bromochloroacetic-acid and Pemphigoid--Bullous

Topics: Biopsy; Epidermis; Epidermolysis Bullosa; Humans; Keratins; Pemphigoid, Bullous; Pemphigus; Peptide Hydrolases; Protease Inhibitors; Skin Diseases, Vesiculobullous

On histologic vertical sections of skin, the epidermis is separated from the dermis by an amorphous thin membrane, the basal lamina. Ultrastructurally, the basal lamina is composed of four areas, including the basal-cell plasma membrane and hemidesmosomes, the lamina lucida, the lamina densa, and the sub-lamina densa fibrillar region. In culture, epidermal keratinocytes are able to produce hemidesmosomes, lamina lucida, and lamina densa. There is no evidence that cultured keratinocytes can produce sub-lamina densa fibrils. Biochemically, the lamina lucida contains two major glycoproteins. One, the bullous pemphigoid antigen, is synthesized by epidermal keratinocytes in vitro. These cells also synthesize laminin, the other glycoprotein of lamina lucida. At the interface between lamina lucida and lamina densa there is probably a heparan sulfate proteoglycan. Whether this proteoglycan is produced by keratinocytes in culture is not known, but the possibility can be considered. Lamina densa contains collagen IV, and this collagen is synthesized by keratinocytes in culture. However, cultured keratinocytes may also synthesize collagen types I, III, and V. Type V is associated with the basal lamina, but its exact location is unknown. Types I and III (if they are produced in vivo) would be situated in the sub-basal lamina region. The problem of fibronectin remains unsolved. There is "some" fibronectin in the lamina lucida, but its origin is not clear.

Topics: Animals; Basement Membrane; Epidermal Cells; Fibronectins; Glycoproteins; Heparitin Sulfate; Keratins; Laminin; Microscopy, Electron; Pemphigoid, Bullous; Protein Biosynthesis; Swine

In epidermal cells, the keratin cytoskeleton interacts with the elements in the basement membrane via a multimolecular junction called the hemidesmosome. A major component of the hemidesmosome plaque is the 230-kDa bullous pemphigoid autoantigen (BP230/BPAG1), which connects directly to the keratin-containing intermediate filaments of the cytoskeleton via its C terminus. A second bullous pemphigoid antigen of 180 kDa (BP180/BPAG2) is a type II transmembrane component of the hemidesmosome. Using yeast two-hybrid technology and recombinant proteins, we show that an N-terminal fragment of BP230 can bind directly to an N-terminal fragment of BP180. We have also explored the consequences of expression of the BP230 N terminus in 804G cells that assemble hemidesmosomes in vitro. Unexpectedly, this fragment disrupts the distribution of BP180 in transfected cells but has no apparent impact on the organization of endogenous BP230 and alpha6beta4 integrin. We propose that the BP230 N terminus competes with endogenous BP230 protein for BP180 binding and inhibits incorporation of BP180 into the cell surface at the site of the hemidesmosome. These data provide new insight into those interactions of the molecules of the hemidesmosome that are necessary for its function in integrating epithelial and connective tissue types.

Topics: Autoantigens; Binding Sites; Carrier Proteins; Cell Membrane; Cloning, Molecular; Collagen Type XVII; Cytoskeletal Proteins; Cytoskeleton; Desmosomes; Dystonin; Humans; Keratins; Nerve Tissue Proteins; Non-Fibrillar Collagens; Pemphigoid, Bullous; Saccharomyces cerevisiae; Transfection

Topics: Adult; Aged; Aged, 80 and over; Autoantibodies; Biomarkers; Cells, Cultured; Epidermis; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Pemphigoid Gestationis; Pemphigoid, Bullous; Pregnancy; Sensitivity and Specificity

