RESUMEN
Antibody-producing plasma cells fuel humoral immune responses. They also contribute to autoimmune diseases such as systemic lupus erythematosus or IgA nephropathy. Interleukin-6 and the tumor necrosis factor (TNF) family ligands BAFF (B cell-activating factor) and APRIL (a proliferation-inducing ligand) participate in plasma cell survival. BAFF binds to three receptors, BAFFR (BAFF receptor), TACI (transmembrane activator and CAML interactor), and BCMA (B cell maturation antigen), while APRIL binds to TACI, BCMA, and proteoglycans. However, which ligand-receptor pair(s) are required to maintain plasma cells in different body locations remains unknown. Here, by combining mouse genetic and pharmacological approaches, we found that plasma cells required BCMA and/or TACI but not BAFFR. BCMA responded exclusively to APRIL, while TACI responded to both BAFF and APRIL, identifying three self-sufficient ligand-receptor pairs for plasma cell maintenance: BAFF-TACI, APRIL-TACI, and APRIL-BCMA. Together, these actors accounted for 90% of circulating antibodies. In BAFF-ko mice, the reduction of plasma cells upon APRIL inhibition indicated that APRIL could function in the absence of BAFF-APRIL heteromers. No evidence was found that in the absence of BCMA and TACI, binding of APRIL to proteoglycans would help maintain plasma cells. IL-6, alone or together with BAFF and APRIL, supported mainly splenic plasmablasts and plasma cells and contributed to circulating IgG but not IgA levels. In conclusion, survival factors for plasma cells can vary with body location and with the antibody isotype that plasma cells produce. To efficiently target plasma cells, in particular IgA-producing ones, dual inhibition of BAFF and APRIL is required.
Asunto(s)
Factor Activador de Células B , Receptor del Factor Activador de Células B , Antígeno de Maduración de Linfocitos B , Interleucina-6 , Proteína Activadora Transmembrana y Interactiva del CAML , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Animales , Factor Activador de Células B/inmunología , Factor Activador de Células B/metabolismo , Factor Activador de Células B/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Antígeno de Maduración de Linfocitos B/inmunología , Antígeno de Maduración de Linfocitos B/metabolismo , Proteína Activadora Transmembrana y Interactiva del CAML/metabolismo , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Proteína Activadora Transmembrana y Interactiva del CAML/inmunología , Interleucina-6/metabolismo , Interleucina-6/inmunología , Ratones , Receptor del Factor Activador de Células B/metabolismo , Receptor del Factor Activador de Células B/inmunología , Receptor del Factor Activador de Células B/genética , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Ratones Noqueados , Células Productoras de Anticuerpos/inmunología , Células Productoras de Anticuerpos/metabolismo , Ratones Endogámicos C57BLRESUMEN
X-linked hypohidrotic ectodermal dysplasia with the cardinal symptoms hypodontia, hypotrichosis and hypohidrosis is caused by a genetic deficiency of ectodysplasin A1 (EDA1). Prenatal EDA1 replacement can rescue the development of skin appendages and teeth. Tabby mice, a natural animal model of EDA1 deficiency, additionally feature a striking kink of the tail, the cause of which has remained unclear. We studied the origin of this phenomenon and its response to prenatal therapy. Alterations in the distal spine could be noticed soon after birth, and kinks were present in all Tabby mice by the age of 4 months. Although their vertebral bones frequently had a disorganized epiphyseal zone possibly predisposing to fractures, cortical bone density was only reduced in vertebrae of older Tabby mice and even increased in their tibiae. Different availability of osteoclasts in the spine, which may affect bone density, was ruled out by osteoclast staining. The absence of hair follicles, a well-known niche of epidermal stem cells, and much lower bromodeoxyuridine uptake in the tail skin of 9-day-old Tabby mice rather suggest the kink being due to a skin proliferation defect that prevents the skin from growing as fast as the skeleton, so that caudal vertebrae may be squeezed and bent by a lack of skin. Early postnatal treatment with EDA1 leading to delayed hair follicle formation attenuated the kink, but did not prevent it. Tabby mice born after prenatal administration of EDA1, however, showed normal tail skin proliferation, no signs of kinking and, interestingly, a normalized vertebral bone density. Thus, our data prove the causal relationship between EDA1 deficiency and kinky tails and indicate that hair follicles are required for murine tail skin to grow fast enough. Disturbed bone development appears to be partially pre-determined in utero and can be counteracted by timely EDA1 replacement, pointing to a role of EDA1 also in osteogenesis.
