Cathepsin B is involved in the trafficking of TNF-α-containing vesicles to the plasma membrane in macrophages

TNF-α is a potent proinflammatory cytpkine, essential for initiating innate immune responses against invading microbes and a key mediator involved in the pathogenesis of acute and chronic inflammatory diseases. To identify molecules involved in the production of TNF-α, we used a functional gene identification method using retroviral integration-mediated mutagenesis, followed by LPS-stimulated TNF-α production analysis in macrophages. We found that cathepsin B, a Iysosomal cysteine proteinase, was required for optimal posttranslational processing of TNF-α in response to the bacterial cell wall component LPS. Mouse bone marrow-derived macrophages from cathepsin B-deflcient mice and macrophages treated with the cathepsin B-specific chemical inhibitor CA074 methyl ester or small interfering RNA against cathepsin B secreted significantly less TNF-α than wild-type or nontreated macrophages. We further showed that the inhibition of cathepsin B caused accumulation of 26-kDa pro-TNF-con-taining vesicles. Ectopic expression of GFP-conjugated pro-TNF further suggests that pro-TNF failed to reach the plasma membrane without intracellular cathepsin B activity. Altogether, these data suggest that intracellular cathepsin B activity is involved in the TNF-α-containing vesicle trafficking to the plasma membrane.