In vitro and in vivo characterisation of anti-murine IL-13 antibodies recognising distinct functional epitopes.

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.

Roles for the interleukin-4 receptor and associated JAK/STAT proteins in human articular chondrocyte mechanotransduction.

OBJECTIVE: To identify functional interleukin-4 (IL4) receptor (IL4R) subtypes and associated Janus kinase/signal transducers and activators of transcription (JAK/STAT) molecules in human articular chondrocytes and assess the role of JAK/STAT proteins in chondrocyte mechanotransduction. METHODS: Expression of IL4R subunits and associated molecules was assessed by immunohistochemistry and western blotting. Functional IL4R were identified by chemical crosslinking of IL4-stimulated chondrocytes and western blotting. JAK and STAT phosphorylation was assessed by western blotting. RESULTS: Chondrocytes from normal and osteoarthritic (OA) cartilage express IL4Ralpha, gammac and IL13Ralpha1 subunits (components of the Type I and Type II IL4R). In the presence of IL4 only functional Type II IL4Rs were identified in normal or OA chondrocytes. With the exception of STAT2, no differences in JAK/STAT expression were detected between normal and OA cartilage. STAT2 was expressed in OA but not normal chondrocytes. Mechanical stimulation (MS) resulted in an IL4R-dependent increase in phosphorylated Tyk2 in normal chondrocytes, which could be abolished by IL1beta preincubation. No phosphorylation of STAT5 or STAT6 was detected in either normal or OA chondrocytes following mechanical stimulation (MS) IL4 stimulation resulted in a decrease in Tyk2 phosphorylation and an increase in phosphorylation of STAT6 in both normal and OA chondrocytes. CONCLUSION: Chondrocytes from normal and OA cartilage signal through a Type II IL4R. This signalling is via a STAT6-independent pathway. Differences in IL4 signalling are likely due to crosstalk between integrin and cytokine signalling pathways, and not differences in IL4R expression.