RESUMEN
Genetic deficiency of ectodysplasin A (EDA) causes X-linked hypohidrotic ectodermal dysplasia, a congenital condition characterized by the absence or abnormal formation of sweat glands, teeth, and several skin appendages. Stimulation of the EDA receptor (EDAR) with agonists in the form of recombinant EDA or anti-EDAR antibodies can compensate for the absence of Eda in a mouse model of Eda deficiency, provided that agonists are administered in a timely manner during fetal development. Here we provide detailed protocols for the administration of EDAR agonists or antagonists, or other proteins, by the intravenous, intraperitoneal, and intra-amniotic routes as well as protocols to collect blood, to visualize sweat gland function, and to prepare skulls in mice.
Asunto(s)
Receptor Edar/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Vías de Administración de Medicamentos , Displasia Ectodérmica/tratamiento farmacológico , Displasia Ectodérmica/genética , Displasia Ectodérmica/metabolismo , Receptor Edar/genética , Ratones , Fenotipo , Proteínas Recombinantes/administración & dosificación , Resultado del TratamientoRESUMEN
BACKGROUND AND PURPOSE: The TNF family ligands, B cell activating factor of the TNF family (BAFF, also known as B lymphocyte stimulator, BLyS) and a proliferation-inducing ligand (APRIL), share the transmembrane activator and calcium-modulator and cyclophilin ligand (CAML)-interactor (TACI) as one of their common receptors. Atacicept, a chimeric recombinant TACI/IgG1-Fc fusion protein, inhibits both ligands. TACI and APRIL also bind to proteoglycans and to heparin that is structurally related to proteoglycans. It is unknown whether the portion of TACI contained in atacicept can bind directly to proteoglycans, or indirectly via APRIL, and whether this could interfere with the anti-coagulant properties of heparin. EXPERIMENTAL APPROACH: Binding of atacicept and APRIL to proteoglycan-positive cells was measured by FACS. Activities of heparin and atacicept were measured with activated factor Xa inhibition and cell-based assays. Effects of heparin on circulating atacicept was monitored in mice. KEY RESULTS: Atacicept did not bind to proteoglycan-positive cells, but when complexed to APRIL could do so indirectly via APRIL. Multimers of atacicept obtained after exposure to cysteine or BAFF 60-mer bound directly to proteoglycans. Atacicept alone, or in complex with APRIL, or in a multimeric form did not interfere with heparin activity in vitro. Conversely, heparin did not influence inhibition of BAFF and APRIL by atacicept and did not change circulating levels of atacicept. CONCLUSIONS AND IMPLICATIONS: Lack of detectable interference of APRIL-bound or free atacicept on heparin activity makes it unlikely that atacicept at therapeutic doses will interfere with the function of heparin in vivo.
