RESUMEN
Laminins are ubiquitous constituents of the basement membranes with major architectural and functional role as supported by the fact that absence or mutations of laminins lead to either lethal or severely impairing phenotypes. Besides genetic defects, laminins are involved in a wide range of human diseases including cancer, infections, and inflammatory diseases, as well as autoimmune disorders. A growing body of evidence implicates several laminin chains as autoantigens in blistering skin diseases, collagenoses, vasculitis, or post-infectious autoimmunity. The current paper reviews the existing knowledge on autoimmunity against laminins referring to both experimental and clinical data, and on therapeutic implications of anti-laminin antibodies. Further investigation of relevant laminin epitopes in pathogenic autoimmunity would facilitate the development of appropriate diagnostic tools for thorough characterization of patients' antibody specificities and should decisively contribute to designing more specific therapeutic interventions.
Asunto(s)
Autoanticuerpos/inmunología , Autoantígenos/inmunología , Autoinmunidad/inmunología , Laminina/inmunología , Animales , Autoantígenos/genética , Autoinmunidad/genética , Membrana Basal/inmunología , Humanos , Laminina/genética , Mutación/inmunología , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Enfermedades de la Piel/genética , Enfermedades de la Piel/inmunologíaRESUMEN
Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease of mucous membranes and the skin caused by autoantibodies against collagen VII. In silico and wet laboratory epitope mapping studies revealed numerous distinct epitopes recognized by EBA patients' autoantibodies within the non-collagenous (NC)1 and NC2 domains of collagen VII. However, the distribution of pathogenic epitopes on collagen VII has not yet been described. In this study, we therefore performed an in vivo functional epitope mapping of pathogenic autoantibodies in experimental EBA. Animals (n = 10/group) immunized against fragments of the NC1 and NC2 domains of collagen VII or injected with antibodies generated against the same fragments developed to different extent experimental EBA. Our results demonstrate that antibodies targeting multiple, distinct epitopes distributed over the entire NC1, but not NC2 domain of collagen VII induce blistering skin disease in vivo. Our present findings have crucial implications for the development of antigen-specific B- and T cell-targeted therapies in EBA.
Asunto(s)
Colágeno Tipo VII/inmunología , Epidermólisis Ampollosa Adquirida/inmunología , Epítopos/inmunología , Animales , Mapeo Epitopo , Femenino , Masculino , Ratones Endogámicos BALB C , Neutrófilos/inmunología , Fragmentos de Péptidos/inmunología , Estructura Terciaria de Proteína , Conejos , Piel/inmunología , Piel/patologíaRESUMEN
Bullous pemphigoid, the most common autoimmune blistering disease in Western Europe and the USA is characterized by the presence of circulating and tissue-bound autoantibodies against the hemidesmosomal proteins BP230 and BP180/collagen XVII. After binding to their target antigens at the basement membrane of the dermal-epidermal junction these autoantibodies are thought to trigger an inflammatory cascade comprising complement- and granulocyte-dependent reactions that result in tissue damage. Whereas the role of anti-BP180 antibodies has been extensively characterized, few and conflicting data is available on the contribution of anti-BP230 antibodies to bullous pemphigoid pathogenesis. Therefore, we addressed in the present study the role of autoantibodies to BP230 in experimental bullous pemphigoid. Rabbit polyclonal antibodies generated against epitopes of the C-terminal fragment of murine BP230 bound to the basement membrane and activated the complement system ex vivo. Affinity-purified antibodies were subsequently subcutaneously transferred into neonatal and adult BALB/c mice. In vivo, we observed a dose-dependent binding of transferred antibodies in the murine skin; however, there was no complement activation and these mice showed no clinical or histological signs of inflammatory disease, in contrast to mice receiving anti-BP180 antibodies. We further conducted ex vivo experiments and demonstrated that rabbit IgG anti-BP230-specific antibodies, in contrast to antibodies from bullous pemphigoid patients or rabbit IgG anti-BP180 antibodies used as positive controls, did not activate human granulocytes to induce dermal-epidermal separation in skin cryosections. Our present findings demonstrate that antibodies against BP230 are non-pathogenic in experimental models of bullous pemphigoid and suggest that proper activation of the complement and granulocytes represent prerequisites for conferring bullous pemphigoid autoantibodies their tissue destructive potential.
Asunto(s)
Epítopos Inmunodominantes/inmunología , Inmunoglobulina G/inmunología , Glicoproteínas de Membrana/inmunología , Penfigoide Ampolloso/inmunología , Dominios y Motivos de Interacción de Proteínas/inmunología , Factores de Edad , Animales , Animales Recién Nacidos , Autoantígenos/inmunología , Proteínas Portadoras , Proteínas del Citoesqueleto , Dermis/inmunología , Dermis/metabolismo , Dermis/patología , Modelos Animales de Enfermedad , Distonina , Epidermis/inmunología , Epidermis/metabolismo , Epidermis/patología , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Epítopos Inmunodominantes/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso , Colágenos no Fibrilares/inmunología , Fenotipo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Colágeno Tipo XVIIRESUMEN
Autoimmunity against laminins has been described in several autoimmune diseases (including mucous membrane pemphigoid, anti-laminin γ1 pemphigoid, and connective tissue diseases), in pregnancy loss, and in infections such as Chagas disease. Except for anti-laminin-332 mucous membrane pemphigoid, adequate evidence has been lacking for the tissue injury potential of laminin-specific antibodies and the pathogenic epitopes. We evaluated the pathogenic potential of antibodies targeting laminin γ1, a major constituent of basement membranes and the main antigen in anti-laminin γ1 pemphigoid. Rabbit antibodies were generated against fragments of the N-terminus and C-terminus of murine laminin γ1, and their ability to disrupt ligand interactions and/or to activate complement and granulocytes was assessed using previously established ex vivo assays. Our findings document a pathogenic potential of antibodies targeting the laminin γ1 N-terminus. These antibodies interfere with the binding of nidogen to laminin and can activate granulocytes and the complement cascade. We detected antibodies with different degrees of reactivity with laminin γ1 N-terminus in patients with anti-laminin γ1 pemphigoid, cutaneous lupus erythematosus, and scleroderma. Our results provide mechanistic insights into the tissue damage associated with laminin autoimmunity and could facilitate development of appropriate diagnostic tools and therapeutic strategies.
