bromochloroacetic-acid and Pemphigus

Autoantibodies binding to the extracellular domains of desmoglein (Dsg) 3 and 1 are critical in the pathogenesis of pemphigus by mechanisms leading to impaired function of desmosomes and blister formation in the epidermis and mucous membranes. Desmosomes are highly organized protein complexes which provide strong intercellular adhesion. Desmosomal cadherins such as Dsgs, proteins of the cadherin superfamily which interact

Topics: Cadherins; Desmogleins; Desmosomes; Humans; Keratinocytes; Keratins; Microscopy, Atomic Force; Pemphigus; Protein Domains

Desmosomes are the most important intercellular adhering junctions that adhere two adjacent keratinocytes directly with desmosomal cadherins, that is, desmogleins (Dsgs) and desmocollins, forming an epidermal sheet. Recently, two cell-cell adhesion states of desmosomes, that is, "stable hyper-adhesion" and "dynamic weak-adhesion" conditions have been recognized. They are mutually reversible through cell signaling events involving protein kinase C (PKC), Src and epidermal growth factor receptor (EGFR) during Ca(2+)-switching and wound healing. This remodeling is impaired in pemphigus vulgaris (PV, an autoimmune blistering disease), caused by anti-Dsg3 antibodies. The antibody binding to Dsg3 activates PKC, Src and EGFR, linked to generation of dynamic weak-adhesion desmosomes, followed by p38MAPK-mediated endocytosis of Dsg3, resulting in the specific depletion of Dsg3 from desmosomes and acantholysis. A variety of pemphigus outside-in signaling may explain different clinical (non-inflammatory, inflammatory, and necrolytic) types of pemphigus. Pemphigus could be referred to a "desmosome-remodeling disease involving pemphigus IgG-activated outside-in signaling events".

Topics: Animals; Desmosomes; Humans; Keratins; Models, Biological; Pemphigus; Signal Transduction

A clinically and genetically heterogeneous group of disorders, known collectively as the palmoplantar keratodermas, are unified by the phenotypic characteristic of a thickening of the skin over the palms and soles. Although spectacular progress has been made in understanding the basis of many genodermatoses, the genetic defects causing many of the keratodermas are still largely unknown. These unusual phenotypes are beginning to capture the attention of investigators in epidermal biology, and several compelling lines of evidence point to the cornified cell envelope and structural components of the desmosome as potential underlying targets of disease. It is anticipated that understanding the molecular basis of the keratodermas will underscore the importance of the integrity of the cell envelope and the desmosome, and provide new insights into the mechanisms of epidermal differentiation and related disorders.

Topics: Animals; Autoantibodies; Cell Differentiation; Cell Membrane; Desmosomes; Epidermal Cells; Esophageal Neoplasms; Genetic Linkage; Humans; Keratins; Keratoderma, Palmoplantar; Keratoderma, Palmoplantar, Diffuse; Keratosis; Membrane Proteins; Mice; Mice, Transgenic; Pemphigus; Protein Precursors

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

Topics: Adrenal Cortex Hormones; Animals; Antigen-Presenting Cells; Antigens, Surface; Autoimmune Diseases; Cells, Cultured; Complement System Proteins; Cytoplasmic Granules; Epidermal Cells; Epidermis; Epitopes; Humans; Immunosuppressive Agents; Keratins; Langerhans Cells; Major Histocompatibility Complex; Mice; Pemphigus; Research

Pemphigus vulgaris is a life-threatening blistering skin disease caused by autoantibodies destabilizing desmosomal adhesion. Current therapies focus on suppression of autoantibody formation and thus treatments directly stabilizing keratinocyte adhesion would fulfill an unmet medical need. We here demonstrate that apremilast, a phosphodiesterase 4 inhibitor used in psoriasis, prevents skin blistering in pemphigus vulgaris. Apremilast abrogates pemphigus autoantibody-induced loss of keratinocyte cohesion in ex-vivo human epidermis, cultured keratinocytes in vitro and in vivo in mice. In parallel, apremilast inhibits keratin retraction as well as desmosome splitting, induces phosphorylation of plakoglobin at serine 665 and desmoplakin assembly into desmosomal plaques. We established a plakoglobin phospho-deficient mouse model that reveals fragile epidermis with altered organization of keratin filaments and desmosomal cadherins. In keratinocytes derived from these mice, intercellular adhesion is impaired and not rescued by apremilast. These data identify an unreported mechanism of desmosome regulation and propose that apremilast stabilizes keratinocyte adhesion and is protective in pemphigus.

Topics: Animals; Autoantibodies; Blister; Cell Adhesion; Desmosomes; Epidermis; gamma Catenin; Humans; Keratinocytes; Keratins; Mice; Pemphigus

Pemphigus vulgaris (PV) is a potentially fatal autoimmune bullous disease primarily caused by acantholysis of keratinocytes attributed to pathogenic desmoglein-3 (Dsg3) autoantibodies. Interleukin-37 (IL-37) reportedly plays important roles in a variety of autoimmune diseases, but its role in PV is not clear.. To investigate whether IL-37 plays a role in the occurrence and progression of PV.. HaCaT keratinocytes were stimulated with anti-Dsg3 antibody to establish an in vitro PV model, which was defined as anti-Dsg3 group. Cells incubated with medium without anti-Dsg3 treatment were used as control. IL-37 was cultured with these cells infected with or without lentiviral vector shRNA-Caveolin-1 (sh-Cav-1-LV). Cell dissociation assay and immunocytofluorescence were performed to assess keratinocyte dissociation, keratin retraction and Dsg3 endocytosis. Real-time PCR was used to detect the mRNA level of Cav-1, and western blot was used to determine the protein expression of Cav-1, Dsg3, STAT3 and phosphorylated-STAT3 (p-STAT3).. The anti-Dsg3 group showed more cell debris, increased keratin retraction, increased Dsg3 endocytosis, reduced Cav-1 expression and co-localization than the control group, while IL-37 treatment neutralized all of these changes. Interestingly, Cav-1 knockdown supressed the inhibitory effect of IL-37 on keratinocyte dissociation and Dsg3 internalization. The protein expression of p-STAT3 was increased in keratinocytes of the PV model but decreased by IL-37. Re-activation of the STAT3 pathway by colivelin supressed the inhibitory effect of IL-37 on keratinocyte dissociation and Dsg3 internalization, along with upregulation of Cav-1 and Dsg3.. IL-37 inhibited keratinocyte dissociation and Dsg3 endocytosis in an in vitro PV model through the upregulating Cav-1 and inhibiting STAT3 pathway.