The bullous pemphigoid antigen BPAG1 is required for keratin filament linkage to the hemidesmosome, an adhesion complex in epithelial basal cells. BPAG1 structural organization is similar to the intermediate filament-associated proteins desmoplakin I (DPI) and plectin. All three proteins have predicted dumbbell-like structure with central alpha-helical coiled-coil rod and regions of N- and C-terminal homology. To characterize the size of the N-terminal globular domain in BPAG1, two polypeptides spanning possible boundaries with the coiled-coil rod domain of BPAG1 were expressed in Escherichia coli. BP-1 (Mr = 111,000), containing amino acids 663-1581 of BPAG1 (Sawamura, D., Li, K., Chu, M.-L., and Uitto, J. (1991) J. Biol. Chem. 266, 17784-17790), and BP-1A, with a 186 amino acid N-terminal deletion, were purified. BP-1 and BP-1A behave as highly asymmetric dimers in aqueous solution according to velocity sedimentation and gel filtration. Both have globular heads with rod-like tails of roughly equal length, 55-60 nm, upon rotary shadowing. BP-1A content of alpha-helix, determined by circular dichroism, is approximately 90%, consistent with alpha-helical coiled-coil formation in the rod-like tails. The estimated rod length, 383 +/- 57 amino acids (0.15 nm/amino acid), implies that globular folding in the BPAG1 N-terminal extends to the end of N-terminal homology with DPI and plectin. These findings support the existence of a common domain structure in the N-terminal regions of the BPAG1/DPI/plectin family.

Topics: Amino Acid Sequence; Autoantigens; Carrier Proteins; Circular Dichroism; Cloning, Molecular; Collagen; Collagen Type XVII; Cytoskeletal Proteins; Desmoplakins; Dystonin; Epithelium; Humans; Intermediate Filament Proteins; Keratins; Microscopy, Electron; Molecular Sequence Data; Nerve Tissue Proteins; Non-Fibrillar Collagens; Pemphigoid, Bullous; Plectin; Protein Conformation; Protein Structure, Secondary; Recombinant Proteins; Sequence Homology, Amino Acid

We used colloidal gold (1-nm diameter) with silver enhancement, in conjunction with a low-temperature post-embedding immunolabeling technique, to localize several antigens in normal skin at both the light and the electron microscopic level within the same tissue blocks. Normal skin subjected to cyrofixation and cryosubstitution and embedded in Lowicryl K11M was used as a substrate. Semi-thin sections (1 micron) were incubated in primary antibody (against epidermal basement membrane zone associated antigens and two keratin sub-types), biotinylated secondary antibodies, and then in 1-nm gold-conjugated streptavidin. Finally, the 1-nm gold label was enhanced using silver staining. Labeling of both basement membrane and keratin antigens was well demonstrated, and the area in the semi-thin sections showing the best structural preservation and the greatest intensity of immunolabeling was used to identify the part of the block to be used for ultra-thin sectioning. Ultra-thin sections were treated using a similar procedure to that employed for semi-thin sections. The labeling with silver-enhanced 1-nm gold probes was intense and readily visible by electron microscopy, even at low magnification. We have found this technique to have a high degree of specificity and sensitivity for labeling both intra- and extracellular antigens in skin, with the added advantage of providing the means for studies at both light microscopic and electron microscopic level.

Topics: Antibodies; Antigens; Autoantigens; Basement Membrane; Carrier Proteins; Collagen; Collagen Type XVII; Colloids; Cytoskeletal Proteins; Dystonin; Gold; Immunohistochemistry; Keratins; Nerve Tissue Proteins; Non-Fibrillar Collagens; Pemphigoid, Bullous; Silver; Skin