Asunto(s)
Autoanticuerpos/inmunología , Laminina/inmunología , Animales , Especificidad de Anticuerpos/inmunología , Adhesión Celular , Línea Celular , Activación de Complemento/inmunología , Simulación por Computador , Reacciones Cruzadas/inmunología , Dermis/metabolismo , Dermis/patología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Técnica del Anticuerpo Fluorescente , Granulocitos/metabolismo , Humanos , Integrinas/metabolismo , Laminina/química , Ligandos , Mediciones Luminiscentes , Glicoproteínas de Membrana/metabolismo , Ratones , Penfigoide Ampolloso/inmunología , Penfigoide Ampolloso/patología , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/metabolismo , Piel/inmunologíaRESUMEN
IgA represents the most prominent Ab class at mucosal surfaces and the second most prevalent Ab in human blood after IgG. We recently demonstrated that cross-linking of the granulocyte IgA FcR (FcαRI) by IgA induces a chemotactic-driven positive-feedback migration loop, hereby amplifying recruitment of granulocytes to IgA deposits. Therefore, we postulated that aberrant IgA-Ag complexes, which can be found in tissues in IgA-mediated diseases, are responsible for tissue damage by inducing continuous granulocyte migration and activation. Using an IgA-dependent skin-blistering disease as a model system, we demonstrated colocalization of FcαRI-positive granulocyte infiltrates with IgA in cryosections of lesional skin of patients suffering from this disease. Furthermore, we showed granulocyte migration to IgA deposits injected in human skin explants and in murine skin of FcαRI transgenic mice in vivo. Importantly, ex vivo migration and tissue damage were inhibited by blocking FcαRI, indicating that these events are dependent on the interaction of IgA autoantibodies with FcαRI. Thus, interrupting the granulocyte migration loop by blocking FcαRI reduces tissue damage in diseases with aberrant IgA-immune complexes. As such, our results may lead to development of new therapies for IgA-mediated chronic inflammatory diseases, hereby decreasing severe morbidity and improving quality of life for these patients.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Granulocitos/inmunología , Inmunoglobulina A/inmunología , Receptores de IgG/inmunología , Animales , Autoanticuerpos/metabolismo , Enfermedades Autoinmunes/metabolismo , Quimiotaxis de Leucocito/inmunología , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Granulocitos/metabolismo , Humanos , Inmunoglobulina A/metabolismo , Inmunohistoquímica , Ratones , Ratones Transgénicos , Receptores de IgG/metabolismo , Enfermedades Cutáneas Vesiculoampollosas/inmunología , Enfermedades Cutáneas Vesiculoampollosas/metabolismoRESUMEN
BACKGROUND: Pemphigoids are rare diseases associated with IgG, IgE and IgA autoantibodies against collagen XVII/BP180. An entity of the pemphigoid group is the lamina lucida-type of linear IgA disease (IgA pemphigoid) characterized by IgA autoantibodies against BP180. While for the detection of IgG and IgE autoantibodies specific to collagen XVII several ELISA systems have been established, no quantitative immunoassay has been yet developed for IgA autoantibodies. Therefore, the aim of the present study was to develop an ELISA to detect IgA autoantibodies against collagen XVII in the sera of patients with pemphigoids. METHODS: We expressed a soluble recombinant form of the collagen XVII ectodomain in mammalian cells. Reactivity of IgA autoantibodies from patients with IgA pemphigoid was assessed by immunofluorescence microscopy and immunoblot analysis. ELISA test conditions were determined by chessboard titration experiments. The sensitivity, specificity and the cut-off were determined by receiver-operating characteristics analysis. RESULTS: The optimized assay was carried out using sera from patients with IgA pemphigoid (n = 30) and healthy donors (n=105). By receiver operating characteristics (ROC) analysis, an area under the curve of 0.993 was calculated, indicating an excellent discriminatory capacity. Thus, a sensitivity and specificity of 83.3% and 100%, respectively, was determined for a cut-off point of 0.48. As additional control groups, sera from patients with bullous pemphigoid (n=31) and dermatitis herpetiformis (n = 50), a disease associated with IgA autoantibodies against epidermal transglutaminase, were tested. In 26% of bullous pemphigoid patients, IgA autoantibodies recognized the ectodomain of collagen XVII. One of 50 (2%) of dermatitis herpetiformis patients sera slightly topped the cut-off value. CONCLUSIONS: We developed the first ELISA for the specific and sensitive detection of serum IgA autoantibodies specific to collagen XVII in patients with pemphigoids. This immunoassay should prove a useful tool for clinical and translational research and should essentially improve the diagnosis and disease monitoring of patients with IgA pemphigoid. Moreover, our findings strongly suggest that IgA pemphigoid and IgG bullous pemphigoid represent two ends of the clinical spectrum of an immunological loss of tolerance against components of hemidesmosomes, which is mediated by both IgG and IgA autoantibodies.