RESUMO
Despite the central role of IFN-γ in vitiligo pathogenesis, systemic IFN-γ neutralization is an impractical treatment option owing to strong immunosuppression. However, most patients with vitiligo present with <20% affected body surface area, which provides an opportunity for localized treatments that avoid systemic side effects. After identifying keratinocytes as key cells that amplify IFN-γ signaling during vitiligo, we hypothesized that tethering an IFN-γâneutralizing antibody to keratinocytes would limit antiâIFN-γ effects on the treated skin for the localized treatment. To that end, we developed a bispecific antibody capable of blocking IFN-γ signaling while binding to desmoglein expressed by keratinocytes. We characterized the effect of the bispecific antibody in vitro, ex vivo, and in a mouse model of vitiligo. Single-photon emission computed tomography/computed tomography biodistribution and serum assays after local footpad injection revealed that the bispecific antibody had improved skin retention, faster elimination from the blood, and less systemic IFN-γ inhibition than the nontethered version. Furthermore, the bispecific antibody conferred localized protection almost exclusively to the treated footpad during vitiligo, which was not possible by local injection of the nontethered antiâIFN-γ antibody. Thus, keratinocyte tethering proved effective while significantly diminishing the off-tissue effects of IFN-γ blockade, offering a safer treatment strategy for localized skin diseases, including vitiligo.
Assuntos
Produtos Biológicos , Vitiligo , Camundongos , Animais , Vitiligo/tratamento farmacológico , Distribuição Tecidual , Queratinócitos/metabolismo , Pele/patologia , Produtos Biológicos/uso terapêuticoRESUMO
Desmosomal cadherins are the pathophysiologic targets of autoimmune or toxin-mediated disruption in the human diseases pemphigus and bullous impetigo (including its generalized form, called staphylococcal scalded skin syndrome). Experiments exploiting the production of both pathogenic and nonpathogenic antidesmoglein antibodies in pemphigus patients' sera have afforded data that make an invaluable contribution towards identifying the functional domains of the desmogleins involved in intercellular adhesion. Conformational epitopes of antidesmoglein autoantibodies in pemphigus patients' sera and the specific cleavage site of desmoglein 1 by exfoliative toxin have been identified, implicating the N-terminal extracellular domains of the desmogleins as critical regions for controlling intercellular adhesion. Furthermore, the development of active autoimmune mouse models for pemphigus allows in vivo characterization of the disease and its pathogenesis. These studies offer new insight into the potential mechanisms of acantholysis in pemphigus and staphylococcal-associated blistering disease, with implications for the role of desmogleins in desmosomal structure and function.
Assuntos
Acantólise/fisiopatologia , Anticorpos/metabolismo , Caderinas/metabolismo , Desmossomos/fisiologia , Impetigo/metabolismo , Pênfigo/metabolismo , Acantólise/metabolismo , Anticorpos/imunologia , Adesão Celular/fisiologia , Epitopos/metabolismo , Humanos , Impetigo/fisiopatologia , Modelos Biológicos , Pênfigo/fisiopatologia , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
In pemphigus foliaceus (PF), autoantibodies against desmoglein 1 (Dsg1) cause blisters. Using Ab phage display, we have cloned mAbs from a PF patient. These mAbs, like those from a previous patient, were directed against mature Dsg1 (matDsg1) on the cell surface of keratinocytes and precursor Dsg1 (preDsg1) in the cytoplasm. To determine whether individuals without pemphigus have B cell tolerance to Dsg1, we cloned mAbs from two patients with thrombotic thrombocytopenic purpura and a healthy person. We found mAbs against preDsg1, but not matDsg1. All but 1 of the 23 anti-preDsg1 mAbs from PF patients and those without PF used the VH3-09 (or closely related VH3-20) H chain gene, whereas no PF anti-matDsg1 used these genes. V(H) cDNA encoding anti-preDsg1 had significantly fewer somatic mutations than did anti-matDsg1 cDNA, consistent with chronic Ag-driven hypermutation of the latter compared with the former. These data indicate that individuals without PF do not have B cell tolerance to preDsg1 and that loss of tolerance to matDsg1 is not due to epitope shifting of anti-preDsg1 B cells (because of different V(H) gene usage). However, presentation of peptides from Dsg1 by preDsg1-specific B cells may be one step in developing autoimmunity in PF.
