Tadashi Karashima

Interaction of periplakin and envoplakin with intermediate filaments

Periplakin is a component of desmosomes and the epidermal cornified envelope. Its N-terminal domain interacts with the plasma membrane; it heterodimerises with envoplakin via its rod domain; and its C-terminus interacts with intermediate filaments. Periplakin has the shortest C-terminus of the plakin family, comprising only the linker domain found in all conventional plakins. By transient transfection of COS7 cells and primary human epidermal keratinocytes with deletion mutants of the periplakin C-terminus we mapped sequences required for intermediate filament interaction to two regions of the linker motif that are most highly conserved amongst the plakins. The results were confirmed by overlay assays of the binding of in vitro translated periplakin constructs to keratins and vimentin. We found that envoplakin and periplakin could still associate with each other when parts of their rod domains were deleted and, surprisingly, that removal of the entire rod domain did not completely inhibit their interaction. Co-transfection of constructs containing the C-termini of envoplakin and periplakin suggested that the periplakin C-terminus may stabilise the interaction of the envoplakin C-terminus with intermediate filaments. We conclude that the periplakin C-terminus plays an important role in linking periplakin and envoplakin to intermediate filaments.

Epidermolysis bullosa acquisita: What's new?

Type VII collagen is an adhesion molecule of the extracellular matrix in epithelial basement membranes, and the main constituent of anchoring fibrils at the dermal–epidermal junction (DEJ). Autoimmunity against this protein is causing the rare organ‐specific epidermolysis bullosa acquisita (EBA). EBA is a rare acquired, heterogeneous, chronic blistering disease of skin disease of skin and mucous membranes characterized by subepidermal blisters and tissue‐bound as well as circulating autoantibodies to the DEJ. EBA has several distinct clinical presentations with other subepidermal bullous diseases, such as mainly dystrophic epidermolysis bullosa or bullous pemphigoid. The circulating immunoglobulin G autoantibodies for EBA react with a 290‐kDa dermal protein, type VII collagen, as detected by immunoblot analysis using dermal extracts. The pathogenicity of these autoantibodies has been demonstrated by experimental animal models, in which anti‐type VII collagen antibodies injected into a mouse produced an EBA‐like blistering disease in the animal. EBA cases often require high doses of systemic corticosteroids and a variety of immunosuppressants. Although treatment for EBA is frequently difficult and unsatisfactory, some therapeutic success has been reported with colchicine, dapsone, infliximab and i.v. immunoglobulin. In this review, we will focus on recent progress in our understanding of the clinical manifestations, the etiopathogenesis as well as the management of EBA.

From anti-p200 pemphigoid to anti-laminin γ1 pemphigoid

Anti‐laminin γ1 pemphigoid is an autoimmune subepidermal bullous disease first described in 1996, and has been distinct from previously known subepidermal blistering diseases, such as bullous pemphigoid and epidermolysis bullosa acquisita. Circulating autoantibodies of the patients do not react to any known autoantigen of the skin, but react to a 200‐kDa molecule (p200) from dermal extracts. The identity of p200 was unmasked as laminin γ1, an extracellular matrix glycoprotein composing several forms of laminin heterotrimers. We renamed this disease from the previously used anti‐p200 pemphigoid to anti‐laminin γ1 pemphigoid, a new entity of an autoimmune bullous disease. In this decade, we have experienced over 70 cases of this disease. Although the number of the cases of anti‐laminin γ1 pemphigoid is half as many as the number of definitely diagnosed cases of epidermolysis bullosa acquisita in the same duration, a considerable number of the cases could be clinically misdiagnosed as epidermolysis bullosa acquisita. Unveiling the pathogenicity and development of a useful diagnostic method is necessary for appropriate management of this new disease.

