1. ¹Department of Dermatology, University of North Carolina, Chapel Hill, North Carolina, USA
2. ²Dermatology Division, Duke University Medical Center, Durham, North Carolina, USA
3. ³Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, USA

Correspondence: DS Rubenstein, E-mail: druben@med.unc.edu

TO THE EDITOR

Pemphigus vulgaris (PV) is an autoimmune disease characterized by epidermal blistering involving mucosa or mucosa and skin (Ding et al., 1997). In PV, autoantibodies directed against the desmosome cadherins desmoglein (dsg) 3 and dsg1 induce loss of cell–cell adhesion (acantholysis) resulting in clinical blisters. Passive transfer experiments using IgG, purified from PV patients (Anhalt et al., 1982) or highly specific anti-dsg3 monoclonal antibodies (Tsunoda et al., 2003; Payne et al., 2005), reproduce the clinical, histologic, and immunologic features of PV in the infused animals. Similarly, pemphigus foliaceus (PF) is an autoimmune blistering disease of the skin in which autoantibodies to dsg1 cause acantholysis in the subcorneal and granular layers of the epidermis. IgG purified from PF patient sera similarly reproduces disease in the passive transfer mouse model (Roscoe et al., 1985; Rock et al., 1989).

We and others have been studying the molecular mechanism by which anti-dsg autoantibodies cause acantholysis in pemphigus. Using keratinocyte tissue culture and murine in vivo models, we have demonstrated that anti-dsg3 PV IgG induce rapid, time- and dose-dependent phosphorylation of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27) (Berkowitz et al., 2005). Activation of keratinocyte p38MAPK and HSP27 by PV IgG may be relevant to the mechanism of acantholysis in PV since (i) both p38MAPK and HSP27 regulate components of the cytoskeleton including actin and intermediate filaments (Lavoie et al., 1993, 1995; Benndorf et al., 1994; Guay et al., 1997; Perng et al., 1999; Geum et al., 2002; Panasenko et al., 2003; Evgrafov et al., 2004) and (ii) in human keratinocyte cultures, inhibitors of p38MAPK prevented PV IgG-induced phosphorylation of HSP27 and the early cytoskeletal changes associated with the loss of cell–cell adhesion (Berkowitz et al., 2005). These observations suggested that inhibition of this signaling pathway in epidermal epithelia could be used to prevent end-organ damage (e.g. blistering) caused by PV autoantibodies. Indeed, in the PV passive transfer mouse model we found that (i) p38MAPK and HSP25, the murine HSP27 homolog, are similarly phosphorylated in vivo, and significantly (ii) p38MAPK inhibitors block blister formation in this vivo model of PV (Berkowitz et al., 2006). These observations in tissue culture and animal models suggest a central role for p38MAPK and HSP27 in the mechanisms of acantholysis in PV. Importantly, targeting this signaling pathway with inhibitors may have a role in treating patients with pemphigus; however, we have yet to determine whether p38MAPK and HSP27 are similarly phosphorylated in lesional epidermis of pemphigus patients.

In this study, we probe for phosphorylation of p38MAPK and HSP27 in skin biopsies of one PF and five PV patients and six specimens from control individuals affected by other skin diseases; this study was approved by the Institutional Review Board at UNC. A diagnosis of PV or PF was established by clinical, histological, and immunological criteria (Diaz and Giudice, 2000). The clinical and serological features of the six patients are shown in Table 1. Biopsies from control individuals showed no immunoreactants by direct immunofluorescence or histologic signs of pemphigus.

Table 1 - Clinical and immunologic characteristics of patients from which the skin biopsies were obtained.

Full table

Extracts were made from frozen archived perilesional skin biopsies in 8 M urea buffer (8 M urea, 4% CHAPS, 10 M pepstatin, 100 M leupeptin, 10 M E-64, 1 mM PMSF, 1 mM Na₃VO₄, 50 mM NaF, 0.5 M okadaic acid). Protein concentration was determined by Bradford method as described previously (Berkowitz et al., 2005). Tissue extracts were separated by 10% SDS-PAGE and immunoblotted using monoclonal antibodies against p38MAPK (Santa Cruz Biotechnology Inc., Santa Cruz, CA), phospho-p38MAPK (Cell Signaling Technology, Beverly, MA), HSP27 (ABR, Golden, CO), and phospho-HSP27 (Cell Signaling Technology). Western blots were developed by ECL reaction and the signal intensity quantified by scanning chemiluminescence on a GeneGnome scanner (Syngene Bio Imaging, Frederick,MD) using GeneSnap software. Compared to controls, increased phosphorylation of p38MAPK was observed in 1 PF and 3 of the 5 PV skin biopsies; whereas, increased HSP27 phosphorylation was observed in all PF and PV patient skin biopsies (Figure 1). In our earlier work with in vivo mouse models, we have noted degradation in the phospho-p38MAPK signal with extended sample storage time even at -70°C, this explains the discrepancy between the phospho-p38MAPK and phospho-HSP27 signals observed in 2 of the 5 PV skin biopsies (PV4 and PV5). Increased phosphorylation of p38MAPK and HSP27 was observed in both mucosal and mucocutaneous variants of PV.

Figure 1.

Increased phospho-p38MAPK and phospho-HSP27 in human pemphigus skin biopsies. (a) Extracts from a PF patient (PF), 5 PV patients (PV1–5), and control (C1–6) human skin biopsies were separated by 10% SDS-PAGE and probed by immunoblot with antibodies to phosphorylated p38MAPK (phos-p38), total p38MAPK (p38), phosphorylated HSP27 (phos-HSP27), and total HSP27. (b) Quantitation of p38 immunoblots; phosphorylated p38MAPK expressed as ratio of phospho-p38MAPK to total p38MAPK. Mean values for pemphigus (Pem) samples and controls (CON) are shown. Error bars show SD. Statistical significance determined by Mann–Whitney test; ^*P-value=0.025. (c) Quantitation of HSP27 immunoblots; phosphorylated HSP27 expressed as ratio of phospho-HSP27 to total HSP27. Mean values for pemphigus (Pem) samples and controls (CON) are shown. Error bars show SD. Statistical significance determined by Mann–Whitney test; ^**P-value=0.037.

Full figure and legend (63K)

The observed activation of intracellular signaling events mediated by p38MAPK and HSP27 in patient skin is analogous to the increased phosphorylation of these two proteins that we have previously reported in human keratinocyte tissue culture and in vivo mouse models. Importantly, a number of p38MAPK inhibitors are currently in early-phase clinical trials for inflammatory and neoplastic diseases (O'Neill, 2006). Along with the earlier in vitro and in vivo mouse model studies, the observation that p38MAPK and HSP27 are phosphorylated in biopsies from perilesional skin of human pemphigus patients provides additional evidence to support the notion that p38MAPK inhibitors may be applicable to the therapy of patients suffering from the pemphigus family of autoimmune blistering diseases.