Recent studies have demonstrated that bullous pemphigoid (BP) antigen, in extracts of cultured human keratinocytes and normal human epidermis, is a 230-kD polypeptide. However, other recent studies have suggested that there is heterogeneity of BP antigen at the molecular level. To demonstrate that this 230-kD polypeptide is the major BP antigen and to further characterize it, we tested multiple BP sera by both immunoprecipitation of extracts of radiolabeled cultured human keratinocytes and immunoblotting of extracts of normal human epidermis. Thirty-six of 37 BP sera immunoprecipitated the 230-kD polypeptide, and 11 of 13 BP sera identified the 230-kD polypeptide by immunoblotting. Eighty-six disease and normal control sera did not bind the 230-kD BP antigen. Immunoprecipitation was a more sensitive assay than was immunoblotting to detect the 230-kD antigen; BP sera that were weak or negative by immunoblotting were strongly positive by immunoprecipitation. To demonstrate that this 230-kD polypeptide shared epitopes, defined by BP sera, with the epidermal basement membrane zone, we showed that BP IgG affinity purified on this polypeptide bound the epidermal basement membrane zone by immunofluorescence. To further characterize the BP antigen and to compare the antigen synthesized by cultured human keratinocytes to the antigen extracted from epidermis, we analyzed the 230-kD BP antigen by two-dimensional gel electrophoresis. BP antigen immunoprecipitated from culture extracts or BP antigen identified by immunoblots of epidermal extracts was a single spot on two-dimensional gels, with a pI of about 8. These data indicate that, although other polypeptides are sometimes identified (e.g., a 166 kD band by immunoprecipitation or a 180 kD band on immunoblots), the major BP antigen identified in cultured human keratinocytes is similar to the antigen found in normal human epidermis and is a basic 230-kD polypeptide.

Topics: Autoantigens; Basement Membrane; Carrier Proteins; Collagen; Collagen Type XVII; Cytoskeletal Proteins; Dystonin; Electrophoresis; Epidermis; Humans; Immunoblotting; Keratins; Molecular Weight; Nerve Tissue Proteins; Non-Fibrillar Collagens; Pemphigoid, Bullous; Peptides; Precipitin Tests; Skin Diseases, Vesiculobullous

Topics: Animals; Cell Line; Epidermal Cells; Flow Cytometry; Histocompatibility Antigens Class II; Immunity, Cellular; Interferon-gamma; Keratins; Mice; Pemphigoid, Bullous; Pemphigus; Skin Diseases, Vesiculobullous; T-Lymphocytes

Human cell lines obtained after Epstein-Barr virus transformation of lymphocytes from seven patients with bullous diseases (Bullous pemphigoid, Pemphigus) and five controls were screened for the production of autoantibodies against skin antigens. In five out of seven patients, the culture supernatants tested by indirect immunofluorescence on frozen sections of normal human skin and rabbit lip showed the production of autoantibodies with different specificities: basal epidermal cells, whole epidermis, Merkel cells, fibroblasts endothelial cells, etc. All autoantibodies were of IgM class and reacted with intracellular structures. Some of them were further tested by immunoblotting against epidermal keratins and were found to react with the main human epidermal keratins (56 to 67 kDa). In contrast, even when patients had circulating autoantibodies, no supernatant showed any reactivity against the antigens usually involved in these diseases, i.e., the dermoepidermal junction or the intercellular spaces of epidermis. Supernatants from controls did not show any reactivity by immunofluorescence. The results demonstrated that human lymphoid cell lines obtained from patients with bullous diseases elicited the production of anti-intermediate filament autoantibodies known to occur spontaneously in normal patients. It is suggested that this phenomenon may be linked to the blistering conditions that provoke tissue destruction.

Topics: Animals; Antibodies, Monoclonal; Autoantibodies; B-Lymphocytes; Cell Transformation, Viral; Cells, Cultured; Fluorescent Antibody Technique; Herpesvirus 4, Human; Humans; Intermediate Filaments; Keratins; Molecular Weight; Pemphigoid, Bullous; Pemphigus; Rabbits; Skin Diseases, Vesiculobullous; Vimentin

The expression and properties of pemphigoid antigen of SV40-transformed human keratinocytes were studied. By indirect immunofluorescence, SV40-transformed keratinocytes in passage 80-85 expressed the pemphigoid antigen as coarsely granular perinuclear fluorescence. To characterize this antigen, NP40 extracts of cells labeled with [14C]amino acids were immunoprecipitated using sera of 8 patients: bullous pemphigoid (6 patients), chronic localized pemphigoid (1 patient), and drug-induced lichen planus pemphigoides (1 patient). These immunoprecipitates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then fluorographed. All 8 sera precipitated a protein of Mr 240K, while normal human sera did not precipitate this protein. These results indicate that SV40-transformed human keratinocytes synthesize pemphigoid antigen, and that autoantibodies in the sera of pemphigoid patients with different clinical features identify the same antigen of Mr 240K in these cells.