Topics: Autoantibodies; Caveolin 1; Desmoglein 3; Endocytosis; Humans; Interleukins; Keratinocytes; Keratins; Pemphigus; STAT3 Transcription Factor; Up-Regulation

Keratins are crucial for the anchorage of desmosomes. Severe alterations of keratin organization and detachment of filaments from the desmosomal plaque occur in the autoimmune dermatoses pemphigus vulgaris and pemphigus foliaceus (PF), which are mainly caused by autoantibodies against desmoglein (Dsg) 1 and 3. Keratin alterations are a structural hallmark in pemphigus pathogenesis and correlate with loss of intercellular adhesion. However, the significance for autoantibody-induced loss of intercellular adhesion is largely unknown. In wild-type (wt) murine keratinocytes, pemphigus autoantibodies induced keratin filament retraction. Under the same conditions, we used murine keratinocytes lacking all keratin filaments (KtyII k.o.) as a model system to dissect the role of keratins in pemphigus. KtyII k.o. cells show compromised intercellular adhesion without antibody (Ab) treatment, which was not impaired further by pathogenic pemphigus autoantibodies. Nevertheless, direct activation of p38MAPK

Topics: Animals; Anisomycin; Autoantibodies; Cell Adhesion; Cells, Cultured; Desmogleins; Humans; Keratinocytes; Keratins; Mice; Mice, Knockout; p38 Mitogen-Activated Protein Kinases; Pemphigus; Protein Binding; Signal Transduction

Topics: Autoantibodies; Autoantigens; Cell Adhesion; Cell Line; Desmoglein 1; Desmoglein 3; Endocytosis; Humans; Immunoglobulin G; Intermediate Filaments; Keratinocytes; Keratins; Pemphigus; Skin

Pemphigus is an autoimmune blistering skin disease caused primarily by autoantibodies against desmoglein (Dsg)1 and 3. Here, we characterized the mechanisms engaged by pemphigus IgG from patients with different clinical phenotypes and autoantibody profiles. All pemphigus vulgaris (PV) and pemphigus foliaceus (PF) IgG and AK23, a monoclonal mouse antibody against Dsg3, caused loss of cell cohesion, cytokeratin retraction and p38MAPK activation. Strong alterations in Dsg3 distribution were caused by mucosal (aDsg3 antibodies), mucocutaneous (aDsg1 + aDsg3) as well as atypical (aDsg3) PV-IgG. All PV-IgG fractions and AK23 compromised Dsg3 but not Dsg1 binding and enhanced Src activity. In contrast, rapid Ca

Topics: Animals; Antibodies, Monoclonal; Autoantibodies; Cell Adhesion; Desmoglein 1; Desmoglein 3; Humans; Immunoglobulin G; Keratinocytes; Keratins; Mice; Pemphigus; Signal Transduction

Plakoglobin (Pg) and desmoplakin (DP) are adapter proteins within the desmosome, providing a mechanical link between desmosomal cadherins as transmembrane adhesion molecules and the intermediate filament cytoskeleton. As in the severe skin blistering disease pemphigus, autoantibodies against desmosomal adhesion molecules induce loss of keratinocyte cohesion at least in part via p38 mitogen-activated protein kinase (p38MAPK) activation and depletion of desmosomal components, we evaluated the roles of Pg and DP in the p38MAPK-dependent loss of cell adhesion. Silencing of either Pg or DP reduced cohesion of cultured human keratinocytes in dissociation assays. However, Pg but not DP silencing caused activation of p38MAPK-dependent keratin filament collapse and cell dissociation. Interestingly, extranuclear but not nuclear Pg rescued loss of cell adhesion and keratin retraction. In line with this, Pg regulated the levels of the desmosomal adhesion molecule desmoglein 3 and tethered p38MAPK to desmosomal complexes. Our data demonstrate a role of extranuclear Pg in controlling cell adhesion via p38MAPK-dependent regulation of keratin filament organization.

Topics: Animals; Cell Adhesion; Cytoskeleton; Desmoplakins; Desmosomes; gamma Catenin; Gene Expression Regulation, Enzymologic; Gene Silencing; Green Fluorescent Proteins; Humans; Keratinocytes; Keratins; Lentivirus; MAP Kinase Signaling System; Mice; p38 Mitogen-Activated Protein Kinases; Pemphigus; RNA, Small Interfering

Desmoplakin (DP) serves to anchor intermediate filaments in desmosomal complexes. Recent data suggest that a specific DP point mutation (S2849G) exhibits increased keratin filament association and fosters Ca(2+) insensitivity of desmosomes in keratinocytes, presumably by rendering DP inaccessible for protein kinase C (PKC) phosphorylation. Previously, we have reported that depletion of the desmosomal adhesion molecule desmoglein (Dsg)3 induced by autoantibodies from patients with the blistering skin disease pemphigus vulgaris (PV) IgG is reduced in maturated desmosomes and dependent on PKC signaling. We investigated the role of DP-S2849G for loss of cell cohesion mediated by PV-IgG. In cell dissociation assays, expression of green fluorescent protein-tagged DP-S2849G (DP-S2849G-GFP) increased cell cohesion in two different human keratinocyte cell lines and ameliorated loss of cell adhesion induced by pemphigus autoantibodies. Depletion of Dsg3 was inhibited by DP-S2849G-GFP in the cytoskeletal (Triton X-100 insoluble) fraction, and keratin filament retraction, a hallmark of PV, was efficiently blocked similar to treatment with the PKC inhibitor Bim-X. We found that DP is phosphorylated after incubation with PV-IgG in a PKC-dependent manner and that DP-S2849G-GFP expression prevents DP phosphorylation and increases association of PKC-α with PKC scaffold receptor for activated C-kinase 1. Taken together, our data indicate that DP phosphorylation at S2849 represents an important mechanism in pemphigus pathogenesis, which, by reversing Ca(2+) insensitivity, promotes Dsg3 depletion.

Topics: Autoantibodies; Autoantigens; Blotting, Western; Cell Adhesion; Cell Line; Desmoglein 3; Desmoplakins; Fluorescent Antibody Technique; Humans; Immunoprecipitation; Keratins; Pemphigus; Point Mutation

The pemphigus family of autoimmune bullous disorders is characterized by autoantibody binding to desmoglein 1 and/or 3 (dsg1/dsg3). In this study we show that EGF receptor (EGFR) is activated following pemphigus vulgaris (PV) IgG treatment of primary human keratinocytes and that EGFR activation is downstream of p38 mitogen-activated protein kinase (p38). Inhibition of EGFR blocked PV IgG-triggered dsg3 endocytosis, keratin intermediate filament retraction, and loss of cell-cell adhesion in vitro. Significantly, inhibiting EGFR prevented PV IgG-induced blister formation in the passive transfer mouse model of pemphigus. These data demonstrate cross-talk between dsg3 and EGFR, that this cross-talk is regulated by p38, and that EGFR is a potential therapeutic target for pemphigus. Small-molecule inhibitors and monoclonal antibodies directed against EGFR are currently used to treat several types of solid tumors. This study provides the experimental rationale for investigating the use of EGFR inhibitors in pemphigus.

Topics: Acantholysis; Animals; Animals, Newborn; Cell Adhesion; Cells, Cultured; Desmogleins; Desmosomes; Detergents; Disease Models, Animal; ErbB Receptors; Female; Gene Expression Regulation; Humans; Immunoglobulin G; Keratinocytes; Keratins; Male; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Microscopy, Fluorescence; p38 Mitogen-Activated Protein Kinases; Pemphigus; RNA, Small Interfering; Signal Transduction

A novel canine epidermal keratinocyte cell line, MSCEK, was developed from skin of a healthy dog. The aim of this study was to determine its expression of desmosomal components and to evaluate its use as a detection tool for circulating autoantibodies in canine pemphigus. Immunofluorescence and western blotting analyses revealed that MSCEK expresses desmoglein (Dsg) 1, Dsg2, Dsg3, desmoplakin, plakoglobin and cytokeratins. Moreover, positive fluorescent reactions on the surface of MSCEK cells were observed when the cells were incubated with sera obtained from four dogs diagnosed with pemphigus complex. These findings indicate that MSCEK should be a useful tool for future research to characterize circulating autoantibodies that recognize desmosomal components in dogs with pemphigus.