Assuntos
Autoanticorpos/biossíntese , Desmogleína 1/imunologia , Proteínas de Membrana/isolamento & purificação , Pênfigo/imunologia , Pênfigo/metabolismo , Precursores de Proteínas/imunologia , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Especificidade de Anticorpos , Autoanticorpos/genética , Clonagem Molecular , Desmogleína 1/isolamento & purificação , Humanos , Tolerância Imunológica , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Pênfigo/patologia , Precursores de Proteínas/antagonistas & inibidores , Precursores de Proteínas/isolamento & purificação , Púrpura Trombocitopênica Trombótica/imunologia , Púrpura Trombocitopênica Trombótica/metabolismoRESUMO
For many years, The Journal of Investigative Dermatology (JID) has been a leader in our understanding of many aspects of the major autoimmune blistering skin diseases, pemphigus and bullous pemphigoid. The purpose of this review is to highlight and summarize those advances by discussing the respective articles, published in the JID from 2015 to 2019. Seminal articles from elsewhere in the literature that set the stage for those advances, or that are "classics" in the area, are also included to provide context and a more complete picture.
Assuntos
Autoanticorpos/imunologia , Autoimunidade , Pênfigo/imunologia , Humanos , Penfigoide Bolhoso/imunologiaRESUMO
Anti-desmoglein (Dsg) 1 and Dsg3 IgG autoantibodies in pemphigus foliaceus and pemphigus vulgaris cause blisters through loss of desmosomal adhesion. It is controversial whether blister formation is due to direct inhibition of Dsg, intracellular signaling events causing desmosome destabilization, or both. Recent studies show that heterophilic binding between Dsg and desmocollin (Dsc) is the fundamental adhesive unit of desmosomes. To eliminate cellular contributions to potential pathogenicity of pemphigus antibodies, bead assays coated with recombinant Dsg1, Dsc1, Dsg3, or Dsc3 ectodomains were developed. A mixture of Dsg beads and Dsc beads formed large aggregates, confirming that the heterophilic binding is dominant. The pathogenic anti-Dsg1 and anti-Dsg3 mAbs, which bind the transadhesive interface, blocked the aggregation of Dsg1/Dsc1 and Dsg3/Dsc3 beads, respectively, whereas nonpathogenic mAbs did not. All sera tested from eight patients with pemphigus foliaceus and eight patients with mucosal pemphigus vulgaris with active disease inhibited the adhesion of Dsg1/Dsc1 and Dsg3/Dsc3 beads, respectively. When paired sera obtained from seven patients with pemphigus foliaceus and six patients with pemphigus vulgaris in active disease and remission were compared, the former inhibited aggregation better than the latter. These findings strongly suggest that steric hindrance of heterophilic transinteraction between Dsg and Dsc is important for disease pathology in both pemphigus foliaceus and pemphigus vulgaris.
