Edward J. O'Keefe

Identification of the Skin Basement-Membrane Autoantigen in Epidermolysis Bullosa Acquisita

Epidermolysis bullosa acquisita is an acquired chronic blistering disease of the skin, in which separation of the skin occurs in the basement-membrane zone between the epidermis and the dermis. There is evidence that blistering is initiated by an immune process. Using serum samples from nine patients as a source of antibodies, we have identified a major protein of the basement membrane of human skin that serves as the antigen (or target) for autoantibodies in this disorder. This previously unrecognized protein, which consists of two components of 290,000 and 145,000 daltons, is distinct from other known components of the basement membrane. These studies provide evidence that epidermolysis bullosa acquisita is a specific disease that is different from other primary bullous diseases, such as bullous pemphigoid and pemphigus vulgaris, and suggest that the basement-membrane component that has been identified may have a role in normal epidermal-dermal adherence.

Human autoantibodies against desmoplakins in paraneoplastic pemphigus

Recently, a previously unrecognized autoantibody mediated blistering disease, paraneoplastic pemphigus has been described. Paraneoplastic pemphigus is associated with lymphoid malignancies, thymomas, and poorly differentiated sarcomas. Serum of affected patients contain pathogenic autoantibodies that immunoprecipitate from normal keratinocytes a characteristic complex of four polypeptides with M(r) of 250, 230, 210, and 190 kD. As our preliminary studies indicated that the 250-kD and the 210-kD antigens comigrated with desmoplakins I and II, we investigated the possibility that autoantibodies against the desmoplakins were a component of this autoimmune syndrome. 11 sera from affected patients were tested by indirect immunofluorescence against desmosome containing tissues, immunoprecipitation of metabolically labeled keratinocytes, and Western immunoblotting of desmoplakins I and II that had been purified to homogeneity from pig tongue epithelium. By indirect immunofluorescence, 9 of 11 sera showed strong binding to epithelial and nonepithelial desmosomes, and 2 were weakly reactive. All 11 immunoprecipitated 250- and 210-kD bands of variable intensity that comigrated with bands identified by a murine monoclonal antidesmoplakin antibody, and immunoblotting confirmed binding of the serum autoantibodies to purified desmoplakins. This demonstrates that paraneoplastic pemphigus is the first human autoimmune syndrome in which autoantibodies against the desmoplakins are a prominent component of the humoral autoimmune response.

Epidermal growth factor receptor number decreases during rat liver regeneration.

The potential role of epidermal growth factor (EGF) in the regulation of rat liver regeneration was examined by assessing the binding of 125I-EGF to hepatic membranes isolated at various times after partial hepatectomy. The results demonstrated a fall in 125I-EGF binding detectable as early as 8 h after partial hepatectomy. The nadir in EGF binding, less than 40% of that observed in sham-operated control rats, was seen 36 and 48 h after partial hepatectomy. Scatchard analysis showed that the decrease in binding capacity was due to a fall in receptor number. The specificity of the observed loss of EGF receptors was substantiated in parallel studies of 125I-insulin and 125I-wheat germ lectin binding; the binding of these ligands did not decrease appreciably during liver regeneration. The data are consistent with the hypothesis that EGF or a similar substance is one component of the complex humoral signal that regulates liver regeneration.

A model of cell cycle control: Sequential events regulated by growth factors

PDGF is a potent mitogen that initiates the proliferation of quiescent fibroblastic cells. EGF and somatomedin C (or insulin) can replace the requirement for plasma to function synergistically with PDGF to stimulate DNA synthesis. PDGF, EGF and somatomedin C control discrete cellular events in the cell cycle. Cyclic AMP can potentiate the effects of polypeptide mitogens. The down-regulation of EGF receptors by PDGF and cyclic AMP brings about a loss of the requirement for exogenous EGF. The transient treatment of density-arrested fibroblasts with PDGF allows better study of synergistic actions of PDGF and plasma-derived factors. These synergistic interactions are important to understand in determining how multiple growth factors regulate cellular proliferation.

Epidermal growth factor (EGF) is required only during the traverse of early G1 in PDGF stimulated density-arrested BALB/c-3T3 cells☆

Density-arrested BALB/c-3T3 cells that had received a transient exposure to PDGF and were then transferred to medium containing only EGF and somatomedin C (Sm-C) began DNA synthesis after the G0/G1 lag. Supraphysiological concentrations of insulin could be employed to replace the Sm-C requirement. This G0/G1 lag phase was bisected by the requirement for the exogenous presence of EGF. Our data indicated that EGF was required during the traverse of only the first half of G0/G1 phase (6 h) and not during the traverse of late G1. Subphysiological serum concentrations of Sm-C were also necessary to be present with EGF for progression through early G0/G1; however, traverse of the final half of G0/G1 and commitment to DNA synthesis required the presence of Sm-C. It was found that physiological concentrations of Sm-C were required for the traverse late G1. The requirement for Sm-C for G0/G1 traverse of BALB/c-3T3 cells as opposed to human fibroblasts or glial cells may be due to a difference in endogenous synthesis of an insulin-like growth factor. Our data are in close agreement with previous reports that EGF is only required for approximately the first 8 h during traverse of the G0/G1 phase. The requirement for EGF to be present for the first 6 h of G0/G1 could result from a continued or repetitious event or by more than one distinct EGF-requiring event.