Topics: Animals; Antibodies, Monoclonal; Autoantibodies; Biopsy; Cell Line; Desmosomes; Dog Diseases; Dogs; Epithelial Cells; Female; Keratinocytes; Keratins; Kidney; Male; Pemphigus; Phenotype; Skin

The autoimmune blistering skin disease pemphigus vulgaris (PV) is caused primarily by autoantibodies against desmosomal cadherins. It was reported that apoptosis can be detected in pemphigus skin lesions and that apoptosis can be induced by PV-IgG in cultured keratinocytes. However, the role of apoptosis in PV pathogenesis is unclear at present. In this study, we provide evidence that apoptosis is not required for acantholysis in PV. In skin lesions from two PV patients, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positivity, but not cleaved caspase-3, was detected in single keratinocytes in some lesions but was completely absent in other lesions from the same patients. In cultures of human keratinocytes (HaCaT and normal human epidermal keratinocytes), PV-IgG from three different PV patients caused acantholysis, fragmented staining of Dsg 3 staining, and cytokeratin retraction in the absence of nuclear fragmentation, TUNEL positivity, and caspase-3 cleavage and hence in the absence of detectable apoptosis. To further rule out the contribution of apoptotic mechanisms, we used two different approaches that are effective to block apoptosis induced by various stimuli. Inhibition of caspases by z-VAD-fmk as well as overexpression of Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory proteins FLIP(L) and FLIP(S) to inhibit receptor-mediated apoptosis did not block PV-IgG-induced effects, indicating that apoptosis was not required. Taken together, we conclude that apoptosis is not a prerequisite for skin blistering in PV but may occur secondary to acantholysis.

Topics: Acantholysis; Amino Acid Chloromethyl Ketones; Anoikis; Apoptosis; Biopsy; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 3; Cell Line; Cell Nucleus Shape; Cysteine Proteinase Inhibitors; Desmoglein 3; DNA Fragmentation; Humans; Immunoglobulin G; Immunohistochemistry; In Situ Nick-End Labeling; Keratinocytes; Keratins; Pemphigus; Transfection

Although it is accepted that pemphigus antibody binding to keratinocytes (KCs) evokes an array of intracellular biochemical events resulting in cell detachment and death, the triggering events remain obscure. It has been postulated that the binding of pemphigus vulgaris IgG (PVIgG) to KCs induces "desmosomal" signaling. Because in contrast to integrins and classical cadherins, desmoglein (Dsg) molecules are not known to elicit intracellular signaling, and because PV patients also produce non-Dsg autoantibodies, we investigated the roles of both Dsg and non-desmoglein PV antigens. The time course studies of KCs treated with PVIgG demonstrated that the activity of Src peaked at 30 min, EGF receptor kinase (EGFRK) at 60 min, and p38 MAPK at 240 min. The Src inhibitor PP2 decreased EGFRK and p38 activities by approximately 45 and 30%, respectively, indicating that in addition to Src, PVIgG evokes other triggering events. The shrinkage of KCs (cell volume reduction) became significant at 120 min, keratin aggregation at 240 min, and an increase of TUNEL positivity at 360 min. Pretreatment of KCs with PP2 blocked PVIgG-dependent cell shrinkage and keratin aggregation by approximately 50% and TUNEL positivity by approximately 25%. The p38 MAPK inhibitor PD169316 inhibited these effects by approximately 15, 20, and 70%, respectively. Transfection of KCs with small interfering RNAs that silenced expression of Dsg1 and/or Dsg3 proteins, blocked approximately 50% of p38 MAPK activity but did not significantly alter the PVIgG-dependent rise in Src and EGFRK activities. These results indicate that activation of p38 MAPK is a late signaling step associated with collapse of the cytoskeleton and disassembly of desmosomes caused by upstream events involving Src and EGFRK. Therefore, the early acantholytic events are triggered by non-Dsg antibodies.

Topics: Acantholysis; Antigens; Autoantibodies; Cell Adhesion; Cell Shape; Cells, Cultured; Cytoskeleton; Desmoglein 1; Desmoglein 2; Desmosomes; DNA Fragmentation; Enzyme Inhibitors; ErbB Receptors; Humans; Imidazoles; Keratinocytes; Keratins; MAP Kinase Signaling System; p38 Mitogen-Activated Protein Kinases; Pemphigus; Pyrimidines; src-Family Kinases; Time Factors

Topics: Animals; gamma Catenin; Humans; Intermediate Filament Proteins; Keratins; Mice; Pemphigus; Signal Transduction

Pemphigus vulgaris (PV) blistering occurs as a result of the disruption of intercellular contacts among keratinocytes, or acantholysis. The hallmark of PV acantholysis in vitro is considered to be the retraction of keratin intermediate filaments (KIF) onto the nucleus, which parallels with loss of cell-cell adhesion and rounding up of keratinocytes. However, the fine morphological changes of keratinocytes as well as the fate of cell adhesion structures cannot be appreciated on immunofluorescence by the simple cytokeratin staining. In this paper, we show that acantholytic dysmorphisms are sharply investigated by using PV IgG as a primary antibody on metabolically quiescent living cells. Indeed, PV IgG recognise a wide spectrum of molecules and enabled us to monitor the main changes occurring in acantholytic keratinocytes, including cell shrinkage with the appearance of prickle-like processes, detachment of keratinocytes from one another and collapse of cytoskeleton-bound proteins along nuclear periphery. This method has wider applications as it could be useful for staining cell periphery of keratinocytes and changes in cell shape. Furthermore, images displayed clear and sharp contours because living cell microscopy allows to avoid antigen distortion due to cell manipulation, which usually precedes the immunolabelling.

Topics: Acantholysis; Cell Line, Transformed; Cell Shape; Cell Size; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Immunoglobulin G; Intermediate Filaments; Keratinocytes; Keratins; Microscopy, Fluorescence; Pemphigus; Staining and Labeling; Time Factors

Desmosomes are adhesive intercellular junctions prominent in the skin and heart. Loss of desmosome function is associated with severe congenital and acquired disorders characterized by tissue fragility. Pemphigus vulgaris (PV) is an autoimmune disorder in which antibodies are directed against the desmosomal adhesion molecule Dsg3, resulting in severe mucosal erosions and epidermal blistering. To define the mechanisms by which Dsg3 autoantibodies disrupt keratinocyte adhesion, the fate of PV IgG and various desmosomal components was monitored in primary human keratinocytes exposed to PV patient IgG. PV IgG initially bound to keratinocyte cell surfaces and colocalized with desmosomal markers. Within 6 h after PV IgG binding to Dsg3, electron microscopy revealed that desmosomes were dramatically disrupted and keratinocyte adhesion was severely compromised. Immunofluorescence analysis indicated that PV IgG and Dsg3 were rapidly internalized from the cell surface in a complex with plakoglobin but not desmoplakin. Dsg3 internalization was associated with retraction of keratin filaments from cell-cell borders. Furthermore, the internalized PV IgG-Dsg3 complex colocalized with markers for both endosomes and lysosomes, suggesting that Dsg3 was targeted for degradation. Consistent with this possibility, biotinylation experiments demonstrated that soluble Dsg3 cell surface pools were rapidly depleted followed by loss of detergent-insoluble Dsg3. These findings demonstrate that Dsg3 endocytosis, keratin filament retraction, and the loss of keratinocyte cell-cell adhesion are coordinated responses to PV IgG.