Assuntos
Autoanticorpos/imunologia , Desmocolinas/imunologia , Desmogleína 1/imunologia , Desmogleína 3/imunologia , Imunoglobulina G/imunologia , Pênfigo/imunologia , Adesão Celular , Desmocolinas/antagonistas & inibidores , Desmocolinas/fisiologia , Desmogleína 1/antagonistas & inibidores , Desmogleína 1/fisiologia , Desmogleína 3/antagonistas & inibidores , Desmogleína 3/fisiologia , Mapeamento de Epitopos , HumanosRESUMO
Autoantibodies in pemphigus foliaceus (PF) and vulgaris (PV) bind to desmoglein (Dsg) 1 and 3, respectively, and cause loss of keratinocyte adhesion. To characterize the pathogenicity and genetics of such antibodies we have used phage display to isolate monoclonal antibodies (mAbs) from patients. PCR is used to clone the heavy and light chain variable region of the peripheral B cells into a vector that creates a phage particle with the antibody expressed on its surface and the cDNA encoding that antibody inside. The library of phage produced from a PF or PV patient are then panned on a plate containing Dsg1 or Dsg3 to isolate clones. The cDNA of each clone is sequenced to characterize the genetics of the expressed mAb. The mAb from each unique clone is tested for pathogenicity either by injecting into normal human skin organ culture or into neonatal mice. Pathogenic antibodies cause typical pemphigus blisters. In both PV and PF patients the heavy chain (VH) genes used for Dsg-binding antibodies are severely restricted. PV and PF patients have both pathogenic and non-pathogenic mAbs. The immunochemical characteristics of the antibodies (including pathogenicity) sort with the VH, not the VL, gene. These monoclonal pathogenic antibodies can be used to screen peptide libraries to find short peptides that block antibody binding. In summary, the antibody response is restricted and, therefore, it may be feasible to target the specific pathogenic antibodies for therapy.
Assuntos
Anticorpos Monoclonais/imunologia , Clonagem Molecular/métodos , Pênfigo/imunologia , Desmogleínas/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina , Biblioteca de Peptídeos , Ligação ProteicaRESUMO
Pemphigus is a life-threatening blistering disorder of the skin and mucous membranes caused by pathogenic autoantibodies to desmosomal adhesion proteins desmoglein 3 (Dsg3) and Dsg1. Mechanisms of antibody pathogenicity are difficult to characterize using polyclonal patient sera. Using antibody phage display, we have isolated repertoires of human anti-Dsg mAbs as single-chain variable-region fragments (scFvs) from a patient with active mucocutaneous pemphigus vulgaris. ScFv mAbs demonstrated binding to Dsg3 or Dsg1 alone, or both Dsg3 and Dsg1. Inhibition ELISA showed that the epitopes defined by these scFvs are blocked by autoantibodies from multiple pemphigus patients. Injection of scFvs into neonatal mice identified 2 pathogenic scFvs that caused blisters histologically similar to those observed in pemphigus patients. Similarly, these 2 scFvs, but not others, induced cell sheet dissociation of cultured human keratinocytes, indicating that both pathogenic and nonpathogenic antibodies were isolated. Genetic analysis of these mAbs showed restricted patterns of heavy and light chain gene usage, which were distinct for scFvs with different desmoglein-binding specificities. Detailed characterization of these pemphigus mAbs should lead to a better understanding of the immunopathogenesis of disease and to more specifically targeted therapeutic approaches.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Autoanticorpos/genética , Autoanticorpos/imunologia , Pênfigo/imunologia , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Caderinas/imunologia , Células Cultivadas , Regiões Determinantes de Complementaridade/genética , Desmogleína 3 , Células Epidérmicas , Epiderme/metabolismo , Mapeamento de Epitopos , Humanos , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Distribuição Aleatória , Alinhamento de SequênciaRESUMO
Identifying previously unknown proteins or detecting the presence of known proteins in research samples is critical to many experiments conducted in life sciences, including dermatology. Sensitive protein detection can help elucidate new intervention targets and mechanisms of disease, such as in autoimmune blistering skin diseases, atopic eczema, or other conditions. Historically, peptides from highly purified single proteins were sequenced, with many limitations, by stepwise degradation from the N-terminus to the C-terminus with subsequent identification by UV absorbance spectroscopy of the released amino acids (i.e., Edman degradation). Recently, however, the availability of comprehensive protein databases from different species (derived from high-throughput next-generation sequencing of those organisms' genomes) and sophisticated bioinformatics analysis tools have facilitated the development and use of mass spectrometry for identification and global analysis of proteins, summarized as mass spectrometry-based proteomics. Mass spectrometry is an analytical technique measuring the mass (m)-to-charge (z) ratio of ionized biological molecules such as peptides. Proteins can be identified by correlating peptide-derived experimental mass spectrometry spectra with theoretical spectra predicted from protein databases. Here we briefly describe how this technique works, how it can be used for identification of proteins, and how this knowledge can be applied in elucidating human biology and disease.
