Effects of Nitrogen Oxidation Metabolism of Murine Macrophages on the Growth of MBT-2 Cell Line

Kim, Sang Woo; Rim, Joung Sik; Chung, Hun Taeg

Abstract

Recently it is known that nitric oxide(NO) generated by an activatedmacrophage plays an important role in tumoricidal or bactericidal activity. Thisstudy was done to know the effects of NO produced by the activated macrophages onthe growth of the murine bladder tumor cell line (MBT-2). For the activation ofmacrophages, RAW 264.7 cell line ( macrophage-derived cell line) and peritonealmacrophages from Balb/c mouse were treated with interferon-r (INF-r),lipopolysaccharide ( LPS) or INF-r plus LPS and for the evaluation of growthinhibition of MBT -2 cell line, tritiated thymidine (3[H] -thymidine)incorporation was measured after coculturing of MBT-2 cell line with macrophageswhich had been activated to produce NO. The results are as follows : l.Peritoneal macrophages from Balb/c mouse could be activated to produce NO onlywhen they are stimulated by the combination of INF-r and LPS (35+/-1.0uM/L). Theycould produce a slight increase amount of NO when they had been stimulated byINF-r (11+/-2.5uM/ L) or LPS (14+/-3.5uM/L) compared to control macrophages(8+/-2.5uM/L). The induction of NO production by INF-r plus LPS could be abrogatedby the use of NG-monomethyl-L- arginine (NGMMA), a competitive inhibitor of NOsynthase (5+/-1.5 M/L). 2. RAW 264.7 cell lines could be activated to produce NOby the treatment of INF-r, LPS, and INF-r plus LPS (40+/-2.5uM/L, 37+/-3.0uM/Land 51+/-2.6uM/L respectively). When they are treated with NGMMA, they produced nomore NO even though they had been stimulated with INF-r plus LPS (14+/-4.0uM/L),and they could produce small .amount of NO(19+/-1.0uM/L) in the absence of thestimulation. 3. The incorporation of 3[H] -thymidine of the MBT-2 and peritonealmacrophages was reduced when the cells had been treated with INF-r plus LPScompared to the control (14,519+/-1,087cpm, 20,716+/-1,474cpm respectively).There was no reduction in the incorporation of 3[H] -thymidine when the cellshad been treated with INF-r or LPS alone. The incorporation of 3[H]-thymidineincreased when the cells had been treated with INF-r plus LPS in the presence ofNGMMA(19,622+/-1,341cpm). 4. The incorporation of 3[H] -thymidine of the MBT-2 and RAW 264.7 cell lines were reduced when the cells had been treated with INF-r, LPS and INF-r plus LPS (19.068+/-144cpm, 15,070+/-122cpm and 7,543+/-85cpm respectively) compared to control( 20,708+/-142cpm). When the cells had been pretreated with NGMMA, the incorporation of 3[H]- thymidine recovered to same degree (12,605+/-108cpm) compared to the cells stimulated with INF-r plus LPS. The above correlation of the NO production from macrophages which had been stimulated by INF-r and/or LPS and the inhibition of growth of the tumor cells suggests that NO produced by stimulated macrophages might be the responsible molecule in the defense system of the body against tumors.