LYSIS OF LEUKEMIC-CELLS BY HUMAN MACROPHAGES - INHIBITION BY 4-(2-AMINOETHYL)-BENZENESULFONYL FLUORIDE (AEBSF), A SERINE-PROTEASE INHIBITOR

Proteases are known to be involved in regulation of macrophage activat ion and killing. We examined the effect of a serine protease inhibitor , 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), on lysis of leuke mic cells by human macrophages. Monocytes, isolated by Histopaque grad ients and centrifugal elutriation, were cultured for 5 days in RPMI-16 40 medium with 5% AB serum, and then activated with interferon-gamma ( IFN-gamma; 100 U/mL) and lipopolysaccharide (LPS) (5 ng/mL), with or w ithout AEBSF, for 2 days. On day 7, macrophages were washed, fresh med ium without AEBSF added, and target cells added for 2 days. Lytic acti vity against two leukemic cell lines (K562 and HL-60) was assessed by an (111)indium-releasing assay. Macrophages treated with IFN-gamma + L PS lysed K562 and HL-60 cells. AEBSF (50-150 mu M) blocked the killing of the se leukemic cells in a concentration-dependent manner. Other p rotease inhibitors were not effective. AEBSF was nontoxic at the conce ntrations used, and did not inhibit tumor necrosis factor-alpha (TNF-a lpha) and interleukin-1 beta (IL-1 beta) secretion from the macrophage s. The lytic activity against leukemic cells was inhibited by anti-TNF -alpha antibody, but not by anti-IL-1 beta, nor by superoxide dismutas e or catalase. However, the leukemic cells were resistant to being kil led by recombinant TNF-alpha alone in the absence of macrophages, indi cating that TNF-alpha was required for killing, but that other factors that were inhibited by AEBSF were also required. Serum-free culture s upernatant of activated macrophages had significant cytotoxic activity against leukemic cells. This cytotoxic activity was not altered by ad dition of AEBSF to the culture supernatant, suggesting that AEBSF affe cted macrophage activation, rather than inhibiting cytotoxic proteases secreted by the macrophages, or affecting the target cells themselves . Thus, a protease, which is susceptible to AEBSF, might be involved i n the activation of macrophages, and might regulate the secretion of a ntitumor effector molecules other than TNF-alpha.