Assuntos
Pesquisa Biomédica/métodos , Dermatologia/métodos , Espectrometria de Massas/métodos , Peptídeos/análise , Dermatopatias/patologia , Humanos , Peptídeos/metabolismo , Proteômica/métodos , Projetos de PesquisaRESUMO
Bullous impetigo due to Staphylococcus aureus is one of the most common bacterial infections of man, and its generalized form, staphylococcal scalded skin syndrome (SSSS), is a frequent manifestation of staphylococcal epidemics in neonatal nurseries. Both diseases are mediated by exfoliative toxins (ETs), which show exquisite pathologic specificity in blistering only the superficial epidermis. We show that these toxins act as serine proteases with extremely focused molecular specificity to cleave mouse and human desmoglein 1 (Dsg1) once after glutamic acid residue 381 between extracellular domains 3 and 4. Mutation of the predicted catalytically active serine to alanine completely inhibits cleavage. The mutated ETs bind specifically to Dsg1 by immunofluorescence colocalization and by coimmunoprecipitation. Thus, ETs, through specific recognition and proteolytic cleavage of one structurally critical peptide bond in an adhesion molecule, cause its dysfunction and allow S. aureus to spread under the stratum corneum, the main barrier of the skin, explaining how, although they circulate through the entire body in SSSS, they cause pathology only in the superficial epidermis.
Assuntos
Vesícula/etiologia , Impetigo/etiologia , Síndrome da Pele Escaldada Estafilocócica/etiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Caderinas/química , Caderinas/genética , Caderinas/metabolismo , Desmogleína 1 , Exfoliatinas/genética , Exfoliatinas/metabolismo , Exfoliatinas/toxicidade , Células HeLa , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Pemphigus foliaceus (PF) is an autoimmune blistering disease caused by autoantibodies (Abs) against desmoglein 1 (Dsg1). PF sera contain polyclonal Abs which are heterogeneous mixture of both pathogenic and non-pathogenic Abs, as shown by isolation of monoclonal Abs (mAbs). OBJECTIVE: To investigate how pathogenic and non-pathogenic anti-Dsg1 Abs contribute to blister formation in PF. METHODS: Using organ-cultured human skin, we compared the effect of a single pathogenic anti-Dsg1 IgG mAb, a single non-pathogenic anti-Dsg1 IgG mAb, and their mixture on blister formation as analyzed by histology, subcellular localization of IgG deposits and desmosomal proteins by confocal microscopy, and desmosomal structure by electron microscopy. In addition, we measured keratinocyte adhesion by an in vitro dissociation assay. RESULTS: 24h after injection, a single pathogenic anti-Dsg1 IgG caused a subcorneal blister with IgG and Dsg1 localized linearly on the cell surface of keratinocytes. A single non-pathogenic anti-Dsg1 IgG bound linearly on the keratinocytes but did not induce blisters. A pathogenic and a non-pathogenic IgG mAb injected together caused an aberrant granular pattern of IgG and Dsg1 in the lower epidermis with blister formation in the superficial epidermis. Electron microscopy demonstrated that the mixture of mAbs shortened desmosomal lengths more than a single mAb in the basal and spinous layers. Furthermore, although Dsg1 clustering required both cross-linking of Dsg1 molecules by the non-pathogenic IgG plus a pathogenic antibody, the latter could be in the form of a monovalent single chain variable fragment, suggesting that loss of trans-interaction of Dsg1 is required for clustering. Finally, a p38MAPK inhibitor blocked Dsg1 clustering. When pathogenic strength was measured by the dissociation assay, a mixture of pathogenic and non-pathogenic IgG mAbs disrupted keratinocyte adhesion more than a single pathogenic mAb. This pathogenic effect was only partially suppressed by the p38MAPK inhibitor. CONCLUSION: These findings indicate that a polyclonal mixture of anti-Dsg1 IgG antibodies enhances pathogenic activity for blister formation associated with p38MAPK-dependent Dsg1 clustering and that not only pathogenic antibodies but also non-pathogenic antibodies coordinately contribute to blister formation in PF.