Topics: Autoantibodies; Biotinylation; Blotting, Western; Cell Adhesion; Cell Line; Cell Membrane; Cytoskeletal Proteins; Desmoglein 3; Desmogleins; Desmosomes; Detergents; Endocytosis; gamma Catenin; Humans; Immunoglobulin G; Keratinocytes; Keratins; Lysosomes; Microscopy, Electron; Microscopy, Fluorescence; Microscopy, Immunoelectron; Models, Biological; Mucous Membrane; Myocardium; Pemphigus; Protein Binding; Skin; Streptavidin; Stress, Mechanical; Time Factors

In the human autoimmune blistering disease pemphigus vulgaris (PV) pathogenic antibodies bind the desmosomal cadherin desmoglein-3 (dsg3), causing epidermal cell-cell detachment (acantholysis). Pathogenic PV dsg3 autoantibodies were used to initiate desmosome signaling in human keratinocyte cell cultures. Heat shock protein 27 (HSP27) and p38MAPK were identified as proteins rapidly phosphorylated in response to PV IgG. Inhibition of p38MAPK activity prevented PV IgG-induced HSP27 phosphorylation, keratin filament retraction, and actin reorganization. These observations suggest that PV IgG binding to dsg3 activates desmosomal signal transduction cascades leading to (i) p38MAPK and HSP27 phosphorylation and (ii) cytoskeletal reorganization, supporting a mechanistic role for signaling in PV IgG-induced acantholysis. Targeting desmosome signaling via inhibition of p38MAPK and HSP27 phosphorylation may provide novel treatments for PV and other desmosome-associated blistering diseases.

Topics: Actins; Cells, Cultured; Cytoskeleton; Desmosomes; Electrophoresis, Gel, Two-Dimensional; Heat-Shock Proteins; Humans; Immunoglobulin G; Keratins; p38 Mitogen-Activated Protein Kinases; Pemphigus; Signal Transduction

Pemphigus vulgaris (PV) is an autoimmune blistering disease caused by IgG autoantibodies against desmoglein 3 (Dsg3). In this study, we characterized the ultrastructural localization of in vivo-bound IgG, Dsg3, and desmoplakin during the process of acantholysis in an active mouse PV model, using post-embedding immunoelectron microscopy. In non-acantholytic areas of keratinocyte contact, IgG labeling was restricted to the extracellular part of desmosomes, and was evenly distributed throughout the entire length of the desmosome. The distribution of in vivo IgG was similar to that of anti-Dsg3 labeling in the control mouse. Within the acantholytic areas, there were abundant split-desmosomes with keratin filaments inserted into the desmosomal attachment plaques. These split-desmosome extracellular regions were also decorated with anti-Dsg3 IgG and were associated with desmoplakin staining in their cytoplasmic attachment plaques. No apparent split-desmosomes, free of IgG-labeling were observed, suggesting that Dsg3 was not depleted from the desmosome before the start of acantholysis in vivo. Desmosome-like structures (without keratin insertion) were found only on the lateral surfaces of basal cells, but not on the apical surfaces at the site of acantholytic splits. These findings indicate that anti-Dsg3 IgG antibodies can directly access Dsg3 present in desmosomes in vivo and cause the subsequent desmosome separation that leads to blister formation in PV.

Topics: Acantholysis; Animals; Autoantibodies; Cadherins; Cytoskeletal Proteins; Desmoglein 3; Desmogleins; Desmoplakins; Desmosomes; Disease Models, Animal; Immunoglobulin G; Keratinocytes; Keratins; Mice; Mice, Mutant Strains; Microscopy, Immunoelectron; Pemphigus

The desmoglein 1 (Dsg1) and desmocollin 1 (Dsc1) isoforms of the desmosomal cadherins are expressed in the suprabasal layers of epidermis, whereas Dsg3 and Dsc3 are more strongly expressed basally. This differential expression may have a function in epidermal morphogenesis and/or may regulate the proliferation and differentiation of keratinocytes. To test this hypothesis, we changed the expression pattern by overexpressing human Dsg3 under the control of the keratin 1 (K1) promoter in the suprabasal epidermis of transgenic mice. From around 12 weeks of age, the mice exhibited flaking of the skin accompanied by epidermal pustules and thinning of the hair. Histological analysis of affected areas revealed acanthosis, hypergranulosis, hyperkeratosis, localized parakeratosis, and abnormal hair follicles. This phenotype has some features in common with human ichthyosiform diseases. Electron microscopy revealed a mild epidermal spongiosis. Suprabasally, desmosomes showed incorporation of the exogenous protein by immunogold labeling but were normal in structure. The epidermis was hyperproliferative, and differentiation was abnormal, demonstrated by expression of K14 in the suprabasal layer, restriction of K1, and strong induction of K6 and K16. The changes resembled those found in previous studies in which growth factors, cytokines, and integrins had been overexpressed in epidermis. Thus our data strongly support the view that Dsg3 contributes to the regulation of epidermal differentiation. Our results contrast markedly with those recently obtained by expressing Dsg3 in epidermis under the involucrin promoter. Possible reasons for this difference are considered in this paper.

Topics: Animals; Autoantigens; Cadherins; Cell Differentiation; Cell Division; Cytoskeletal Proteins; Desmocollins; Desmoglein 1; Desmoglein 3; Desmogleins; Desmoplakins; Desmosomes; Epidermis; Humans; Keratinocytes; Keratins; Mice; Mice, Transgenic; Pemphigus

The desmoglein compensation hypothesis, namely that one desmoglein can compensate for loss of function of another, has been proposed to explain the tissue specificity of the autoantibody-induced loss of cell adhesion in pemphigus. To validate this hypothesis genetically, we used desmoglein-3 knockout mice (DSG3-/-) that lose their telogen hair prematurely due to loss of adhesion between keratinocytes of the telogen hair club and the outer root sheath, where the only desmoglein expressed in normal mice is desmoglein-3. To determine if desmoglein-1 could substitute for the function of desmoglein-3 in telogen hair, we produced transgenic mice that express desmoglein-1 driven off the keratin 14 promoter, and then bred the transgene (TG) into DSG3-/- mice. Immunoblotting showed transgene expression in skin, and immunofluorescence showed desmoglein-1 in the telogen club of DSG3-/-TG+ but not DSG3-/-TG- mice. DSG3-/-TG- mice lost telogen hair with each wave of telogen, whereas DSG3-/-TG+ mice had markedly delayed and decreased hair loss. DSG3-/- mice also show low weights due to blisters in the oral mucosa. Surprisingly, DSG3-/-TG+ mice showed similar low weights, because the transgene, although expressed in skin, was not well expressed in oral mucous membranes. These studies show that desmoglein-1 can compensate for loss of desmoglein-3-mediated adhesion, and provide genetic evidence confirming the desmoglein compensation hypothesis.

Topics: Animals; Blister; Breeding; Cadherins; Cell Adhesion; Desmoglein 1; Desmoglein 3; Desmosomes; Female; Gene Expression; Hair Follicle; Keratin-14; Keratinocytes; Keratins; Mice; Mice, Inbred Strains; Mice, Knockout; Mouth Mucosa; Pemphigus; Phenotype; Pregnancy; Promoter Regions, Genetic; Transgenes; Weight Loss

Topics: Acantholysis; Actins; Cell Communication; Connexin 43; Desmosomes; Gap Junctions; Humans; Intercellular Junctions; Keratins; Pemphigus; Skin

In pemphigus vulgaris (PV), autoantibody binding to desmoglein (Dsg) 3 induces loss of intercellular adhesion in skin and mucous membranes. Two hypotheses are currently favored to explain the underlying molecular mechanisms: (a) disruption of adhesion through steric hindrance, and (b) interference of desmosomal cadherin-bound antibody with intracellular events, which we speculated to involve plakoglobin. To investigate the second hypothesis we established keratinocyte cultures from plakoglobin knockout (PG-/-) embryos and PG+/+ control mice. Although both cell types exhibited desmosomal cadherin-mediated adhesion during calcium-induced differentiation and bound PV immunoglobin (IgG) at their cell surface, only PG+/+ keratinocytes responded with keratin retraction and loss of adhesion. When full-length plakoglobin was reintroduced into PG-/- cells, responsiveness to PV IgG was restored. Moreover, in these cells like in PG+/+ keratinocytes, PV IgG binding severely affected the linear distribution of plakoglobin at the plasma membrane. Taken together, the establishment of an in vitro model using PG+/+ and PG-/- keratinocytes allowed us (a) to exclude the steric hindrance only hypothesis, and (b) to demonstrate for the first time that plakoglobin plays a central role in PV, a finding that will provide a novel direction for investigations of the molecular mechanisms leading to PV, and on the function of plakoglobin in differentiating keratinocytes.

