RESUMEN
Interleukin-13 (IL-13) sequentially binds to IL-13Ralpha1 and IL-4Ralpha forming a high affinity signalling complex. This receptor complex is expressed on multiple cell types in the airway and signals through signal transducer and activator of transcription factor-6 (STAT-6) to stimulate the production of chemokines, cytokines and mucus. Antibodies have been generated, using the UCB Selected Lymphocyte Antibody Method (UCB SLAM), that block either binding of murine IL-13 (mIL-13) to mIL-13Ralpha1 and mIL-13Ralpha2, or block recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. Monoclonal antibody (mAb) A was shown to bind to mIL-13 with high affinity (K(D) 11 pM) and prevent binding of mIL-13 to mIL-13Ralpha1. MAb B, that also bound mIL-13 with high affinity (K(D) 8 pM), was shown to prevent recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. In vitro, mAbs A and B similarly neutralised mIL-13-stimulated STAT-6 activation and TF-1 cell proliferation. In vivo, mAbs A and B demonstrated equipotent, dose-dependent inhibition of eotaxin generation in mice stimulated by intraperitoneal administration of recombinant mIL-13. In an allergic lung inflammation model in mice, mAbs A and B equipotently inhibited muc5ac mucin mRNA upregulation in lung tissue measured two days after intranasal allergen challenge. These data support the design of therapeutics for the treatment of allergic airway disease that inhibits assembly of the high affinity IL-13 receptor signalling complex, by blocking the binding of IL-13 to IL-13Ralpha1 and IL-13Ralpha2, or the subsequent recruitment of IL-4Ralpha.
Asunto(s)
Anticuerpos Bloqueadores/inmunología , Anticuerpos Monoclonales/inmunología , Subunidad alfa1 del Receptor de Interleucina-13/antagonistas & inhibidores , Subunidad alfa2 del Receptor de Interleucina-13/antagonistas & inhibidores , Interleucina-13/antagonistas & inhibidores , Receptores de Superficie Celular/antagonistas & inhibidores , Animales , Línea Celular , Línea Celular Tumoral , Quimiocina CCL11/análisis , Quimiocina CCL11/inmunología , Modelos Animales de Enfermedad , Epítopos/inmunología , Humanos , Hipersensibilidad/inmunología , Interleucina-13/inmunología , Subunidad alfa1 del Receptor de Interleucina-13/inmunología , Subunidad alfa2 del Receptor de Interleucina-13/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Mucina 5AC/inmunología , Mucina 5AC/metabolismo , Ovalbúmina/inmunología , Neumonía/inmunología , Neumonía/metabolismo , Conejos , Receptores de Superficie Celular/inmunología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT6/inmunología , Factor de Transcripción STAT6/metabolismoRESUMEN
Bacillus thuringiensis protein delta-endotoxins are toxic to a variety of different insect species. Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin. Here, we investigated the role of proteolytic processing in determining the potency of the B. thuringiensis Cry1Ac delta-endotoxin towards Pieris brassicae (family: Pieridae) and Mamestra brassicae (family: Noctuidae). In bioassays, Cry1Ac was over 2,000 times more active against P. brassicae than against M. brassicae larvae. Using gut juice purified from both insects, we processed Cry1Ac to soluble forms that had the same N terminus and the same apparent molecular weight. However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate. With proteases from P. brassicae, the Cry1Ac-susceptible insect, Cry1Ac was processed to an insoluble product with a molecular mass of approximately 56 kDa, whereas proteases from M. brassicae, the non-susceptible insect, generated products with molecular masses of approximately 58, approximately 40, and approximately 20 kDa. N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, but M. brassicae proteases also cleaved the toxin at Arg423 in domain II. A similar pattern of processing was observed in vivo. When Arg423 was replaced with Gln or Ser, the resulting mutant toxins resisted degradation by M. brassicae proteases. However, this mutation had little effect on toxicity to M. brassicae. Differential processing of membrane-bound Cry1Ac was also observed in qualitative binding experiments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.