

Complement System Proteins - immunology

Basement Membrane - metabolism

Cell Adhesion

Case-Control Studies

Immunoglobulin E - blood

Pemphigoid, Bullous - pathology

Flow Cytometry

Biopsy

HL-60 Cells

Neurotoxins - metabolism

Eosinophils - cytology

Fluorescent Antibody Technique, Indirect

Microscopy, Fluorescence

Pemphigoid, Bullous - immunology

Skin - pathology

Details

Title: Subtitle: Eosinophil localization to the basement membrane zone is autoantibody- and complement-dependent in a human cryosection model of bullous pemphigoid
Creators: Kelly N Messingham - Department of Dermatology, University of Iowa, Iowa City, IA, USA
Jeffrey W Wang - Department of Dermatology, University of Iowa, Iowa City, IA, USA
Heather M Holahan - Department of Dermatology, University of Iowa, Iowa City, IA, USA
Rupasree Srikantha - Department of Dermatology, University of Iowa, Iowa City, IA, USA
Samantha C Aust - Department of Dermatology, University of Iowa, Iowa City, IA, USA
Janet A Fairley - Veterans Administration Medical Center, Iowa City, IA, USA
Resource Type: Journal article
Publication Details: Experimental dermatology, Vol.25(1), pp.50-55
DOI: 10.1111/exd.12883
PMID: 26475989
PMCID: PMC4730885
NLM abbreviation: Exp Dermatol
ISSN: 0906-6705
eISSN: 1600-0625
Publisher: Denmark
Grant note: I01 BX001680 / BLRD VA T35 HL007485 / NHLBI NIH HHS I01 CX000317 / CSRD VA P30 CA086862 / NCI NIH HHS
Language: English
Date published: 01/2016
Academic Unit: Dermatology
Record Identifier: 9984025482102771

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