Assuntos
Autoanticorpos/imunologia , Desmogleína 1/imunologia , Imunoglobulina G/imunologia , Queratinócitos/fisiologia , Pênfigo/imunologia , Anticorpos de Cadeia Única/imunologia , Pele/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adesão Celular , Desmogleína 1/metabolismo , Desmossomos/ultraestrutura , Imunofluorescência , Humanos , Imidazóis/farmacologia , Queratinócitos/imunologia , Microscopia Eletrônica , Técnicas de Cultura de Órgãos , Pênfigo/sangue , Cultura Primária de Células , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
In autoantibody-mediated diseases such as pemphigus, serum antibodies lead to disease. Genetic analysis of B cells has allowed characterization of antibody repertoires in such diseases but would be complemented by proteomic analysis of serum autoantibodies. Here, we show using proteomic analysis that the serum autoantibody repertoire in pemphigus is much more polyclonal than that found by genetic studies of B cells. In addition, many B cells encode pemphigus autoantibodies that are not secreted into the serum. Heavy chain variable gene usage of serum autoantibodies is not shared among patients, implying targeting of the coded proteins will not be a useful therapeutic strategy. Analysis of autoantibodies in individual patients over several years indicates that many antibody clones persist but the proportion of each changes. These studies indicate a dynamic and diverse autoantibody response not revealed by genetic studies and explain why similar overall autoantibody titers may give variable disease activity.
Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Pênfigo/genética , Pênfigo/imunologia , Proteômica/métodos , Sequência de Aminoácidos , Técnicas de Visualização da Superfície Celular , Cromatografia Líquida , Células Clonais , Regiões Determinantes de Complementaridade/genética , Desmogleínas/metabolismo , Humanos , Mutação/genética , Pênfigo/sangue , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Espectrometria de Massas em TandemRESUMO
Pemphigus and bullous pemphigoid are autoantibody-mediated blistering skin diseases. In pemphigus, keratinocytes in epidermis and mucous membranes lose cell-cell adhesion, and in pemphigoid, the basal keratinocytes lose adhesion to the basement membrane. Pemphigus lesions are mediated directly by the autoantibodies, whereas the autoantibodies in pemphigoid fix complement and mediate inflammation. In both diseases, the autoantigens have been cloned and characterized; pemphigus antigens are desmogleins (cell adhesion molecules in desmosomes), and pemphigoid antigens are found in hemidesmosomes (which mediate adhesion to the basement membrane). This knowledge has enabled diagnostic testing for these diseases by enzyme-linked immunosorbent assays and dissection of various pathophysiological mechanisms, including direct inhibition of cell adhesion, antibody-induced internalization of antigen, and cell signaling. Understanding these mechanisms of disease has led to rational targeted therapeutic strategies.