Topics: Animals; Armadillo Domain Proteins; Autoantibodies; Cell Adhesion; Cell Differentiation; Cell Membrane; Cells, Cultured; Cytoskeletal Proteins; Desmogleins; Desmoplakins; Desmosomes; Drosophila Proteins; Fetus; gamma Catenin; Immunoglobulin G; Insect Proteins; Keratinocytes; Keratins; Mice; Mice, Knockout; Pemphigus; Protein Binding; Signal Transduction; Trans-Activators

To determine the assembly pathway of desmoglein 3 (Dsg3) into desmosomes and the subsequent effects of pemphigus vulgaris immunoglobulin G (PV-IgG) on such, we employed a time-lapsed labeling for FITC/Rhodamine (Rod) double-stained immunofluorescence and 5-nm/10-nm gold double-stained immunoelectron microscopy by using PV-IgG, which was confirmed to react specifically Dsg3. Cells from a human squamous cell carcinoma cell line (DJM-1) were first treated briefly with PV-IgG (3 min), then incubated in either anti-human IgG-FITC or 5-nm gold antibody-containing medium (5 min), followed by a 60-minute chase in normal medium without antibodies. The same cells were reincubated with PV-IgG medium for 3 minutes, followed by either anti-human IgG-Rod or 10-nm gold antibodies for 5 minutes. Using this method, FITC and 5-nm gold particles show the fate of Dsg3-PV-IgG complexes during the following 60-minute chase. IgG-Rod or 10-nm gold particles, which are bound during the last 5 minutes of the chase, show Dsg3 molecules newly expressed on the cell surface during the 60-minute-chase period. Initially, Dsg3 formed two types of small clusters on the nondesmosomal plasma membrane, ie, either half-desmosome-like clusters with keratin intermediate filament (KIF) attachment or simple clusters without KIF attachment. The PV-IgG binding to Dsg3 caused the internalization of the simple clusters into endosomes, but not the half-desmosome-like clusters. After the 60-minute-chase period, both types of cell surface Dsg3 clusters were labeled with only 10-nm gold, suggesting that new Dsg3 molecules were being delivered to the cell surface. Desmosomes were labeled with both 5-nm gold and 10-nm gold, whereas the half-desmosome-like clusters were labeled with only 10-nm gold, suggesting that the desmosomes themselves were not split. These results suggest that Dsg3 first forms simple clusters, followed by KIF-attachment, and then becomes integrated into desmosomes, and that PV-IgG-induced internalization of the nondesmosomal simple clusters of Dsg3 may represent the primary effects of PV-IgG on keratinocytes.

Topics: Autoantigens; Cadherins; Calcium; Desmoglein 3; Desmosomes; Humans; Immunoglobulin G; Keratinocytes; Keratins; Microscopy, Immunoelectron; Pemphigus; Tumor Cells, Cultured

Topics: Animals; Antigen-Antibody Reactions; Autoantibodies; Autoantigens; Autoimmune Diseases; Cadherins; Cell Adhesion; Cell Adhesion Molecules; Cytoskeletal Proteins; Desmoglein 3; Desmoplakins; Desmosomes; Humans; Keratins; Membrane Proteins; Paraneoplastic Syndromes; Pemphigus; Plakins; Protein Precursors

Topics: Actin Cytoskeleton; Desmosomes; Epidermolysis Bullosa; Humans; Intercellular Junctions; Keratins; Pemphigus; Skin

In order to clarify the molecular mechanism of blister formation in oral mucosa in pemphigus vulgaris (PV) comparing with that in epidermis, we analyzed the effects of PV serum on the distribution of keratin intermediate filaments (KIFs) and desmoplakins in oral as well as epidermal cultured keratinocytes by immunofluorescence microscopy using anti-keratin and anti-desmoplakin I/II monoclonal antibodies. After incubation with PV serum for 96 h at 37 degrees C, clusters of anti-keratin positive dots were formed around the nucleus in some of the keratinocytes from normal gingiva and soft palate but not in keratinocytes from tongue and skin, and desmoplakins also changed their distribution from linear arrangement at cell-cell contacts to clusters of dots around the nucleus in gingiva but not in epidermal keratinocytes. The dotted structures similar to those induced by pemphigus serum were formed also by incubation with human plasmin in gingival keratinocytes. However, no dot-formation of keratins was induced in these cells after incubation with trypsin. Furthermore, in epidermal keratinocytes, no keratin-dot formation was observed even after incubation with plasmin or trypsin. These results suggest that the dotted structures of KIFs caused by PV serum and plasmin might be a feature characteristic for the response of oral keratinocytes to PV serum and that there are some distinct differences in susceptibility to, and mode of, bulla formation between oral epithelium and epidermis.

Topics: Antibodies; Antibodies, Monoclonal; Cells, Cultured; Cytoskeletal Proteins; Desmoplakins; Epidermal Cells; Epidermis; Fibrinolysin; Fluorescent Antibody Technique; Humans; Intermediate Filaments; Keratinocytes; Keratins; Mouth Mucosa; Pemphigus; Trypsin

We have studied the immunohistochemical localization of desmosomal and cytoskeletal proteins in the epidermis of healthy individuals and patients with Hailey-Hailey's disease. It was found that the localization of desmosomal proteins (desmoplakin, desmoglein) and keratin filaments in the involved epidermis of patients with Hailey-Hailey's disease was almost same as that in the normal epidermis of healthy individuals. In contrast, the localization of actin filaments in the involved epidermis of patients with Hailey-Hailey's disease differed from that in the normal epidermis of healthy individuals.

Topics: Adult; Cytoskeletal Proteins; Desmosomes; Humans; Immunohistochemistry; Keratins; Microfilament Proteins; Middle Aged; Pemphigus; Skin

To investigate the mechanisms by which cutaneous plasminogen activator (PA) may be regulated, we have tested cultured keratinocytes for the presence of PA inhibitors. Using biosynthetic labeling experiments with 35S-methionine in conjunction with specific antibody precipitation, we have shown that human keratinocytes in culture synthesized and secreted both PA inhibitor 1 and PA inhibitor 2. PA inhibitor 1 was present in conditioned media in the inactive form, but it could be detected with reverse phase autography. PA inhibitor 2 was detected by its ability to form complexes with 125I-uPA. Potential therapeutic relevance for cutaneous PA inhibitor 2 was suggested in skin organ culture experiments which demonstrated that purified PA inhibitor 2 from human placenta was able to prevent the acantholytic changes induced by pemphigus IgG.