Lesional Th17 cells and regulatory T cells in bullous pemphigoid

Abstract:  Th17 cells play crucial roles in the pathogenesis of autoimmune diseases. We previously reported that Th17 cells are recruited to the lesional skin in pemphigus vulgaris (PV) and pemphigus foliaceus (PF). The aim of this study was to evaluate lesional Th17 cells and Treg cells in bullous pemphigoid (BP). Correlations between these cells and disease severity of BP were also evaluated. Immunohistochemical studies showed that both IL‐17+ and Foxp3+ cells were present in higher numbers in BP lesions, compared with control skin. IL‐17/CD4 ratio in BP was significantly higher than that in PF. Foxp3/CD4 ratio in BP was significantly less than that in either PV or PF. There were no obvious correlations between these cells and disease severity of BP. This study suggests that, compared with pemphigus, BP shows more Th17 cell‐related inflammation and less Treg‐related regulation.

FK506 and cyclosporin A inhibit growth factor-stimulated human keratinocyte proliferation by blocking cells in the G0/G1 phases of the cell cycle

FK506, a new immunosuppressive agent, is effective in treating patients with psoriasis. A major feature of psoriasis vulgaris is the hyperproliferation of keratinocytes together with inflammation. To determine the effect of FK506 or cyclosporin A (CsA) on the keratinocyte cell cycle, flow cytometry and the growth factor free normal human keratinocyte-arrested system were used to assess keratinocyte proliferation. FK506 and CsA inhibit keratinocyte proliferation induced by EGF, TGF-alpha or IL-6. The antiproliferative effects of FK506 and CsA directly correlated with blockade of the keratinocyte cell cycle at the G0/G1 phases. These findings might indicate that the effects of FK506 and CsA on proliferation of cultured normal human keratinocytes are probably related to direct effects on growth regulation of keratinocytes via EGF, TGF-alpha or IL-6 stimulation.

How do keratinizing disorders and blistering disorders overlap

Inherited keratinizing disorders are caused by mutations in the genes encoding cornified cell envelope proteins, enzymes and their inhibitors, adhesion molecules, cytoskeletal proteins and others in the epidermis. These molecules are known to regulate differentiation, proliferation and cell adhesions. Intriguingly, some keratinizing disorders show blistering skin lesions, while some inherited blistering disorders show abnormal keratinization. Therefore, hereditary keratinizing and blistering diseases are closely related and show overlapping genetic backgrounds. In this review, we overviewed keratinizing and blistering disorders in terms of overlapping of the two disease groups. Gene mutations in desmosomal components cause striate keratoderma, Naxos disease, epidermolytic palmoplantar keratoderma and plakophilin deficiency, which first show skin fragility and blisters and later hyperkeratosis. Gene mutations in hemidesmosomal components cause various forms of epidermolysis bullosa, some of which show hyperkeratosis on the nails, palms and soles, in addition to blister formation. Diseases with gene mutations in calcium pump proteins are Darier disease and Hailey–Hailey disease, which show clinicopathological overlaps and develop both keratinizing and blistering skin lesions. Finally, gene mutations in epidermal keratins cause epidermolysis bullosa simplex, epidermolytic ichthyosis, superficial epidermolytic ichthyosis, epidermolytic palmoplantar keratoderma and pachyonychia congenita/focal palmoplantar keratoderma, which show thickening of the palms and soles with underlying blister formation. In general, responsible proteins for diseases developing both keratinizing and blistering conditions are adhesion molecules, calcium pump proteins and keratins, but not connexins, cornified cell envelop proteins, enzymes or inhibitors. It is still unknown how particular keratinizing diseases develop blisters and vice versa.

Five Japanese cases of antidesmoglein 1 antibody‐positive and antidesmoglein 3 antibody‐negative pemphigus with oral lesions

Background  Oral mucosal lesions develop in pemphigus vulgaris, but not in pemphigus foliaceus. This clinical phenomenon is explained by the ‘desmoglein (Dsg) compensation theory’. Dsg3 and Dsg1 are major autoantigens for pemphigus vulgaris and pemphigus foliaceus, respectively. Dsg3 is overexpressed and Dsg1 is weakly expressed on the oral mucosa. Thus, on the oral mucosa, suppression of Dsg3 function by anti‐Dsg3 autoantibodies is not compensated by weakly expressed Dsg1 in pemphigus vulgaris, while suppression of Dsg1 function by anti‐Dsg1 autoantibodies is perfectly compensated by richly expressed Dsg3 in pemphigus foliaceus.

