Cell junction protein armadillo repeat gene deleted in velo-cardio-facial syndrome is expressed in the skin and colocalizes with autoantibodies of patients affected by a new variant of endemic pemphigus foliaceus in Colombia

Abstract

Background: We previously described a new variant of endemic pemphigus foliaceus in El Bagre, Colombia, South America (El Bagre-EPF, or pemphigus Abreu-Manu). El Bagre-EPF differs from other types of EPF clinically, epidemiologically, immunologically and in its target antigens. We reported the presence of patient autoantibodies colocalizing with armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), a catenin cell junction protein colocalizing with El Bagre-EPF autoantibodies in the heart and within pilosebaceous units along their neurovascular supply routes. Here we investigate the presence of ARVCF in skin and its possibility as a cutaneous El Bagre-EPF antigen.

Methods: We used a case-control study, testing sera of 45 patients and 45 controls via direct and indirect immunofluorescence (DIF/IIF), confocal microscopy, immunoelectron microscopy and immunoblotting for the presence of ARVCF and its relationship with El Bagre-EPF autoantibodies in the skin. We also immunoadsorbed samples with desmoglein 1 (Dsg1) ectodomain (El Bagre-EPF antigen) by incubating with the positive ARVCF samples from DIF and IIF.

Results: ARVCF was expressed in all the samples from the cases and controls. Immunoadsorption with Dsg1 on positive ARVCF immunofluorescence DIF/IIF cases showed that the immune response was present against non-desmoglein 1 antigen(s). Overall, 40/45 patients showed colocalization of their autoantibodies with ARVCF in the epidermis; no controls from the endemic area displayed colocalization.

Conclusions: We demonstrate that ARVCF is expressed in many areas of human skin, and colocalizes with the majority of El Bagre-EPF autoantibodies as a putative antigen.

Keywords: ARCVF; autoimmunity; cell junctions; endemic pemphigus foliaceus; skin.

Figure 1

(a) An El Bagre EPF clinical blister (black arrow). (b) H&E staining of one of multiple patterns seen in El Bagre EPF. In this case, note the loss of the upper layers of the epidermis (black arrow), and a dilated dermal blood vessel (blue arrow) with dermal edema and inflammation. (c) DIF of the skin from a patient affected by El Bagre-EPF, showing positive double staining in the epidermis (in orange stain, as result of the patient’s autoantibodies in green and the ARVCF in red) (+++) (black arrow), basement membrane zone (greenish stain) (+++) (white arrow) and a dermal neurovascular bundle (yellowish stain) (+++) (blue arrow) using FITC conjugated anti-human IgG (green staining) and Alexa 555 conjugated ARVCF (red staining). The staining observed represents overlap staining of different strength from yellowish-orange to green (400X). (d) Confocal microscopy, with multiple channels of fluorescence utilized. In the presented case we used a FITC channel (green peaks) (Excitation/Emission (nm):495/519), a DAPI channel (blue peaks) (Excitation/Emission (nm):360/460), and an Alexa Fluor^®555 channel (red peaks) (Excitation/Emission (nm):555/568). The graphic shows the colocalization of the peaks of the immunofluorescence of the patient’s antibodies (green peaks; white arrow) with the ARVCF antibody (red peaks; white arrow). Both green and red are aligned, demonstrating colocalization. The blue peaks represent DAPI (nuclear counterstaining). (e) and (f) IEM photographs of patient skin, showing ARVCF antibodies labelled with 10 nm Gold-conjugated protein A antibodies (tiny black dots). In (e), the antibodies are located in the epidermal cells junctions (black arrow), at the cutaneous basement membrane zone (black dots; red arrow) and in the upper papillary dermis (tiny black dots; blue arrow) (100kV). (f) Positive IEM staining in the upper epidermal keratinocytes cells junction showing ARVCF antibody gold labeled grouped in some areas (tiny black dots; red arrows) (100kV). [Copyright: ©2017 Abreu Velez et al.]