Assuntos
Penfigoide Bolhoso/imunologia , Penfigoide Bolhoso/patologia , Pênfigo/imunologia , Pênfigo/patologia , Animais , HumanosRESUMO
Pemphigus vulgaris (PV) is a prototypic tissue-specific autoantibody-mediated disease, in which anti-desmoglein 3 (Dsg3) IgG autoantibodies cause life-threatening blistering. We characterized the autoimmune B-cell response over 14 patient years in two patients with active and relapsing disease, then in one of these patients after long-term remission induced by multiple courses of rituximab (anti-CD20 antibody). Characterization of the anti-Dsg3 IgG(+) repertoire by antibody phage display (APD) and PCR indicated that six clonal lines persisted in patient 1 (PV3) over 5.5 years, with only one new clone detected. Six clonal lines persisted in patient 2 (PV1) for 4 years, of which five persisted for another 4.5 years without any new clones detected. However, after long-term clinical and serologic remission, â¼11 years after initial characterization, we could no longer detect any anti-Dsg3 clones in PV1 by APD. Similarly, in another PV patient, â¼4.5 years after a course of rituximab that induced long-term remission, anti-Dsg3 B-cell clones were undetectable. These data suggest that in PV a given set of non-tolerant B-cell lineages causes autoimmune diseases and that new sets do not frequently or continually escape tolerance. Therapy such as rituximab, aimed at eliminating these aberrant sets of lineages, may be effective for disease because new ones are unlikely to develop.
Assuntos
Envelhecimento/patologia , Autoanticorpos/metabolismo , Linfócitos B/imunologia , Linfócitos B/patologia , Desmogleína 3/imunologia , Imunoglobulina G/imunologia , Pênfigo/patologia , Adulto , Envelhecimento/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais Murinos/uso terapêutico , Linhagem da Célula , Relação Dose-Resposta a Droga , Feminino , Humanos , Fatores Imunológicos/uso terapêutico , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Pênfigo/tratamento farmacológico , Pênfigo/imunologia , Indução de Remissão , Rituximab , Resultado do TratamentoRESUMO
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.
Assuntos
Caderinas/genética , Queratinócitos/citologia , Pênfigo/genética , Pênfigo/patologia , Animais , Vesícula/patologia , Cruzamento , Adesão Celular/fisiologia , Desmogleína 1 , Desmogleína 3 , Desmossomos/patologia , Feminino , Expressão Gênica/fisiologia , Folículo Piloso/citologia , Queratina-14 , Queratinas/genética , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Mucosa Bucal/patologia , Fenótipo , Gravidez , Regiões Promotoras Genéticas/fisiologia , Transgenes/fisiologia , Redução de Peso/genéticaRESUMO
Within stratified squamous epithelia, such as the epidermis, desmogleins are generally expressed in a differentiation-specific manner. Similar to the epidermis, the hair follicle is compartmentalized into a hierarchy of cell types based on their level of differentiation. Relatively undifferentiated stem cells in the bulge can generate epidermis, sebaceous gland, and hair bulb matrix cells. The latter give rise to at least six different cell types that keratinize as they move up the hair shaft and inner root sheath. Here, we examined expression patterns of the desmoglein isotypes, desmogleins 1, 2, and 3 in the cutaneous epithelium, and discovered that desmoglein 1 and 2 expression correlated with the state of differentiation of defined populations within the hair follicle. Desmoglein 2 was highly expressed by the least differentiated cells of the cutaneous epithelium, including the hair follicle bulge of the fetus and adult, bulb matrix cells, and basal layer of the outer root sheath. In contrast, desmoglein 1 defined more differentiated cell populations, and was expressed in epidermal suprabasal cells, the inner root sheath, and the innermost layers of the outer root sheath. We found that the expression pattern of desmoglein 3 correlated with different types of keratinization. In areas of trichilemmal keratinization in the follicle, and in cysts arising from these areas, desmoglein 3 was expressed throughout all layers of the outer root sheath and cyst wall. In areas of epidermal-like keratinization, such as in the infundibulum and in epidermal inclusion cysts, desmoglein 3 expression was limited mainly to the basal layer. We conclude that desmoglein expression patterns define compartments of cells in similar states of differentiation within the cutaneous epithelium, and reveal a hierarchy of differentiation among these compartments.