Topics: Acantholysis; Cells, Cultured; Culture Techniques; Epidermal Cells; Epidermis; Glycoproteins; Humans; Immunoglobulin G; Keratins; Pemphigus; Plasminogen Activators; Plasminogen Inactivators; Skin Diseases; Urokinase-Type Plasminogen Activator

The role of plasminogen activator (PA) in the pathogenesis of acantholysis in canine pemphigus vulgaris (PV) was evaluated using differentiated cultures of canine oral keratinocytes. Both the secreted and cell-associated PA activity in cultured canine keratinocytes were completely inhibited by specific anti-urokinase antibodies. Anti-tissue type PA antibodies did not inhibit either secreted or cell-associated PA activity. Immunoblots and fibrin zymography revealed a single 57,000 molecular weight urokinase-type PA in the conditioned media of the canine oral keratinocytes. Incubation of the differentiated cultures with PV Ig resulted in a significant increased in the levels of PA activity and both canine and human PV Ig were effective at inducing acantholysis typical of that seen in the clinical disease. The addition of urokinase inhibitor to the cultures treated with PV Ig prevented the development of acantholysis. These data strongly support the conclusion that PA is involved in acantholysis which is the cardinal feature of PV.

Topics: Acantholysis; Animals; Antibodies; Cells, Cultured; Dogs; Epidermis; Immunoglobulins; Keratins; Pemphigus; Plasminogen Activators; Plasminogen Inactivators; Urokinase-Type Plasminogen Activator

Pemphigus is an intraepidermal autoimmune blistering disease of humans caused by circulating IgGs. We have investigated the binding mode and the fate of bound antibodies from Pemphigus sera (P-IgG) on guinea pig keratinocytes in suspension in order to find clues to the loss of cell adhesion in vivo (acantholysis). Flow cytometry, following indirect immunofluorescent labeling of the keratinocytes, and dead cells' staining with ethidium bromide, demonstrated the specific surface binding of P-IgG onto living keratinocytes only. This was shown with several Pemphigus sera or purified P-IgG. This technique, used with various Pemphigus sera, showed that the specific binding is increased when the serum titer is higher, and "Km" values for P-IgG were roughly and inversely correlated to the titers. Upon saturation the same average number of Pemphigus IgG sites per cell were found for the sera of different patients. Analysis of the specific binding of [125I]-P-IgG onto Percoll-separated (living) keratinocytes showed the existence of two classes of sites: 2 x 10(6) sites/cell high-affinity sites (Kd = 1.5 x 10(-6) M total IgG) and 25 x 10(6) sites/cell low-affinity sites (Kd = 6 x 10(-5) M total IgG). Cell sorting and flow cytometry of individual cells allowed us to correlate the light-scattering signal, the RNA content, the size and morphology, and the P-IgG binding to the cells. The results indicated that P-IgG binding is homogeneous within the living keratinocytes and increases with cell size (cell maturity). Cell-sorter analysis of cells with membrane-bound P-IgG, coupled to direct determination of P-IgG released in the medium, revealed the fate of bound P-IgG: 40-60% of the P-IgGs were released in the medium within 30 minutes at 37 degrees C. This was accompanied and followed by a much slower, metabolic energy-dependent, internalization process of the membrane-bound P-IgG. The internalization has been confirmed by electron microscopy of bound P-IgG labeled with protein A-gold. Internalized IgGs were seen in the cells in coated membranous vesicles and other endocytic compartments. Similar behavior was also observed with two other membrane ligands: i.e., concanavalin A and multispecific rabbit "antisurface" antibodies.

Topics: Animals; Antigens, Differentiation; Cell Adhesion; Epidermal Cells; Epidermis; Flow Cytometry; Guinea Pigs; Humans; Immunoglobulin G; In Vitro Techniques; Keratins; Microscopy; Pemphigus; Receptors, Fc; Receptors, IgG

Ultrastructural localization of pemphigus vulgaris antigen-antibody complexes and their fate during acantholysis were studied in epidermal sheets obtained from the area surrounding the bullae and in acantholytic cells in blister fluid. The distribution of pemphigus vulgaris antibodies already bound to the keratinocytes in early acantholytic lesions was detected with ferritin-conjugated goat antihuman IgG. Ferritin particles were observed on the surface of keratinocytes with particular affinity for desmosomal structures. The acantholytic cells in the blister fluid bound only a small number of ferritin particles on their surface. During incubation at 37 degrees C, pemphigus vulgaris antigen-antibody complexes on the surface of separated desmosomes were internalized and recognized in cytoplasmic vesicles. Endocytosis of separated desmosomes also was observed in vivo when freshly obtained epidermal sheets were immediately processed for routine electron microscopic study. These findings suggest that pemphigus vulgaris antibodies are densely located on desmosomes and that the antigen-antibody complexes, together with other serum proteins on the keratinocyte surface, are internalized by a process of endocytosis.

Topics: Acantholysis; Binding Sites, Antibody; Epidermis; Humans; Keratins; Pemphigus; Skin Diseases

Using the Combi-ring-dish (CRD), a new culture device, organotypic cultures of human epidermal keratinocytes were grown on bovine eye lens capsules. In these highly differentiated cultures, typical suprabasal acantholysis was induced by pemphigus vulgaris antibodies. This in vitro pemphigus vulgaris model may be used to analyse keratinocyte-derived factors causing acantholysis in experimental pemphigus.

Topics: Acantholysis; Animals; Antibodies; Cattle; Cells, Cultured; Cytological Techniques; Epidermis; Humans; Keratins; Microscopy, Electron; Pemphigus

In both the endemic and sporadic forms of pemphigus foliaceus (PF), antiepidermal autoantibodies against desmoglein I are present. Desmoglein I is a highly insoluble 160-kD transmembrane glycoprotein of the desmosomal core. The detailed immunochemical characterization of the epitope(s) recognized by the PF autoantibodies is hampered by its large molecular weight and the insolubility of desmoglein I in nondenaturing buffers. This study was designed to identify alternative methods that could yield soluble immunoreactive PF antigen (Ag) from normal human epidermis. The presence of PF Ag in human epidermis and in its soluble or insoluble fractions was monitored by indirect immunofluorescence, immunoadsorption of PF sera, and immunoprecipitation of radiolabeled fractions. The PF Ag from trypsin-resistant, radiolabeled cell envelope preparations was cleaved by papain and immunoprecipitated by PF sera. A 50-kD peptide, isoelectric at pH 5.5-5.8, was immunoprecipitated by sera from all patients with endemic PF (n = 15) or idiopathic PF (n = 4), and by two of four pemphigus vulgaris sera, but by no control sera (n = 7). This study shows that a significant fraction of the PF Ag is insoluble, trypsin-resistant, and is associated with the cornified cell envelope fraction, but an Ag fragment can be obtained in a small molecular weight, soluble, and immunoreactive form by papain digestion. This 50-kD papain fragment is more amenable to detailed chemical and immunologic characterization than the native molecule.

Topics: Antigens; Cell Differentiation; Electrophoresis, Polyacrylamide Gel; Epidermal Cells; Epidermis; Humans; Keratins; Papain; Pemphigus; Peptide Fragments; Solubility; Trypsin

To gain preliminary data on the location of a 195 kD cell peripheral protein of epidermal keratinocytes recognized by monoclonal antibody AE11, immunofluorescence staining was carried out on cultured human epidermal cells. B11 antidesmosomal polyclonal antibody and AE3 antikeratin monoclonal antibody staining were used as comparative reference controls. Desmosomal antigens span the cell membrane and keratins constitute a family of cytoplasmic proteins. The cultured cells were systematically used either unfixed, fixed with 4% paraformaldehyde, or fixed with methanol at 4 degrees C. The former two preparations were designed to expose only the surface antigens, whereas the latter method permeated the membrane to allow cytoplasmic staining. Differences in staining patterns were observed in the basal layer as compared with the upper layers with all three antibodies. B11 antidesmosomal antibody staining was consistent in pattern after either paraformaldehyde or methanol fixation. In addition to the punctate staining along adjacent cell surfaces as shown in the basal layer, the surface planes of the upper cells exhibited parallel arrays of linear streaks, demonstrating the distribution of desmosomal proteins. Antikeratin staining by AE3 showed the typical filamentous staining in basal cells. However, a homogeneous patchy staining of suprabasal cells was observed. The presence of punctate surface staining using antikeratin antibody on paraformaldehyde fixed cells suggests leakage of keratin to the cell surface. AE11 showed stronger staining in the top cells on methanol treated cultures and a punctate surface staining of the cells fixed with paraformaldehyde. These observations provide useful preliminary information in localizing peripheral proteins in epidermal keratinocytes.

