Neutrophil elastase (NE) and cathepsin G (CG), the proteolytic enzymes localized in azurophil granules of neutrophils (PMN), are involved in PMN responses to various stimuli. When released at sites of inflammation, they participate in the degradation of numerous proteins involved in the regulation of the immune response. In this study, we employed ADAM17(-/-) fibroblasts stably transfected with cDNA of human pro-tumor necrosis factor alpha (proTNFalpha) (ADAM17(-/-)TNF(+)) to investigate the effects of NE and CG on shedding and degradation of TNFalpha. Both NE and CG were found to diminish the level of membrane TNFalpha (mTNFalpha) as measured by flow cytometry. This process was accompanied by the accumulation of biologically active soluble TNFalpha (sTNFalpha) in the culture medium, as determined by an increase in both the cytotoxic activity of TNFalpha and its ability to serve as a co-stimulator in the induction of inducible nitric oxide synthase (iNOS). However, in contrast to CG, NE at high concentrations was able to degrade sTNFalpha released from the cell surface. Using soluble recombinant human TNFalpha, we identified Val(93)-Ala(94) and Val(117)-Glu(118) as the NE cleavage sites within the sTNFalpha molecule. Taken together, the ability of NE and CG to modulate levels of membrane and soluble forms of TNFalpha may contribute to the proinflammatory activity of neutrophils.