Assuntos
Caderinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Cisto Folicular/metabolismo , Doenças do Cabelo/metabolismo , Folículo Piloso/metabolismo , Queratinas/metabolismo , Adulto , Diferenciação Celular , Desmogleína 1 , Desmogleína 2 , Desmogleína 3 , Desmogleínas , Desmoplaquinas , Feto/metabolismo , Cisto Folicular/patologia , Doenças do Cabelo/patologia , Folículo Piloso/citologia , Folículo Piloso/embriologia , HumanosRESUMO
Anchorage of the hair to its follicle is of paramount importance for survival of rodents in the wild, and is aberrant in some human alopecias. Little is understood about the mechanisms responsible for hair shaft anchorage. Desmoglein (Dsg)3-/- (knockout) mice lose hair during telogen, but their anagen hairs remain anchored to the follicle. We hypothesized that Dsg1 compensates for the loss of Dsg3 in the anagen hair follicles of these Dsg3-/- mice. Consistent with this hypothesis, we found Dsg1 and Dsg3 expression overlapping in the companion layer. To functionally address this hypothesis, we used exfoliative toxin A (ETA) to inactivate Dsg1 in Dsg3-/- mice. Four hours after injection of ETA, Dsg3-/- mice, but not Dsg3+/+ or Dsg3+/- mice, showed striking loss of anagen hair, which was confirmed and quantitated by gentle tape stripping. Histology of the skin of these mice as well as of the tape-stripped hair showed separation between the outer root sheath and inner root sheath of the hair follicle, at the plane of the companion layer. Immunostaining for trichohyalin and K6, which highlights the companion layer, in skin and stripped hair confirmed the plane of separation. Labeling of proliferating cells with bromodeoxyuridine demonstrated that the matrix keratinocytes responsible for producing the hair shaft were below the split and remained in the follicle after loss of the anagen hair. These findings demonstrate the importance of the companion layer, and particularly the Dsg1 and Dsg3 in this layer, in anchoring the anagen hair to the follicle.
Assuntos
Caderinas/genética , Caderinas/metabolismo , Folículo Piloso/fisiologia , Cabelo/fisiologia , Animais , Desmogleína 1 , Desmogleína 3 , Exfoliatinas/farmacologia , Imuno-Histoquímica/métodos , Camundongos , Camundongos Knockout , Coloração e Rotulagem/métodosRESUMO
In bullous impetigo, Staphylococcus aureus spreads under the stratum corneum of skin by elaboration of exfoliative toxin, which hydrolyzes only one peptide bond in a highly structured calcium-binding domain of desmoglein 1, resulting in loss of its function. We investigated the basis of this exquisite specificity. Exfoliative toxin cannot cleave desmoglein 1 pretreated at 56 degrees C or higher or at low or high pH, suggesting that the proper conformation of desmoglein 1 is critical for its cleavage. Because cleavage occurs in an area of desmoglein 1 stabilized by calcium, we determined if the conformation necessary for cleavage is calcium-dependent. Depletion of calcium from desmoglein 1 completely inhibited its cleavage by exfoliative toxin, even after calcium was added back. A change in conformation of desmoglein 1 by calcium depletion was shown, with immunofluorescence and enzyme-linked immunoassay, by loss of binding of PF sera, which recognize conformational epitopes. This change in conformation was confirmed by tryptophan fluorometry and circular dichroism, and was irreversible with repletion of calcium. These data suggest that the specificity of exfoliative toxin cleavage of desmoglein 1 resides not only in simple amino acid sequences but also in its calcium-dependent conformation.