Topics: Cells, Cultured; Epidermis; Fluorescent Antibody Technique; Formaldehyde; Histological Techniques; Humans; Keratins; Methanol; Pemphigus; Polymers; Proteins

We have investigated expression of pemphigus vulgaris antigen(s) in cultured human keratinocytes induced by the addition of extracellular calcium. Cycloheximide (10(-4) M) inhibited pemphigus antigen expression and stratification but actinomycin D (2 micrograms/ml) had no effect. Tunicamycin, which inhibits dolichol pyrophosphate-mediated glycosylation of asparaginyl residues specifically, was used to study the role of glycosylation. When calcium switching was carried out in the presence of tunicamycin, human keratinocytes did not stratify, and the expression of pemphigus antigen was partially inhibited and limited to cell-cell contact areas. Analysis of biosynthetically labeled proteins showed that the synthesis of high-molecular-weight proteins was markedly reduced in the tunicamycin-treated cells. A reciprocal blocking test demonstrated that concanavalin A and wheat germ agglutinin receptor share an epitope with pemphigus vulgaris antigen(s). These results suggest that Ca++, newly synthesized protein, and N-asparaginyl glycosylation are required for normal pemphigus antigen expression and epidermal stratification in vitro. Pemphigus vulgaris antigen may have a highly glycosylated, high-molecular-weight protein chain with carbohydrates playing an important role in epidermal cell morphology, adhesion, and stability of cell surface antigens.

Topics: Anti-Bacterial Agents; Antigens; Calcium; Cells, Cultured; Cycloheximide; Dactinomycin; Epidermal Cells; Epidermis; Glycoproteins; Humans; Keratins; Lectins; Membrane Proteins; Pemphigus; Tunicamycin

This study was undertaken to determine if Brazilian Pemphigus foliaceus (BPF) autoantibodies will fix complement as do P. vulgaris (PV) autoantibodies. When sections of human skin were examined following indirect immunofluorescence (IF) staining, BPF autoantibodies reacted with the upper layers of epidermis, while PV autoantibodies were reactive with the lower layers. When tissue culture epidermal cells were used for indirect IF, BPF autoantibodies were not detected after plating until 24 h, while PV autoantibodies reacted within 18 h of plating. Using complement IF staining methods, BPF autoantibodies were found to fix C1q, C4 and C3 to whole skin sections in an intercellular pattern and to the surface of cultured keratinocytes. Although reactive with different epidermal cell surface antigens, autoantibodies in BPF will fix complement in a fashion similar to autoantibodies in PV.

Topics: Animals; Autoantibodies; Cells, Cultured; Complement Activating Enzymes; Complement C1; Complement C1q; Complement C3; Complement C4; Complement Fixation Tests; Epidermal Cells; Fluorescent Antibody Technique; Humans; Immunoglobulin G; Keratins; Mice; Mice, Inbred BALB C; Pemphigus; Skin

Pemphigus foliaceus is an uncommon dermatologic disorder occurring in several species and has been reported in horses during the past decade. An ultrastructural analysis of affected skin of horses presenting to our clinics has revealed early cytopathologic features of pemphigus-like disease, some of which closely resemble pemphigus foliaceus in the human, calve, and guinea pig. Prior to complete acantholysis and bullae formation, the intercellular spaces enlarged, but intercellular bridges and desmosomes remained intact. A novel finding was presence of aggregates of electron dense granular material which were seen in intercellular spaces of the epidermal basal cell layer, and may represent antigen-autoantibody complexed material or deranged cement substances. Other changes preceding acantholysis consisted of mild dyskeratosis, reduction of peripheral tonofilaments, enlargement of rough endoplasmic reticula, cytoplasmic vacuolization, and mitochondrial damage in epidermal cells. In more severe lesions where bullae were present and acantholysis was observed, bacterial invasion and leucocytic infiltration were evident in all epidermal layers, and corneal cells displayed cytoplasmic vacuolization and retention of nuclei. Basal cells remained intact, though intercellular spaces were enlarged on apical and lateral boundaries. The pathogenesis of this disease in the horse appeared morphologically similar to a pemphigus autoimmune disorder and its variants in other species, and morphologic evidence is provided to suggest that some cellular metabolic derangements may be concurrent with the extracellular events or cell peripheral changes that precede acantholysis and bullae formation.

Topics: Animals; Autoimmune Diseases; Corneal Diseases; Dermatitis; Epidermal Cells; Female; Horse Diseases; Horses; Keratins; Male; Pemphigus

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

Immunohistological study of dermal infiltrate from two pemphigus- and one captopril-induced lichenoid eruption showed the presence of a CD8 (T suppressor) infiltrate that resembled that found in skin eruptions induced by graft-versus-host disease. It is suggested that a T cell cytotoxic reaction occurs in the epidermis that might be triggered by captopril-induced modification of keratinocytic antigens.

Topics: Aged; Aged, 80 and over; Captopril; Drug Eruptions; Epidermis; Female; Fluorescent Antibody Technique; HLA-DR Antigens; Humans; Keratins; Lichen Planus; Male; Pemphigus; T-Lymphocytes, Regulatory; Time Factors

Pemphigus is an autoimmune blistering disease of epidermal cells in which autoantibodies to the surface develop. The present study was performed to determine whether the binding of pemphigus antibodies to the surface of keratinocytes can inhibit the regeneration of cell-cell contact induced by altering from low to normal Ca++ concentration medium. Human keratinocytes (a cell line of squamous cell carcinoma, DJM-1 cell) were grown in low Ca++ medium for 4 days, then the cells were incubated in normal Ca++ medium containing 10% pemphigus (4 patients with pemphigus vulgaris and 4 patients with pemphigus foliaceus) or normal serum (treated at 56 degrees C, for 30 min) for various incubation periods (2, 6, 12, 24 h). The cells were fixed and stained with antikeratin antibody by the indirect immunofluorescence method so that the detachment of cell-cell contact was able to be clearly visualized by observing the cytoskeletal arrays of keratin filaments. The cells grown in normal Ca++ medium showed detachments of cell-cell contact 24-36 h after addition of any one of the pemphigus sera used in this study. The cells grown in low Ca++ medium formed no cell-cell contacts and expressed no pemphigus antigens. However, re-formation of cell-cell contacts and reexpression of the antigens were confirmed by immunofluorescence microscopy 30 min after the addition of Ca++ to the medium. The addition of any pemphigus vulgaris and foliaceus sera with Ca++ did not inhibit the regeneration of cell-cell contact and exerted no effects on the contact during the subsequent 12 h. However, after 24 h, these cells again lost the contact. These results indicate that pemphigus antibody and antigen reaction on the cell surface did not directly inhibit the Ca++-induced re-formation of cell-cell contact.