Asunto(s)
Linfocitos B/efectos de los fármacos , Citocinas/antagonistas & inhibidores , Heparina/farmacología , Proteínas Recombinantes de Fusión/farmacología , Animales , Linfocitos B/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Factor Xa/metabolismo , Femenino , Células HEK293 , Heparina/administración & dosificación , Heparina/sangre , Humanos , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/sangre , Relación Estructura-ActividadRESUMEN
B cell activating factor of the TNF family (BAFF, also known as BLyS), a cytokine that regulates homeostasis of peripheral B cells, is elevated in the circulation of patients with autoimmune diseases such as systemic lupus erythematosus (SLE). BAFF is synthetized as a membrane-bound protein that can be processed to a soluble form after cleavage at a furin consensus sequence, a site that in principle can be recognized by any of the several proteases of the pro-protein convertase family. Belimumab is a human antibody approved for the treatment of SLE, often cited as specific for the soluble form of BAFF. Here we show in different experimental systems, including in a monocytic cell line (U937) that naturally expresses BAFF, that belimumab binds to membrane-bound BAFF with similar EC50 as the positive control atacicept, which is a decoy receptor for both BAFF and the related cytokine APRIL (a proliferation inducing ligand). In U937 cells, binding of both reagents was only detectable in furin-deficient U937 cells, showing that furin is the main BAFF processing protease in these cells. In CHO cells expressing membrane-bound BAFF lacking the stalk region, belimumab inhibited the activity of membrane-bound BAFF less efficiently than atacicept, while in furin-deficient U937 cells, belimumab inhibited membrane-bound BAFF and residual soluble BAFF as efficiently as atacicept. These reagents did not activate complement or antibody-dependent cell cytotoxicity upon binding to membrane-bound BAFF in vitro. In conclusion, our data show that belimumab can inhibit membrane-bound BAFF, and that BAFF in U937 cells is processed by furin.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Factor Activador de Células B/antagonistas & inhibidores , Membrana Celular/inmunología , Factor Activador de Células B/inmunología , Furina/inmunología , Células HEK293 , Humanos , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunología , Células U937RESUMEN
Hypohidrotic ectodermal dysplasias (HED) are hereditary differentiation disorders of multiple ectodermal structures including the mammary gland. The X-linked form of HED (XLHED) is caused by a lack of the secreted signaling molecule ectodysplasin A1 (EDA1) which is encoded by the gene EDA and belongs to the tumor necrosis factor (TNF) superfamily. Although male patients (hemizygous) are usually more severely affected by XLHED, heterozygous female carriers of an EDA mutation may also suffer from a variety of symptoms, in particular from abnormal development of their breasts. In Tabby mice, a well-studied animal model of XLHED, EDA1 is absent. We investigated the effects of prenatal administration of Fc-EDA, a recombinant EDA1 replacement protein, on mammary gland development in female Tabby mice. Intra-amniotic delivery of Fc-EDA to fetal animals resulted later in improved breastfeeding and thus promoted the growth of their offspring. In detail, such treatment led to a normalization of the nipple shape (protrusion, tapering) that facilitated sucking. Mammary glands of treated female Tabby mice also showed internal changes, including enhanced branching morphogenesis and ductal elongation. Our findings indicate that EDA receptor stimulation during development has a stable impact on later stages of mammary gland differentiation, including lactation, but also show that intra-amniotic administration of an EDA1 replacement protein to fetal Tabby mice partially corrects the mammary gland phenotype in female adult animals.
Asunto(s)
Displasia Ectodermal Anhidrótica Tipo 1/terapia , Glándulas Mamarias Animales/patología , Animales , Mama/patología , Lactancia Materna/métodos , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Ectodisplasinas/genética , Femenino , Terapias Fetales/métodos , Fragmentos Fc de Inmunoglobulinas/genética , Lactancia/genética , Ratones , Ratones Endogámicos C57BL , Morfogénesis/genética , Morfogénesis/fisiología , Mutación/genética , Embarazo , Proteínas Recombinantes de Fusión/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Genetic deficiency of ectodysplasin A (EDA) causes X-linked hypohidrotic ectodermal dysplasia (XLHED), in which the development of sweat glands is irreversibly impaired, an condition that can lead to life-threatening hyperthermia. We observed normal development of mouse fetuses with Eda mutations after they had been exposed in utero to a recombinant protein that includes the receptor-binding domain of EDA. We administered this protein intraamniotically to two affected human twins at gestational weeks 26 and 31 and to a single affected human fetus at gestational week 26; the infants, born in week 33 (twins) and week 39 (singleton), were able to sweat normally, and XLHED-related illness had not developed by 14 to 22 months of age. (Funded by Edimer Pharmaceuticals and others.).