Assuntos
Caderinas/química , Caderinas/efeitos dos fármacos , Cálcio/fisiologia , Exfoliatinas/farmacologia , Autoanticorpos/farmacologia , Cálcio/farmacologia , Dicroísmo Circular , Desmogleína 1 , Ensaio de Imunoadsorção Enzimática , Fluorometria , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Conformação Molecular , Pênfigo/imunologia , Peptídeo Hidrolases/farmacologia , Desnaturação Proteica , TriptofanoRESUMO
Staphylococcal scalded skin syndrome and its localized form, bullous impetigo, show superficial epidermal blister formation caused by exfoliative toxin A or B produced by Staphylococcus aureus. Recently we have demonstrated that exfoliative toxin A specifically cleaves desmoglein 1, a desmosomal adhesion molecule, that when inactivated results in blisters. In this study we determine the target molecule for exfoliative toxin B. Exfoliative toxin B injected in neonatal mice caused superficial epidermal blisters, abolished cell surface staining of desmoglein 1, and degraded desmoglein 1 without affecting desmoglein 3 or E-cadherin. When adenovirus-transduced cultured keratinocytes expressing exogenous mouse desmoglein 1 or desmoglein 3 were incubated with exfoliative toxin B, desmoglein 1, but not desmoglein 3, was cleaved. Furthermore, cell surface staining of desmoglein 1, but not that of desmoglein 3, was abolished when cryosections of normal human skin were incubated with exfoliative toxin B, suggesting that living cells were not necessary for exfoliative toxin B cleavage of desmoglein 1. Finally, in vitro incubation of the recombinant extracellular domains of desmoglein 1 and desmoglein 3 with exfoliative toxin B demonstrated that both mouse and human desmoglein 1, but not desmoglein 3, were directly cleaved by exfoliative toxin B in a dose-dependent fashion. These findings demonstrate that exfoliative toxin A and exfoliative toxin B cause blister formation in staphylococcal scalded skin syndrome and bullous impetigo by identical molecular pathophysiologic mechanisms.
Assuntos
Caderinas/metabolismo , Exfoliatinas/metabolismo , Síndrome da Pele Escaldada Estafilocócica/metabolismo , Animais , Animais Recém-Nascidos , Vesícula/metabolismo , Vesícula/patologia , Caderinas/genética , Células Cultivadas , Desmogleína 1 , Desmogleína 3 , Epiderme/metabolismo , Epiderme/patologia , Exfoliatinas/farmacologia , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Síndrome da Pele Escaldada Estafilocócica/patologiaAssuntos
Autoanticorpos/fisiologia , Desmogleínas/metabolismo , Impetigo , Pênfigo , Síndrome da Pele Escaldada Estafilocócica , Adesão Celular , Desmogleínas/química , Desmogleínas/imunologia , Humanos , Imunoglobulina G/metabolismo , Impetigo/imunologia , Impetigo/patologia , Queratinócitos/metabolismo , Pênfigo/imunologia , Pênfigo/patologia , Pênfigo/terapia , Síndrome da Pele Escaldada Estafilocócica/imunologia , Síndrome da Pele Escaldada Estafilocócica/patologiaRESUMO
Many children suffer from the bacterial skin diseases bullous impetigo and staphylococcal scalded skin syndrome (SSSS). Staphylococcus aureus, which produces exfoliative toxins (ETs), causes these diseases. Recently, it was proven that ETs cleave the cell adhesion molecule desmoglein (Dsg) 1, which plays an important role in maintaining the proper structure and barrier function of the epidermis. Surprisingly, Dsg1 is also the antibody target in the autoimmune disease pemphigus foliaceus. Skin biopsies from pemphigus foliaceus patients show the same pathology as those from bullous impetigo and SSSS patients. The crystal structure of ET suggests that it is a serine protease with an inactive catalytic site, which may become activated when ET binds a specific receptor. This receptor binding is thought to cause a change in conformation that exposes the catalytic site. It has recently been shown that Dsg1 specifically binds and activates ET, which in turn cleaves the bound Dsg1 at only one peptide bond. This process is absolutely dependent on the calcium-dependent conformation of Dsg1. These data suggest that ETs have a very high specificity for human Dsg1, and that S. aureus uses ETs to disrupt the barrier of the human epidermis in order to survive and proliferate on the human body.