Topics: Antibodies; Antigens, Surface; Calcium; Cell Communication; Cell Line; Epidermal Cells; Epidermis; Fluorescent Antibody Technique; Histocytochemistry; Humans; Intermediate Filaments; Keratins; Pemphigus

We have identified a novel IgG antikeratin autoantibody in the serum of a Brazilian pemphigus foliaceus patient (Cascas-42). This antibody is specific for the 59 kD acidic murine keratin and its 56.5 kD human counterpart (Moll's catalogue #10), and is distinct from the pemphigus antibody system. Antikeratin autoantibodies present in the Cascas-42 serum were purified by affinity chromatography with a 59 kD murine keratin-agarose column (IAP-Cascas-42 antibodies). The specificity of the IAP-Cascas-42 antibodies was tested by indirect immunofluorescence and immunoelectron microscopy against epidermal cryosections, trypsin-dissociated keratinocytes, and epidermal cell cultures. The serum was also tested with extracts from unlabeled and surface 125I-labeled keratinocytes (Iodo-Gen method) by immunoblot analysis of one- and two-dimensional polyacrylamide gel electrophoresis. The IAP-Cascas-42 antibodies bind the intercellular spaces of murine epidermis, and the cell surfaces of viable, dissociated murine keratinocytes, as well as murine epidermal cells in culture by immunofluorescence and immunoelectron microscopy. These autoantibodies did not stain cytoplasmic keratins and did not react with parallel human epidermal substrates. The Cascas-42 serum identified the 59 kD murine acidic keratin and its 56.5 kD human counterpart in epidermal extracts by two-dimensional polyacrylamide gel electrophoresis and immunoblot analysis. In addition, surface radioiodination of viable murine keratinocytes selectively labeled the 59 kD keratin suggesting that a domain of this molecule is exposed on the cell surface. The 125I-labeled 59 kD keratin was also recognized by the Cascas-42 serum by immunoblotting and autoradiography. These studies suggest that in murine epidermis, the 59 kD keratin is a transmembrane protein with an extracellular domain recognized by the IAP-Cascas-42 antibodies.

Topics: Animals; Autoantibodies; Dermatitis; Epidermal Cells; Epidermis; Extracellular Space; Fluorescent Antibody Technique; Humans; Immunologic Techniques; Iodine Radioisotopes; Keratins; Mice; Molecular Weight; Pemphigus

HLA-DR expression on human keratinocytes (KC) was induced in vivo by intradermal injection of purified protein derivative of tuberculin (PPD). Neither preceding nor subsequent exposure of the PPD injection site to a dose of approximately 2 MED of UVB radiation abolished KC HLA-DR, though subsequent irradiation caused a slight diminution in the intensity of the antigen expression. By contrast, epidermal Langerhans cell (LC) HLA-DR and T6 expressions in normal epidermis were greatly reduced by an identical dose of UVB. Pemphigus antigen on the surface of KC was not affected by irradiation or PPD injection.

Topics: Adult; Epidermal Cells; Epidermis; Female; Fluorescent Antibody Technique; HLA-D Antigens; HLA-DR Antigens; Humans; Keratins; Langerhans Cells; Male; Pemphigus; Ultraviolet Rays

The expression of HLA-DR and OKT6 antigens by epidermal cells in pemphigus was examined by immunohistochemical methods using skin biopsy specimens from human patients with pemphigus vulgaris. Changes in the distribution and subpopulations of Langerhans cells were observed in lesional (involved) skin. HLA-DR-positive, OKT6-negative dendritic cells were numerous and generally confined to the margins of epidermal clefts. OKT6-positive dendritic cells were rare or absent in lesional skin. The HLA-DR-positive, OKT6-negative dendritic cells associated with intraepidermal clefts are most likely functionally active antigen-presenting cells, which may play a role in lesion development. Aberrant expression of HLA-DR molecules by keratinocytes was observed in both involved and uninvolved skin, and may function to facilitate the recognition of surface-bound pemphigus antigen(s) by immunocompetent cells.

Topics: Antibodies, Monoclonal; Antigens, Surface; Epidermis; HLA-D Antigens; HLA-DR Antigens; Humans; Immunoenzyme Techniques; Keratins; Langerhans Cells; Pemphigus

It has been suggested that pemphigus antibodies (PA) react with the surface molecules on keratinocytes, and induce the production and release of proteases resulting in acantholysis. If this is the case, the immunoreactions on the cell surface may send signals to the interior of the cell across the membrane. The present study was carried out to determine whether or not cytoskeletons [microtubules (MT) and keratin-intermediate filaments (KIF)] respond to PA-immunoreactions in cultured human keratinocytes, by indirect immunofluorescence microscopy using anti-keratin and anti-alpha-tubulin antibodies. During incubation for 30 min to 72 h in a PA-containing medium with a normal concentration of Ca2+ (1.2 mM), no changes in MT or KIF organization were detected. Alterations in the organization of these filaments were observed 96 h after addition of PA. When cells grown in a normal medium for 5-7 days were transferred to a medium containing PA and a low level of Ca2+ (0.07-0.14 mM) the reorganization of KIFs and MTs occurred after 1 h incubation. However, no reorganization of the cytoskeletons was detected in the absence of cell detachment. These observations suggest that the pemphigus antibody-induced reorganization of MTs and KIFs does not precede acantholysis and is probably secondary to it, but is not a direct transmembrane response. The present study also showed that immunofluorescence microscopy of KIFs may be one of the most sensitive methods for detecting early cell-to-cell dissociation in cultured keratinocytes.

Topics: Autoantibodies; Calcium; Cells, Cultured; Epidermal Cells; Fluorescent Antibody Technique; Humans; Intermediate Filaments; Keratins; Microtubules; Pemphigus

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

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

Pemphigus is a human disease that causes extensive blistering of the skin. This blistering is related to a loss of epidermal cell cohesion and is accompanied by circulating autoantibodies that stain epidermal cell surfaces, as shown by immunofluorescence microscopy. One of the major components involved in epidermal cell cohesion is the desmosome. The pathological changes that accompany pemphigus led us to determine whether the autoantibodies are specific for desmosomes. Incubation of cultured mouse keratinocytes in medium containing pemphigus antiserum leads to cell separation at cell-cell contact sites, which possess desmosomes. Tissue sections of mouse skin processed for indirect immunofluorescence, using pemphigus antiserum or a rabbit antiserum directed against components of desmosomes, show similar punctate cell-surface staining patterns within the epidermis. Cultured mouse keratinocytes possessing well-defined intermediate filament bundles (tonofilaments) and desmosomes were processed for double indirect immunofluorescence, using a monoclonal antibody directed against mouse skin keratin and either pemphigus antiserum or the desmosome antiserum. The keratinocytes exhibit a complex system of keratin-containing tonofilaments. Tonofilaments in contacting cells are separated by thin dark bands at the cell surface, which correspond precisely to desmosomal plaques seen by phase-contrast microscopy. These bands specifically stain with both pemphigus antiserum and the desmosome antiserum. Double indirect immunofluorescence of the cultured mouse keratinocytes, using pemphigus antiserum and the desmosome antiserum, reveals that the pemphigus autoantibodies stain the same areas of cell-cell contact as the desmosome antibodies. Our evidence supports the idea that pemphigus blisters form, at least in part, from a specific antibody-induced disruption of desmosomes in the epidermis.

Topics: Animals; Autoantibodies; Autoimmune Diseases; Cells, Cultured; Desmosomes; Epidermis; Humans; Keratins; Mice; Pemphigus

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

Topics: Erythema Multiforme; Fluorescent Antibody Technique; Gingiva; Humans; Keratins; Lichen Planus; Mouth Diseases; Mouth Mucosa; Pemphigus; Skin

Topics: Cytoplasmic Granules; Darier Disease; Humans; Hyalin; Keratins; Pemphigus; Skin

Topics: Acantholysis; Carcinoma in Situ; Electrons; Humans; Keratins; Microscopy, Electron; Pemphigus