Neutrophil elastase (NE) activity is increased in many diseases. Other families of proteases, including cathepsins and matrix metalloproteases (MMPs), are also present at elevated levels in similar disease conditions. We postulated that NE could induce expression of cathepsins and MMPs in human macrophages. NE exposure resulted in macrophages, producing significantly greater amounts of cathepsin B and latent and active MMP-2. Cathepsin B and MMP-2 activities were decreased in Pseudomonas-infected NE knockout mice compared with wild-type littermates. We also demonstrate that NE can activate NF-κB in macrophages, and inhibition of NF-κB resulted in a reduction of NE-induced cathepsin B and MMP-2. Also, inhibition of TLR-4 or transfection of macrophages with dominant-negative IL-1R-associated kinase-1 resulted in a reduction of NE-induced cathepsin B and MMP-2. This study describes for the first time a novel hierarchy among proteases whereby a serine protease up-regulates expression of MMPs and cathepsins. This has important implications for therapeutic intervention in protease-mediated diseases.