Isolation and Initial Characterization of Tumoricidal Monokine(s) From the Human Monocytic Leukemia Cell Line THP-1<xref ref-type="fn" rid="FN2">2</xref>

Abstract

-A cloned subline of the human monocytic leukemia cell line, THP-1, was induced to produce high levels of cytotoxic activity following an 18-hour phorbol myristate acetate (CAS: 16561-29-8) stimulation in vitro. This activity, termed monocyte cell line cytotoxin(s) (MCCT), was tested in vitro on different human continuous cell lines (Chang, ESH-7, GM3104A, HeLa, HT-1080, K562, Mel, T-24) and on primary human fibroblasts (GM3468, Manz). The continuous cell lines exhibited a spectrum of sensitivity to MCCT-containing supernatants whereas the primary fibroblasts were resistant to lysis. Enzymatic degradation and heat denaturation studies indicate that MCCT is a protein. Its bioactivity can be resolved into three lytic peaks after molecular sieving on Ultrogel AcA 44. The major peak, designated aMCCT, eluted with a molecular weight of 100,000-140,000 daltons. A minor peak, {3MCCT, was seen at 60,000-80,000 daltons, and a third, unstable minor peak,,,MCCT, eluted at less than 10,000 daltons. The a-lytic peak was examined further and was found to migrate as a single peak in 7% native polyacrylamide gel electrolysis tube gels with an r, of 0.25-0.30. None of the MCCT forms were immunologically cross-reactive with human a-Iymphotoxin. Various protease inhibitors known to inhibit monokine-and macrophage-mediated direct cell lysis in vitro were tested for their inhibitory effects on aMCCT activity. The irreversible binding inhibitor Na-p-tosyl-L-Iysyl chloromethyl ketone inhibited the biologic activity of aMCCT. The reversible binding inhibitors Na-p-tosyl-L-arginine methyl ester and soybean trypsin inhibitor were able to block in vitro lytic activity when added to aMCCT in the presence of the target cell. In contrast, the inhibitors phenylmethylsulfonyl fluoride, L-1-tosylamide, 2-phenylethyl chloromethyl ketone, and Na-acetyl-L-Iysine methyl ester were not effective in blocking cytolysis. Finally, the hydrogen peroxide scavenger catalase inhibited aMCCT lytic activity in vitro; however, the hypochlorous acid scavengers alanine, serine, and valine were without effect. -JNCI 1985; 74:1-9. Human peripheral blood monocytes and alveolar macrophages can be raised to a cell-lytic state by treatment in vitro with lymphokines and/or bacterial components (1-5). These activated human mononuclear cells exhibit a greater capacity to lyse neoplastic cells rather than primary cells in vitro. In addition, recent reports have demonstrated the release of eLM from peripheral blood monocyte and alveolar macrophage cultures [(6-8); Klostergaard J: In preparation; Sone S, Lopez-Dertein G, Fidler IJ: In preparation). Supernatants containing eLM from these cultures were also found in general to preferentially lyse continuous cells over primary cells in vitro.For the in-depth investigation of eLM, the production of large amounts of human supernatant would be necessary from a homogeneous monocyte-macrophage source, preferably from a single donor. Primary cultures 1 of monocytes from many individual donors do not fulfill these crit...