Common and distinct signalling cascades in the production of tumour necrosis factor‐α and interleukin‐13 induced by lipopolysaccharide in RBL‐2H3 cells

Summary Background Activation of mast cells by lipopolysaccharide (LPS) results in the production of TNF‐α and IL‐13. TNF‐α and IL‐13 are key mediators in the development of neutrophilic and allergic inflammation, respectively. LPS‐induced TNF‐α and IL‐13 production in mast cells has been reported to be mediated by Toll‐like receptor 4 (TLR4) signalling, but differences in signal transduction mechanisms leading to the production of these cytokines are not clearly defined. Objective We investigated the molecular mechanisms responsible for LPS‐induced TNF‐α and IL‐13 production in mast cells. Methods TNF‐α and IL‐13 production by LPS was assessed by transfecting RBL‐2H3 cells with dominant‐negative (DN) expression vectors. Results Transfection of RBL‐2H3 cells with plasmids encoding DN mutants of myeloid differentiation protein (MyD88) and TNFR‐associated factor (TRAF6) inhibited both LPS‐induced TNF‐α and IL‐13 production. IκBα‐DN inhibited LPS‐induced production of TNF‐α, but not IL‐13. We also found that inhibition of p38 kinase suppressed both TNF‐α and IL‐13 induction by LPS, and inhibition of JNK reduced IL‐13 production, but not TNF‐α. Furthermore, we found that protein kinase R (PKR) was activated by LPS in these cells. Treatment with 2‐aminopurine, a PKR inhibitor, attenuated LPS‐induced nuclear factor‐κB activation and TNF‐α production, whereas inhibition of PKR had little effect on IL‐13 production. Conclusion These findings indicate that the production of TNF‐α and IL‐13 by LPS required TLR4/MyD88/TRAF6 signalling as a common pathway of mast cell‐mediated inflammation. We furthermore found that TNF‐α and IL‐13 production were differentially regulated by signalling cascades through PKR and mitogen‐activated protein kinases downstream of TRAF6 in mast cells.