INACTIVATION OF INTERLEUKIN-6 IN-VITRO BY MONOBLASTIC U937 CELL PLASMA-MEMBRANES INVOLVES BOTH PROTEASE AND PEPTIDYL-TRANSFERASE ACTIVITIES

Human promonocytic U937 cells have previously been shown to possess at their cell surface specific transmembrane serine proteases and N-term inal amino acid proteases as well as associated enzymes including elas tase and cathepsin G. In this study, purified plasma membranes from U9 37 cells are reported to degrade the recombinant 21-kDa I-125-interleu kin-6 (I-125-IL-6) into 8-kDa products with loss of biological activit y, as monitored by polyacrylamide gel electrophoresis and a cell-proli feration bioassay. Degradation of I-125-IL-6 by plasma membranes was c ompletely prevented by the serine-protease inhibitor diisopropyl fluor ophosphate, but was only partially impaired by alpha1-protease inhibit or and antibody against cathepsin G. A similar incubation of I-125-IL- 6 with cathepsin G purified from U937 cells caused hydrolysis of the c ytokine into similar inactive 8-kDa fragments, whereas incubation with purified U937 cell elastase failed to degrade the peptide. These find ings indicate that U937 cells hydrolyze IL-6 using cell-associated ser ine-protease activity and that cathepsin G partially participates in t his degradation. Prolonged incubation of 8-kDa I-125-IL-6 fragments wi th purified U937 plasma membranes, led to a complete loss of IL-6 acti vity related to the transformation of the 8-kDa forms into a higher-mo lecular-mass complex (16 kDa). This complex was stable in SDS and 2-me rcaptoethanol at 100-degrees-C and was not dissociated by hydroxylamin e treatment, indicating the formation of a covalent non-ester bond bet ween the 8-kDa I-125-IL-6-derived peptide and an undetermined acceptor . An initial oxidative treatment of I-125-IL-6 partially prevented com plex formation, suggesting the presence of one or more oxidizable meth ionine residues at the binding site of 8-kDa I-125-IL-6 peptide. The k inetics of complex formation (time dependence and plasma-membrane-conc entration dependence), as well as its inhibition by a specific inhibit or of N-aminopeptidase activity, bestatin, suggest the participation o f peptidyl-transferase activity in complex formation. Finally, a plasm a-membrane fraction, corresponding to a molecular mass greater-than-or -equal-to 30 kDa, was able to convert the 8-kDa I-125-IL-6 forms into the I-125-labeled 16-kDa complex, suggesting that a greater-than-or-eq ual-to 30-kDa peptidyl-transferase enzyme catalyzes the reaction and p rovides the I-125-labeled 16-kDa peptide by dimerization of 8-kDa I-12 5-IL-6-derived intermediates. Further identification of the plasma-mem brane-associated peptidyl transferase as a regulator of IL-6 proteolys is may be of physiological relevance for the control of IL-6 biologica l activity.