Pemphigus Antibodies Identify a Cell Surface Glycoprotein Synthesized by Human and Mouse Keratinocytes

Abstract

A B S T R A C T Pemphigus is an antibody-mediated autoimmune skin disease in which loss of cell-to-cell contacts in the epidermis results in blister formation. Patients with pemphigus develop antibodies that bind to the keratinocyte cell surface, the site of primary pathology. The purpose of this study was to characterize the antigen(s) to which pemphigus antibodies bind. Because we could detect pemphigus antigen by indirect immunofluorescence on the surface of multiply-passaged cells in cultures of both a spontaneously transformed mouse keratinocyte cell line (Pam) and normal human epidermal cells, we used these cells as a source of antigen. In order to demonstrate biosynthesis of antigen and to characterize the antigen(s), we radiolabeled cell cultures with ['4C]glucosamine or D-[2-3H]mannose and used different pemphigus sera to immunoprecipitate antigen from nonionic detergent extracts of these labeled cells. Specifically precipitated radiolabeled molecules were identified using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and fluorography. Sera from five of seven pemphigus patients specifically precipitated (from extracts of both Pam cells and human epidermal cells) a molecule that, when reduced, was -130 kD, whereas seven normal human sera and two pemphigoid sera did not precipitate this molecule. The findings that (a) these precipitated molecules comigrated on SDS-PAGE and that (b) the 130-kD molecule could no longer be precipitated from cell extracts that had been previously reacted with a pemphigus serum, indicate that reactive pemphigus sera bind the same molecule. The molecule was not detected in the culture medium of these cells. This finding, along with the cell surface

Multiplexed autoantigen microarrays identify HLA as a key driver of anti-desmoglein and -non-desmoglein reactivities in pemphigus

“…Although early immunofluorescence studies demonstrated the presence of autoAbs in patient sera that bound to the surface of keratinocytes, a direct role of autoAbs in disease pathogenesis was not established until purified patient IgG (PVIgG) was shown to elicit blister formation upon passive transfer in mice (3). The main targets of these autoAbs were identified as Desmoglein (Dsg) 3 and 1, cadherin proteins that constitute key components of desmosomes, protein complexes responsible for maintaining cell-cell adhesion (4)(5)(6)(7). Later experiments in which patient sera depleted of anti-Dsg3 Abs failed to produce blisters when passively transferred to mice (8) seemingly cemented the notion that these autoAbs alone were responsible for blister formation.…”

Human autoantibodies against desmosomes: possible causative factors in pemphigus.

“…The keratinocytes can be induced to produce desmosomes and subsequently to stratify by adjusting the Ca2+ concentration in the culture medium to normal levels (-1.2 mM). Under these cell culture conditions, Stanley et al (10) have been able to immunoprecipitate a Mr 130,000 polypeptide from mouse keratinocytes with some, but not all, pemphigus antisera. More recently, Stanley and Yuspa (11) have used the mouse keratinocyte culture system to show that there is an induction of pemphigus antigen when keratinocytes are switched from medium containing low Ca2+ to medium containing normal Ca2+ levels (11).…”

Is Endoplasmic Reticulum Stresslinked to the Pathogenesis of Pemphigus Vulgaris?

“…In the late 1970s to early 1980s, studies showing that pemphigus autoantibodies related to blister formation in skin organ culture systems as well as by passively transferring IgG fractions of patients with pemphigus vulgaris into neonatal mice contributed to our understanding of disease pathogenesis (Schiltz and Michel 1976;Anhalt et al 1986). In the midto late 1980s, target antigens underwent immunochemical characterization (Stanley et al 1982;Hashimoto et al 1990) while in 1991, studies revealed that target antigens were the cadherin-type adhesion molecules of desmosomes desmoglein 1 (DSG1) and DSG3, (Koch et al 1990, Amagai et al 1991).…”