SERINE-PROTEASE INHIBITORS BLOCK PRIMING OF MONOCYTES FOR ENHANCED RELEASE OF SUPEROXIDE

Monocytes freshly isolated from human blood produced large amounts of superoxide when triggered by phorbol ester. After monocytes were cultu red for 18-24 hr in endotoxin-free, non-adherent conditions, they prod uced low amounts of superoxide. Addition of lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) , or platelet-activating factor (PAF) at the beginning of culture 'pri med' the monocytes, causing them to maintain a high superoxide respons e for at least 96 hr. Also, in response to LPS, monocytes secreted TNF -alpha. The ability of LPS, IFN-gamma, TNF-alpha or PAF to maintain th e high superoxide response was blocked by addition of inhibitors of se rine proteases, either 4-(2-aminoethyl)-benzenesulpho nyl flue ride (A EBSF) or 3,4-dichloroisocoumarin. AEBSF was most effective at 200 mu M , and required 6 hr for maximum effect. AEBSF did not affect phorbol-t riggered superoxide release by unprimed monocytes. AEBSF did not affec t cell viability, nor did it interfere with the TNF-a secretion in res ponse to LPS. An analogue of AEBSF that lacked ability to inhibit prot eases did not affect monocyte responses. 3,4-Dichloroisocoumarin block ed priming at a low concentration, 1 mu M. We conclude that activity o f a monocyte serine protease is required to maintain the high superoxi de response in monocytes primed with LPS, IFN-gamma, TNF-alpha, or PAF .