Asunto(s)
Antígenos CD/uso terapéutico , Displasia Ectodermal Anhidrótica Tipo 1/terapia , Ectodisplasinas/genética , Ectodisplasinas/uso terapéutico , Terapias Fetales/métodos , Terapia Genética/métodos , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Diagnóstico Prenatal , Receptores Fc/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Adulto , Líquido Amniótico , Displasia Ectodermal Anhidrótica Tipo 1/diagnóstico por imagen , Displasia Ectodermal Anhidrótica Tipo 1/genética , Ectodisplasinas/deficiencia , Femenino , Humanos , Inyecciones , Masculino , Mutación , Embarazo , Radiografía , Proteínas Recombinantes/uso terapéutico , Glándulas Sudoríparas/anomalías , Glándulas Sudoríparas/diagnóstico por imagen , Germen Dentario/diagnóstico por imagenRESUMEN
The B cell survival factor (TNFSF13B/BAFF) is often elevated in autoimmune diseases and is targeted in the clinic for the treatment of systemic lupus erythematosus. BAFF contains a loop region designated the flap, which is dispensable for receptor binding. Here we show that the flap of BAFF has two functions. In addition to facilitating the formation of a highly active BAFF 60-mer as shown previously, it also converts binding of BAFF to TNFRSF13C (BAFFR) into a signaling event via oligomerization of individual BAFF-BAFFR complexes. Binding and activation of BAFFR can therefore be targeted independently to inhibit or activate the function of BAFF. Moreover, structural analyses suggest that the flap of BAFF 60-mer temporarily prevents binding of an anti-BAFF antibody (belimumab) but not of a decoy receptor (atacicept). The observed differences in profiles of BAFF inhibition may confer distinct biological and clinical efficacies to these therapeutically relevant inhibitors.
Asunto(s)
Factor Activador de Células B/química , Factor Activador de Células B/fisiología , Receptor del Factor Activador de Células B/química , Linfocitos B/citología , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Factor Activador de Células B/genética , Diferenciación Celular , Supervivencia Celular , Reactivos de Enlaces Cruzados/química , Femenino , Técnicas de Sustitución del Gen , Células HEK293 , Humanos , Fragmentos de Inmunoglobulinas/química , Linfopenia/metabolismo , Masculino , Ratones , Ratones Transgénicos , Mutación , Unión Proteica , Dominios Proteicos , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
B cell activating factor of the TNF family (BAFF), also known as B lymphocyte stimulator, is a ligand required for the generation and maintenance of B lymphocytes. In this study, the ability of different monoclonal antibodies to recognize, inhibit, or activate mouse BAFF was investigated. One of them, a mouse IgG1 named Sandy-2, prevented the binding of BAFF to all of its receptors, BAFF receptor, transmembrane activator and calcium modulating ligand interactor, and B cell maturation antigen, at a stoichiometric ratio; blocked the activity of mouse BAFF on a variety of cell-based reporter assays; and antagonized the prosurvival action of BAFF on primary mouse B cells in vitro A single administration of Sandy-2 in mice induced B cell depletion within 2 weeks, down to levels close to those observed in BAFF-deficient mice. This depletion could then be maintained with a chronic treatment. Sandy-2 and a previously described rat IgG1 antibody, 5A8, also formed a pair suitable for the sensitive detection of endogenous circulating BAFF by ELISA or using a homogenous assay. Interestingly, 5A8 and Sandy-5 displayed activities opposite to that of Sandy-2 by stimulating recombinant BAFF in vitro and endogenous BAFF in vivo These tools will prove useful for the detection and functional manipulation of endogenous mouse BAFF and provide an alternative to the widely used BAFF receptor-Fc decoy receptor for the specific depletion of BAFF in mice.
Asunto(s)
Anticuerpos/farmacología , Factor Activador de Células B/antagonistas & inhibidores , Linfocitos B/inmunología , Inmunoglobulina G/farmacología , Animales , Anticuerpos/inmunología , Factor Activador de Células B/genética , Factor Activador de Células B/inmunología , Linfocitos B/patología , Supervivencia Celular/efectos de los fármacos , Hiperplasia , Inmunoglobulina G/inmunología , Depleción Linfocítica/métodos , Ratones , Ratones NoqueadosRESUMEN
The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.