Topics: Antigen-Antibody Reactions; Antigens; Autoantigens; Cell Transformation, Viral; Epidermis; Fluorescent Antibody Technique; Humans; Infant, Newborn; Keratins; Lichen Planus; Pemphigoid, Bullous; Pemphigus; Precipitin Tests; Simian virus 40

Autoantibodies to a normal component of stratified squamous epithelia, the bullous pemphigoid antigen (BPA), are synthesized in patients with the disease bullous pemphigoid. We have used these sera to study the distribution of BPA in vivo and in vitro. At low magnification, indirect immunofluorescent staining for BPA is linear at the basement membrane zone (BMZ) of skin and many other epithelial tissues. At higher magnification however, we observed a punctate staining pattern for BPA which was regular in appearance and suggested localization of BPA to discrete structures at the BMZ. Subsequent immunoelectron microscopy using both peroxidase and colloidal gold labeling techniques with patients' sera or IgG, revealed that BPA is associated with hemidesmosomes--putative adhesion structures at the BMZ, based on their similarity in ultrastructure to desmosomes. More specifically BPA was immunolocalized to the cytoplasmic face of hemidesmosomes and was not observed extracellularly in the basement membrane. In stratifying and nonstratifying cultures of rat keratinocytes, BPA is expressed intracellularly and not in the cell-derived matrix, unlike other known basement membrane components. These cells also synthesize BPA in vitro, and immunoprecipitation from metabolically labeled cultures revealed a 220 kD polypeptide under reducing conditions. From these observations we conclude (1) that BPA is a 220 kD polypeptide component either of or associated with hemidesmosomes, and (2) that it is localized intracellularly both in vivo and in vitro. We suggest that BPA is not normally a lamina lucida component, but that it may form part of a linkage between the cytoskeleton and the basement membrane.

Topics: Animals; Antigens; Autoantigens; Basement Membrane; Carrier Proteins; Cells, Cultured; Chemical Precipitation; Collagen; Collagen Type XVII; Cytoskeletal Proteins; Desmosomes; Dystonin; Epidermis; Fluorescent Antibody Technique; Histocytochemistry; Humans; Immunologic Techniques; Keratins; Microscopy, Electron; Nerve Tissue Proteins; Non-Fibrillar Collagens; Pemphigoid, Bullous; Rats; Skin; Skin Diseases, Vesiculobullous

Vitamin A and its derivatives (retinoids) have both profound effects on epidermal differentiation and beneficial therapeutic effects in various dermatologic diseases. In order to understand these effects, much work has been done with cultured keratinocytes, which show specific morphologic, cellular, and biochemical changes modulated by retinoids. In an attempt to further define specific molecular effects of retinoids in cultured human keratinocytes, we studied the expression of pemphigus (P) and pemphigoid (BP) antigens by human keratinocytes cultured with retinoic acid (RA) in concentrations which modulated differentiation. Cultures of human keratinocytes in medium with 10% delipidized fetal bovine serum (vitamin A-depleted medium) demonstrated areas of extensive differentiation with flattened stratifying cells, keratohyaline granules, and an anucleate stratum corneum-like superficial layer. These cells also synthesized a 67 kd keratin, characteristic of well-differentiated epidermis. In contrast, cultures of human keratinocytes in the same medium supplemented with (10(-7) M, 3 X 10(-7) M, or 10(-6) M) RA demonstrated less differentiated small cuboidal cells that were stratified but did not form an anucleate layer or keratohyaline granules, and did not synthesize the 67 kd keratin. In order to detect P and BP antigens in these cultures, we used indirect immunofluorescence. In vitamin A-depleted cultures, P antigen either was not detected or was seen focally on the cell surface of basal cells. BP antigen was seen on the basal pole of the basal cells, approximating its in vivo location. In RA-treated cells, P antigen was seen on the cell surface of most of the cells, and BP antigen was seen throughout the cytoplasm of the basal cells. In order to study the expression of newly synthesized antigens, we radiolabeled cultures with 14C-amino acids and quantitatively immunoprecipitated the antigens, which were then identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We detected a major decrease in newly synthesized P antigen precipitated from extracts of vitamin A-depleted cells compared to RA-supplemented cells, whereas amounts of newly synthesized BP antigen were about the same. Taken together these data demonstrate that RA, at concentrations that decrease differentiation of cultured human keratinocytes, increases the expression of P antigen and changes the subcellular location of BP antigen.

