The Journal of Immunology, Vol 134, Issue 5 3524-3531, Copyright © 1985 by American Association of Immunologists

ARTICLES

Rapid killing of actinomycin D-treated tumor cells by human monocytes. II. Cytotoxicity is independent of secretion of reactive oxygen intermediates and is suppressed by protease inhibitors

F Colotta, L Bersani, A Lazzarin, G Poli and A Mantovani

Pretreatment with Actinomycin D (ActD, 1 microgram/ml for 3 hr) rendered WEHI 164 tumor cells susceptible to killing by human monocytes in a 6-hr 51Cr release assay. The present study was designed to elucidate the role of reactive oxygen intermediates (ROI) and of proteolytic enzymes in this reactivity. ActD-treated WEHI 164 cells did not trigger any measurable release of O-2 or H2O2 from monocytes. Monocytes exposed to phorbol-12-myristate-13-acetate, which enhanced release of ROI, did not show augmented killing of ActD-treated tumor cells. Scavengers of oxygen metabolites (catalase, superoxide dismutase, gluthatione, and mannitol), which inhibited ROI-mediated PMA- induced monocyte cytotoxicity against erythrocytes, did not affect monocyte killing of ActD-treated WEHI 164 cells. Enzymatically generated ROI with xanthine/xanthine-oxidase glucose/glucose-oxidase did not show preferential killing of ActD-treated WEHI 164 cells. Two patients with chronic granulomatous disease had normal levels of monocyte cytotoxicity against ActD-treated tumor cells. To determine the possible role of proteolytic enzymes in mediating this reactivity, we studied various antiproteases. Organophosphorous agents (DFP and PMSF), chloromethyl-ketone derivatives of tosylamino acids (TLCK and TPCK), Actinomyces products (pepstatin and chymostatin), and the synthetic protease substrate TAME inhibited monocyte-mediated cytotoxicity against ActD-treated WEHI 164 cells. The macromolecular protease inhibitors alpha-1 antitrypsin, bovine pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor, and the synthetic protease substrate ATEE had little effect on monocyte cytotoxicity. When monocytes were preincubated with drugs for 1 hr and washed, TLCK, TPCK, and PMSF inhibited cytolysis, whereas the less effective chymostatin and TAME and the inactive BPTI had no effect under these conditions. Inhibition by preincubation with TLCK, PMSF, and TPCK was completely reversed after 6 hr of culture. Supernatants of monocyte cultures had lytic activity against ActD-treated WEHI 164 but not against untreated cells. Antiproteases inhibited the lytic activity of monocyte supernatants. These results strongly suggest that ROI do not play a critical role in monocyte-mediated rapid killing of drug-treated tumor cells, and that proteolytic enzymes are involved in this reactivity.

This article has been cited by other articles:

				P. Cui, K. Tani, H. Kitamura, Y. Okumura, M. Yano, D. Inui, T. Tamaki, S. Sone, and H. Kido A novel bioactive 31-amino acid endothelin-1 is a potent chemotactic peptide for human neutrophils and monocytes J. Leukoc. Biol., August 1, 2001; 70(2): 306 - 312. [Abstract] [Full Text] [PDF]

				O. Chertov, H. Ueda, L. L. Xu, K. Tani, W. J. Murphy, J. M. Wang, O.M. Z. Howard, T. J. Sayers, and J. J. Oppenheim Identification of Human Neutrophil-derived Cathepsin G and Azurocidin/CAP37 as Chemoattractants for Mononuclear Cells and Neutrophils J. Exp. Med., August 29, 1997; 186(5): 739 - 747. [Abstract] [Full Text] [PDF]

				X. Gong, W. Gong, D. B. Kuhns, A. Ben-Baruch, O. M. Z. Howard, and J. M. Wang Monocyte Chemotactic Protein-2 (MCP-2) Uses CCR1 AND CCR2B as Its Functional Receptors J. Biol. Chem., May 2, 1997; 272(18): 11682 - 11685. [Abstract] [Full Text] [PDF]

				M. Cota, M. Mengozzi, E. Vicenzi, P. Panina-Bordignon, F. Sinigaglia, P. Transidico, S. Sozzani, A. Mantovani, and G. Poli Selective inhibition of HIV replication in primary macrophages but not T lymphocytes by macrophage-derived chemokine PNAS, August 1, 2000; 97(16): 9162 - 9167. [Abstract] [Full Text] [PDF]