Asunto(s)
Factor Activador de Células B/metabolismo , Antígeno de Maduración de Linfocitos B/metabolismo , Proteína Activadora Transmembrana y Interactiva del CAML/metabolismo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Animales , Factor Activador de Células B/química , Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/metabolismo , Antígeno de Maduración de Linfocitos B/genética , Dimerización , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Unión Proteica , Transducción de Señal , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/química , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genéticaRESUMEN
Impaired ectodysplasin A (EDA) receptor (EDAR) signaling affects ectodermally derived structures including teeth, hair follicles, and cutaneous glands. The X-linked hypohidrotic ectodermal dysplasia (XLHED), resulting from EDA deficiency, can be rescued with lifelong benefits in animal models by stimulation of ectodermal appendage development with EDAR agonists. Treatments initiated later in the developmental period restore progressively fewer of the affected structures. It is unknown whether EDAR stimulation in adults with XLHED might have beneficial effects. In adult Eda mutant mice treated for several weeks with agonist anti-EDAR antibodies, we find that sebaceous gland size and function can be restored to wild-type levels. This effect is maintained upon chronic treatment but reverses slowly upon cessation of treatment. Sebaceous glands in all skin regions respond to treatment, although to varying degrees, and this is accompanied in both Eda mutant and wild-type mice by sebum secretion to levels higher than those observed in untreated controls. Edar is expressed at the periphery of the glands, suggesting a direct homeostatic effect of Edar stimulation on the sebaceous gland. Sebaceous gland size and sebum production may serve as biomarkers for EDAR stimulation, and EDAR agonists may improve skin dryness and eczema frequently observed in XLHED.
Asunto(s)
Receptor Edar/fisiología , Glándulas Sebáceas/anatomía & histología , Glándulas Sebáceas/fisiología , Transducción de Señal/efectos de los fármacos , Envejecimiento , Animales , Proliferación Celular , Displasia Ectodérmica/tratamiento farmacológico , Receptor Edar/agonistas , Ratones , Tamaño de los Órganos , Transducción de Señal/fisiologíaRESUMEN
The TNF family ligand ectodysplasin A (EDA) regulates the induction, morphogenesis and/or maintenance of skin-derived structures such as teeth, hair, sweat glands and several other glands. Deficiencies in the EDA - EDA receptor (EDAR) signalling pathway cause hypohidrotic ectodermal dysplasia (HED). This syndrome is characterized by the absence or malformation of several skin-derived appendages resulting in hypotrychosis, hypodontia, heat-intolerance, dry skin and dry eyes, susceptibility to airways infections and crusting of various secretions. The EDA-EDAR system is an important effector of canonical Wnt signalling in developing skin appendages. It functions by stimulating NF-κB-mediated transcription of effectors or inhibitors of the Wnt, Sonic hedgehog (SHH), fibroblast growth factor (FGF) and transforming growth factor beta (TGFß) pathways that regulate interactions within or between epithelial and mesenchymal cells and tissues. In animal models of Eda-deficiency, soluble EDAR agonists can precisely correct clinically relevant symptoms with low side effects even at high agonist doses, indicating that efficient negative feedback signals occur in treated tissues. Hijacking of the placental antibody transport system can help deliver active molecules to developing foetuses in a timely manner. EDAR agonists may serve to treat certain forms of ectodermal dysplasia.
Asunto(s)
Displasia Ectodermal Anhidrótica Tipo 1/genética , Ectodisplasinas/genética , Receptor Edar/genética , Fenómenos Fisiológicos de la Piel/genética , Piel/crecimiento & desarrollo , Animales , Perros , Displasia Ectodermal Anhidrótica Tipo 1/tratamiento farmacológico , Receptor Edar/agonistas , Células Epiteliales/citología , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Proteínas Hedgehog/antagonistas & inhibidores , Humanos , Mesodermo/citología , Ratones , Morfogénesis , FN-kappa B/genética , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Proteínas Wnt/antagonistas & inhibidores , Vía de Señalización WntRESUMEN
Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated.