Immunology Letters 122 (2009) 208–213 Contents lists available at ScienceDirect Immunology Letters journal homepage: www.elsevier.com/locate/ High-dose pemphigus antibodies against linear epitopes of desmoglein 3 (Dsg3) can induce acantholysis and depletion of Dsg3 from keratinocytes Nicola Cirillo a,b,c,∗ , Felice Femiano b , Fernando Gombos a,b , Alessandro Lanza a,b a Regional Center on Craniofacial Malformations-MRI, Italy b AISS Accademia Italiana di Stomatologia Sperimentale, 1st School of Medicine and Surgery, II University of Naples, Naples, Italy c Department of Oral and Dental Science, University of Bristol, Bristol, United Kingdom article info Article history: Received 8 August 2008 Received in revised form 18 December 2008 Accepted 14 January 2009 Available online 4 February 2009 Keywords: Desmoglein 3 Pemphigus Keratinocytes Autoantibodies Linear epitopes abstract We have previously demonstrated that serum autoantibodies of patients with pemphigus vulgaris (PV) may affect desmoglein 3 (Dsg3)-mediated adhesion by decreasing its half-life and inducing Dsg3 cleavage. Here we sought to gain more insights into the role of Dsg3-targetting IgG in acantholysis. To do so, alterations of keratinocyte morphology and cell–cell adhesion strength were investigated in the presence of PV serum, PV IgG, and IgG purified from PV patients’ sera against linear epitopes of Dsg3 (anti-Dsg3-L IgG). Changes in Dsg3 protein levels were assessed by Western blotting. Results showed that both PV serum and PV IgG were able to induce acantholysis and decrease the total amount of Dsg3 in cell lysates. Polyclonal anti-Dsg3-L IgG displayed Dsg3-depleting activity solely when used at 1 g/ml, i.e. under non-physiologic conditions. Furthermore, cell–cell detachment induced by PV IgG and anti-Dsg3-L IgG seemed to precede the loss of Dsg3 from keratinocytes, suggesting that depletion/degradation of Dsg3 represents a late event in acantholysis. Collectively, the data presented here demonstrate that PV IgG recognizing non-conformational epitopes of Dsg3 are pathogenic when administered on doses largely exceeding those found in PV sera. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Pemphigus vulgaris (PV) is a potentially fatal autoimmune blistering disease affecting stratified squamous epithelia [1]. Autoantibodies in PV recognize receptor-like molecules regulating epidermal cohesion. Among the array of keratinocyte self antigens immunoprecipitated by circulating PV IgG [2,3], the best charac- terized is desmoglein (Dsg) 3, a desmosomal glycoprotein which is critically involved in ensuring calcium-dependent intercellular adhesion among keratinocytes [4]. Indeed, PV associates with the presence of circulating IgG against linear and conformational epi- topes of Dsg3 and, less frequently, Dsg1 [5,6]. In PV, blisters develop as a consequence of the loss of cell–cell adhesion, or acantholysis. Although PV IgG against non-desmoglein antigens as well as serum factors other than IgG can exert pathogenic effects on keratinocytes [7–9], the key role of Dsg3- targeting antibodies in PV pathophysiology is widely accepted. Binding of IgG to Dsg3 is thought to affect Dsg3 function by steric hindrance (reviewed in ref. [9]) or by inducing desmosomal sig- ∗ Corresponding author at: Department of Oral and Dental Science, University of Bristol, Lower Maudlin Street, BS1 2LY Bristol, United Kingdom. Tel.: +44 117 3423453; fax: +44 117 3424428. E-mail address: cirillo.sun@libero.it (N. Cirillo). nalling [7]. Alternatively, or additionally, binding of autoantibodies to Dsg3 could modulate its own synthesis, leading to the forma- tion of aberrant desmosomes lacking Dsg3 [10–13]. In this regard, we previously reported that PV sera can decrease the cell content of Dsg3 by reducing its half-life and perturbing the subsequent incorporation of Dsg3 into desmosomes [11]. Consistently, it has been suggested that PV IgG may work through the depletion of the Triton X-100 soluble pool of Dsg3, thus depriving cells from free Dsg3 before its assembly into desmosomes [10]. This phenomenon could depend on the down-regulation of Dsg3 at the transcrip- tional level [13]. A recent study from Kitajima’s lab reported the presumptive depletion of Dsg3 from desmosomes to depend on the pathogenicity of anti-Dsg3 IgG against conformational epitopes of Dsg3 [12]. This finding would be in agreement with previous papers demonstrating that the dominant autoimmune epitopes in PV are found in the N-terminal adhesive surfaces of Dsg3. By using domain-swapped Dsg3 recombinants, Futei et al. showed indeed that the majority of autoimmune IgG in PV sera reacted with epi- topes formed by aa 1–161 of the NH 2 -terminus of Dsg3 [14]. IgG against the amino-terminal adhesive interface of Dsg3 were subse- quently found to be pathogenic [15]. In general, the role of IgG against conformational sequences of the extracellular domain of Dsg3 in PV has received great atten- tion. In marked contrast, participation of Dsg3 linear epitopes in PV remains largely unknown. The recent finding that IgG titres 0165-2478/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2009.01.002