Two concurrent effects of serine protease inhibition by TAME (N alpha-p- tosyl-L-arginine methyl ester) on IL-1 beta release were observed in our studies by ELISA, 1) an inhibition of IL-1 beta synthesis, resulting in a decrease in the total amount of IL-1 beta available for release from the cell and 2) an increase in the percentage of total synthesized IL-1 beta that is actually released. Studies utilizing Western blot techniques to further elucidate these effects with respect the greatest inhibition occurring with the 31 kDA precursor IL-1 beta (pIL-1 beta) inside the cell. LPS alone induced production and release of the 17 kDA mature IL-1beta (mIL-1 beta), while the presence of >1 mM TAME for 6 or 18 hours resulted in release of the 33 kDA precursor IL-1 beta (pIL-1 beta) as well. This effect was also observed when TAME was added 18 hours after PLS stimulation. Since the monoclonal antibodies utilized in the ELISA recognize both IL-1 beta species, this could explain why the percentage of IL-1 beta released actually increased. The release of IL-1 beta with TAME was not accompanied by release of the cytosolic marker lactate dehydrogenase (LDH), indicating the pIL-1 beta release was unrelated to toxicty resulting in cell lysis. TNF alpha release was inhibited by 10mM TAME, and this effect was probably due to an inhibition of protein synethesis and as a result, monokine production. These results indicate that serine proteases may be involved in proteolysis of the 33 kDA pIL-1 beta and that proteolysis is probably not mechanistically involved in the signalling of mIL-1 beta release. Results further suggest that comprehensive characterization of the role of proteases in pIL-1 beta proteolysis and IL-1 beta release are required before a valid and specifc therapeutic intervention in IL-1- related disease states can be successfully developed.
