Pamela Hawley-Nelson

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.

Pemphigus Antibodies Identify a Cell Surface Glycoprotein Synthesized by Human and Mouse Keratinocytes

Pemphigus is an antibody-mediated autoimmune skin disease in which loss of cell-to-cell contacts in the epidermis results in blister formation. Patients with pemphigus develop antibodies that bind to the keratinocyte cell surface, the site of primary pathology. The purpose of this study was to characterize the antigen(s) to which pemphigus antibodies bind. Because we could detect pemphigus antigen by indirect immunofluorescence on the surface of multiply-passaged cells in cultures of both a spontaneously transformed mouse keratinocyte cell line (Pam) and normal human epidermal cells, we used these cells as a source of antigen. In order to demonstrate biosynthesis of antigen and to characterize the antigen(s), we radiolabeled cell cultures with [(14)C]glucosamine or d-[2-(3)H]mannose and used different pemphigus sera to immunoprecipitate antigen from nonionic detergent extracts of these labeled cells. Specifically precipitated radiolabeled molecules were identified using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and fluorography. Sera from five of seven pemphigus patients specifically precipitated (from extracts of both Pam cells and human epidermal cells) a molecule that, when reduced, was approximately 130 kD, whereas seven normal human sera and two pemphigoid sera did not precipitate this molecule. The findings that (a) these precipitated molecules comigrated on SDS-PAGE and that (b) the 130-kD molecule could no longer be precipitated from cell extracts that had been previously reacted with a pemphigus serum, indicate that reactive pemphigus sera bind the same molecule. The molecule was not detected in the culture medium of these cells. This finding, along with the cell surface immunofluorescence pattern, suggests that the antigen is bound to the cell surface. Cultured mouse and human fibroblasts do not synthesize the antigen. The antigen contains protein because it was degraded by V8 protease and chymotrypsin, and it could also be labeled with [(14)C]amino acids. It is probably not a sulfated proteoglycan because it did not label with (35)SO(4). Taken together, these data indicate that some, but not all, pemphigus sera bind a specific cell surface glycoprotein that is synthesized by keratinocytes.

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.