John R. Stanley

Characterization of bullous pemphigoid antigen: A unique basement membrane protein of stratified squamous epithelia

Abstract Bullous pemphigoid (BP) antigen is a normal basement membrane zone antigen of epidermis and other stratified squamous epithelia. It is defined immunologically by antibodies in the sera of patients with the subepidermal blistering disease BP. In this study we sought to demonstrate that epidermal cells synthesize this antigen, to determine the immunological specificity of BP antibodies and to characterize this antigen. Cultured human epidermal cells (HEC) and a spontaneously transformed mouse epidermal cell line (Pam) both demonstrated BP antigen by indirect immunofluorescence. To characterize the antigen, these cells were radiolabeled with 35 S-methionine or 14 C-amino acids and extracts were immunoprecipitated using nine different BP sera. Immunoprecipitated proteins were identified using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and fluorography. All nine BP sera precipitated a protein with disulfide-linked chains of apparent molecular weight approximately 220 kd. Eight normal human sera and six pemphigus vulgaris sera, as well as antibodies directed against fibronectin and laminin, did not precipitate this protein. Furthermore, it was not precipitated by BP sera from radiolabeled extracts of fibroblasts. The protein was soluble in Tris-HCI buffered saline but was not secreted into the culture medium. These studies demonstrate that BP antigen is synthesized by epidermal cells in culture, different patients with BP have antibodies against the same protein, and BP antigen can be identified on SDS-PAGE as a high molecular weight protein consisting of disulfide-linked chains of approximate molecular weight 220 kd.

Cell Adhesion Molecules as Targets of Autoantibodies in Pemphigus and Pemphigoid, Bullous Diseases Due to Defective Epidermal Cell Adhesion

Publisher Summary This chapter focuses on cell adhesion molecules as targets of autoantibodies in pemphigus and pemphigoid, bullous diseases. In various autoimmune blistering diseases of skin, autoantibodies are directed against epidermal cell adhesion molecules or molecules found in cell adhesion junctions. The blisters and dysfunction of the epidermal barrier in these diseases result from loss of adhesion of cells either to the basement membrane or to other cells. This theme is developed for three diseases, bullous pemphigoid (BP), pemphigus foliaceus (PF), and pemphigus vulgaris (PV). Studies of the autoantibodies from patients with these diseases have shed light on potential pathophysiologic mechanisms and have enabled the start of the understanding of the way the epidermis maintains its intact state. BP, PF, and PV are all bullous diseases of skin. Each has a distinctive clinical appearance and the blisters in each result from loss of adhesion at a specific level of the epidermis. BP and pemphigus also have characteristic immunofluorescence findings; however, the immunofluorescence findings in PF and PV are very similar. BP is a subepidermal blistering disease in which autoantibodies localize to the site of pathology and define a normal antigen of stratified squamous and complex epithelial basement membranes.

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis.

Abstract Studies were conducted to determine the effects of the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured human epidermal cells for comparison with known effects on mouse keratinocytes. In contrast to its effect on mouse cells, TPA did not stimulate human epidermal cell DNA synthesis. TPA stimulated differentiation in human keratinocytes resulting in sloughing of many cells by the 3rd day after exposure. Quantitative assays revealed that 50% of the TPA-exposed population was composed of cornified cells as opposed to 8% in untreated controls. A morphologically distinct cell type (TT cell) emerged after TPA treatment which was triangular in shape, did not stratify, appeared to proliferate rapidly and at most TPA concentrations became the predominant cell type within 1–2 weeks. Cultures composed predominantly of TT cells formed few cornified envelopes, grew well in the absence of TPA and formed colonies at low cell input. In contrast to its effect on keratinocytes, TPA enhanced TT colony formation 3–4-fold and decreased the doubling time of TT cells. Studies were performed to determine the origin of TT cells. Immunofluorescent staining indicated that TT cells lacked the keratinocyte antigens keratin, pemphigus and pemphigoid. Tonofilaments and desmosomes were not seen by electron microscopy. The lack of both melanosomes and standard histochemical DOPA oxidase staining indicated that TT cells were probably not of melanocyte origin. Tests used to identify Langerhans cells were negative. Whereas TT cells, as well as dermal fibroblasts, yielded positive immunofluorescence with antibodies to vimentin, TT cells gave a weak histochemical leucine aminopeptidase reaction, while the reaction of fibroblasts exposed to TPA was strong. Treatment of human dermal fibroblasts with TPA did not yield TT cells. The endothelial cell antigen factor VIII-associated protein was absent by immunofluorescence. These results suggest that the primary effect of TPA on cultured human epidermis is to accelerate terminal differentiation in the keratinocyte population and to stimulate growth of an as yet unidentified cell type.

Epidermal autoantibodies in erythema multiforme

We report a case of refractory erythema multiforme associated with atypical epidermal autoantibodies. The patient was a 63-year-old woman with clinical and histologic features of erythema multiforme and a large mediastinal T cell lymphoma. Immunofluorescence studies disclosed high serum titers of cell surface antibodies against epidermal keratinocytes in a pattern typical of pemphigus vulgaris. The epidermal antigen reacting with these circulating antibodies was characterized by incubating 14C-labeled keratinocytes with serum. The patients serum pattern precipitated multiple proteins keratinocytes antigens), but not the 130,000- and 85,000-dalton polypeptides characteristic of pemphigus vulgaris. Thus autoantibodies reacting selectively with non-pemphigus vulgaris epidermal keratinocyte antigens may be the cause of a false-positive immunofluorescence test for pemphigus vulgaris.

Erythema Annulare-like Acantholytic Dermatosis (EAAD): Nonbullous Pemphigus or a New Entity?

This article describes a case of unusual annular erythema-like dermatosis, with histological features of pemphigus foliaceus (subcorneal acantholysis) and IgG antibodies in circulation and bound in vivo to the keratinocyte surface. The reactivity of the antibodies, restricted to human squamous epithelium, was unique, differing from that of all known forms of pemphigus. This also was confirmed by immunoprecipitation. The problem is that these circulating antibodies could be missed if not determined on human substrate. It is to be established whether such cases present a new type of pemphigus or a unknown dermatosis with an autoimmune response of a pemphigus type.

Is pemphigus an anti-adhering junction autoimmune disease?

The skin as an organ hardly ever fails. Even in extreme old age it almost always performs flawlessly its functions of protection from infection and fluid retention. Therefore, it is a tribute to the potentially terrible power of the immune system that its autoantibodies, in patients with pemphigus, can wreak havoc on the normal structure and function of the skin, and cause death through skin failure. How do these antibodies cause this severe and debilitating disease? How do they interfere with the normal function of the skin? Not all the answers are available now, but this review will discuss how these antibodies bind components of structures crucial for keratinocyte cell-cell adhesion, thus providing one possible mechanism of interference in the normal integrity of the epidermis.