Topics: Antigens; Cells, Cultured; Fluorescent Antibody Technique; Humans; Keratins; Pemphigoid, Bullous; Pemphigus; Skin; Skin Diseases, Vesiculobullous; Tretinoin; Vitamin A

The alterations of keratinocyte surface and basement membrane markers by treatment with 8-methoxypsoralen (8-MOP) plus long-wave ultraviolet (UVA) light (PUVA) were examined in guinea pig skin using immunofluorescence (IF) microscopy. Following a single PUVA treatment (UVA doses: 1.8 - 7.2 J/cm2), reactivity with pemphigus and pemphigoid sera, rabbit anti-guinea pig epidermal cell serum, and fluorescein-conjugated concanavalin A was decreased in intensity in a dose-dependent fashion. Fluorescence became distinctly less pronounced by day 2 postirradiation and recovered between days 6-10. IF with rabbit antiglomerular basement membrane antibodies, which cross-reacted with the basement membrane of the skin, apparently was unaltered within this energy dose range. 8-MOP or UVA alone did not change the reactivity with any reagent. These results suggest that PUVA treatment affects keratinocyte membrane markers and pemphigoid antigens but not some of the basement membrane antigens.

Topics: Animals; Basement Membrane; Epidermis; Fluorescent Antibody Technique; Guinea Pigs; Keratins; Male; Pemphigoid, Bullous; Photochemotherapy; PUVA Therapy; Skin Diseases, Vesiculobullous

Isolated adult human keratinocytes were grown either on plastic coverslips or a nonviable basement membrane surface containing intact laminin, type IV and V collagens, and heparan sulfate proteoglycan and examined by indirect immunofluorescence for the expression of bullous pemphigoid, pemphigus and Prlh antigens. Initial cell suspensions had a mean of 23% and 30%, respectively, of bullous pemphigoid and Prlh positive staining cells, while those stained with pemphigus serum were usually negative (19 of 22 series). Pemphigus antigen was expressed as intercellular staining between keratinocytes within 24 hr in both cultures on plastic and basement membrane. Likewise, Prlh antigen was expressed within 24 hr as a homogeneous cytoplasmic fluorescence leaving the basement membrane zone unstained. In contrast, pemphigoid antigen was expressed as a linear fluorescent band at the basement membrane zone between days 3 and 4 of culture. Systematic cell counts of bullous pemphigoid antigen positive cells from trypsin disrupted primary cultures made on plastic over time showed a nadir (8%) of positive cells in early cultures after which the percentage rapidly rose to a peak of 58% between days 14 and 21 of culture. In subcultures repeatedly disrupted at short intervals, the percentage of bullous pemphigoid positive cells remained low when compared to those interrupted and passaged over longer intervals. The percentage of bullous pemphigoid antigen bearing cells in culture over time is similar, but not identical, to the percentage of basal cells and is related to the age and known growth kinetics of the cultures system. Bullous pemphigoid, pemphigus and Prlh antigens are synthesized by the epidermal cell whether cultured on basement membrane or plastic.

Topics: Antigens; Autoantigens; Cell Count; Cells, Cultured; Fluorescent Antibody Technique; Humans; In Vitro Techniques; Keratins; Pemphigoid, Bullous; Pemphigus; Skin; Skin Diseases, Vesiculobullous; Time Factors

Topics: Antigens; Cells, Cultured; Epidermal Cells; Epidermis; Fibroblasts; Fluorescent Antibody Technique; Humans; Keratins; Pemphigoid, Bullous; Pemphigus; Skin Diseases, Vesiculobullous