Epidermolysis bullosa acquisita antigen is synthesized by human keratinocytes cultured in serum-free medium

The epidermolysis bullosa acquisita antigen is a newly discovered major extracellular matrix component within basement membranes beneath stratified squamous epithelia. Human keratinocytes cultured without mesenchymal cells synthesize the major 290 kd chain of the epidermolysis bullosa acquisita antigen.

Mitogenic response to epidermal growth factor (EGF) modulated by platelet-derived growth factor in cultured fibroblasts

The mitotic effects of epidermal growth factor (EGF) were investigated in two cultured fibroblast lines, BALB/c-3T3 and C3H 10T1/2 cells. EGF (30 ng/ml) added to quiescent 3T3 cells in medium containing either platelet-poor plasma or 10(-5) M insulin caused only minimal increases in the percentage of cells stimulated to initiate DNA synthesis. In contrast, EGF acted synergistically with either insulin or plasma to stimulate DNA synthesis in quiescent cultures of 10T1/2 cells, although the maximum effects of EGF were measured at concentrations several-fold greater than those found in either serum or plasma. In either 3T3 or 10T1/2 cells a transient preexposure to platelet-derived growth factor (PDGF) caused over a 10-fold increase in the sensitivity to the mitogenic effects of EGF. It is therefore possible that a primary action of PDGF is to increase the sensitivity of fibroblasts to EGF, independent of whether EGF alone is found to be mitogenic.

Cyclic AMP potentiates down regulation of epidermal growth factor receptors by platelet-derived growth factor

Abstract Pretreatment of Balb c -3T3 cells with platelet-derived growth factor (PDGF) has been shown to decrease binding sites for 125I-labelled epidermal growth factor (EGF) (1,2,3). Agents which elevate cellular cyclic AMP concentrations enhance this ability, and the change in EGF binding is inversely proportional to the elevation of cyclic AMP. In quiescent density arrested cells, the sensitivity of cells to down regulation of EGF receptors by PDGF is proportional to the cyclic AMP content of the cultures in three different cell lines. Agents which elevate cyclic AMP and which potentiate PDGF mediated heterologous down regulation of EGF receptors are able, like cholera toxin (3), to stimulate cells to synthesize DNA in defined medium in the absence of EGF. Down regulation of EGF receptors by PDGF in combination with agents elevating cyclic AMP effectively mimics the action of EGF.

Specific affinity between fibronectin and the epidermolysis bullosa acquisita antigen.

Autoantibodies in the skin and sera of patients with epidermolysis bullosa acquisita bind to a large matrix molecule within the lamina densa region of skin basement membrane. At the site of these immune complexes, the epidermis separates from the dermis, which creates a subepidermal blister just below the lamina densa. The target molecule for the autoantibodies is in close apposition to fibronectin, a major extracellular matrix molecule that is abundant in the upper dermis of skin. In this report, we show specific affinity between fibronectin and the 290,000-D chain of the epidermolysis bullosa acquisita antigen, and that this affinity is mediated by the gelatin/collagen-binding domain of fibronectin (Mr = 60,000). Since blistering in epidermolysis bullosa acquisita often occurs in the absence of clinical and histological inflammation, a direct interruption in the fibronectin-epidermolysis bullosa acquisita antigen bond may be involved in the pathogenesis of epidermal-dermal disadherence that occurs in this bullous disease.

Placental keratinocyte growth factor: Partial purification and comparison with epidermal growth factor

A water-soluble extract of term human placenta, which was previously shown to promote proliferative growth of human keratinocytes in defined medium, enhanced both cellular attachment and proliferative growth. We have partially purified the activity which enhanced cell growth and examined its action in keratinocytes. Activity was precipitated from the crude extract by (NH4)2SO4 between 33 and 60% saturation and chromatographed by gel filtration. The activity did not bind to heparin-Sepharose at low ionic strength but was adsorbed to DEAE-cellulose from which it was eluted with NaCl and then passed over phenyl-HPLC to remove bovine serum albumin previously added to protect the activity. The active fraction was applied to gel exclusion HPLC in the presence of 0.02% octyl-beta-D-glucopyranoside, which yielded an apparent Mr 35,000 for the factor. Purification was approximately 200-fold with approximately 4% recovery. The factor appears to be a protein, since activity is destroyed by trypsin. Autoradiography of cultures treated with the placental factor or epidermal growth factor (EGF) revealed that approximately 50% of cells were labeled after treatment with either growth factor compared to 9% in control cultures after a [3H]thymidine pulse. Protein synthesis was increased by about 50% 42 h after treatment with either agent, consistent with a 50% increase in nuclear labeling. Cell number was increased fivefold after 6 days in the presence of the partially purified factor, whereas EGF increased cell number eightfold. Stimulation of [3H]thymidine incorporation by the partially purified factor, in contrast, was about twice that produced by EGF, indicating that thymidine incorporation is preferentially stimulated by the placental factor and does not correlate well with other parameters of proliferative growth. The placental keratinocyte growth factor is a unique factor with a novel effect on incorporation of thymidine into DNA.