A clinical study of patients with pemphigus vulgaris and pemphigus foliaceous: an 11‐year retrospective study (1996–2006)

Only a few reports have been published of detailed clinical studies of pemphigus in Japan. The aim of this study was to determine the clinical characteristics of patients with pemphigus vulgaris (PV) and pemphigus foliaceous (PF), who were newly diagnosed in the dermatology department of Kurume University Hospital, Japan, over the past 11 years. The primary site of involvement was the oral mucosa in 21 patients (75%) with PV. At the initial visit, most of the patients with PV had moderate to severe disease. With regard to management, systemic corticosteroids were the mainstay of treatment for patients with PV, and plasmapheresis was the most frequently used adjuvant therapy. Dapsone was the mainstay of treatment for the patients with PF. The patients were investigated for any association with an underlying malignancy; in patients with PV, lung, stomach and uterine cancers (one patient each) were seen.

Novel non-radioisotope immunoprecipitation studies indicate involvement of pemphigus vulgaris antigen in paraneoplastic pemphigus

We have developed two different novel immunoprecipitation assays in which radioisotopes are not used, and have examined antigens for four cases of paraneoplastic pemphigus (PNP) including three new patients. The PNP sera showed a clear reactivity with transitional epithelia of rat urinary bladder by immunofluorescence, and reacted with a characteristic doublet of the 210 and 190 kD proteins by immunoblotting of normal human epidermal extract, confirming the diagnosis of PNP. In addition, by immunoprecipitation using silver-stain to detect immunoprecipitated proteins, the PNP sera detected the 250, 210 and 190 kD proteins, while control bullous pemphigoid sera detected only the 230 kD bullous pemphigoid antigen. Furthermore, with another immunoprecipitation using cell surface biotinylation, three of the four PNP sera specifically reacted with the 130 kD pemphigus vulgaris antigen (Dsg3), indicating that pemphigus vulgaris antigen may be involved in PNP. This reactivity was further suggested by the immunoblot analysis using recombinant pemphigus vulgaris antigen. In future, these non-radioisotope immunoprecipitation assays should become a useful tool not only to unravel the complex situation for the PNP antigens, but also to study antigens in other autoimmune bullous skin diseases.

Anti-Alpha-2-Macroglobulin-Like-1 Autoantibodies Are Detected Frequently and May Be Pathogenic in Paraneoplastic Pemphigus

Paraneoplastic pemphigus (PNP) shows autoantibodies mainly to plakin and desmosomal cadherin family proteins. We have recently identified alpha-2-macroglobulin-like-1 (A2ML1), a broad range protease inhibitor, as a unique PNP antigen. In this study, we tested a large number of PNP sera by various methods. Forty (69.0%) of 58 PNP sera recognized A2ML1 recombinant protein expressed in COS7 cells by immunofluorescence (IF) and/or immunoprecipitation (IP)/immunoblotting (IB). IP/IB showed higher sensitivity than IF. In addition, 22 (37.9%) PNP sera reacted with A2ML1 by IB of cultured normal human keratinocytes (NHKs) under non-reducing conditions. Statistical analyses using various clinical and immunological data showed that the presence of anti-A2ML1 autoantibodies was associated with early disease onset and absence of ocular lesions. Next, to investigate the pathogenic role of anti-A2ML1 antibody, we performed additional functional studies. Addition of anti-A2ML1 polyclonal antibody to culture media decreased NHK cell adhesion examined by dissociation assay, and increased plasmin activity detected by casein zymography, suggesting that anti-A2ML1 antibody may decrease NHK cell adhesion through plasmin activation by inhibition of A2ML1. This study demonstrates that autoantibodies to A2ML1 are frequently and specifically detected and may have a pathogenic role in PNP.