Lysis of leukemic cells by human macrophages: inhibition by 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor

Abstract

Proteases are known to be involved in regulation of macrophage activation and killing. We examined the effect of a serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), on lysis of leukemic cells by human macrophages. Monocytes, isolated by Histopaque gradients and centrifugal elutriation, were cultured for 5 days in RPMI-1640 medium with 5% AB serum, and then activated with interferon-gamma (IFN-gamma; 100 U/mL) and lipopolysaccharide (LPS) (5 ng/mL), with or without AEBSF, for 2 days. On day 7, macrophages were washed, fresh medium without AEBSF added, and target cells added for 2 days. Lytic activity against two leukemic cell lines (K562 and HL-60) was assessed by an 111indium-releasing assay. Macrophages treated with IFN-gamma + LPS lysed K562 and HL-60 cells. AEBSF (50-150 microM) blocked the killing of these leukemic cells in a concentration-dependent manner. Other protease inhibitors were not effective. AEBSF was nontoxic at the concentrations used, and did not inhibit tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion from the macrophages. The lytic activity against leukemic cells was inhibited by anti-TNF-alpha antibody, but not by anti-IL-1 beta, nor by superoxide dismutase or catalase. However, the leukemic cells were resistant to being killed by recombinant TNF-alpha alone in the absence of macrophages, indicating that TNF-alpha was required for killing, but that other factors that were inhibited by AEBSF were also required. Serum-free culture supernatant of activated macrophages had significant cytotoxic activity against leukemic cells. This cytotoxic activity was not altered by addition of AEBSF to the culture supernatant, suggesting that AEBSF affected 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 in the activation of macrophages, and might regulate the secretion of antitumor effector molecules other than TNF-alpha.