Synergistic Effect of LPS, IFN- and Iron on Apoptosis of Balb/c Mice Macrophages Following Nitric Oxide Production

Document Type : Original Article

Authors

1 College of Medicine and Medical Sciences, Islamic Azad University, Ardebil Branch, Ardebil, Iran

2 Department of Immunology, School of Medicine, Tarbiat Modares University, Tehran, Iran

Abstract

Objective(s)
Previous studies have demonstrated that the nitric oxide (NO) dependent death of murine peritoneal macrophages activated in vitro with IFN-g and LPS is mediated through apoptosis. In the present study, we investigated the synergistic effect of LPS, IFN-g and iron on NO production and apoptosis.
Materials and Methods
After determination of iron cytotoxicity, the peritoneal macrophages of Balb/c mice were cultured with iron, LPS, and IFN-g separately, or a mixture of these for 18 hr at 37C. Then after 18 hr incubation, the level of NO in supernatant was measured by the Griess method. At the same time, after incubation with ethidium bromide and acridine orange dye, the apoptotic macrophages were detected by fluorescence microscopy.
Results
NO production was significantly greater than the control group in macrophages exposed to iron,LPS, or IFN-g  alone (P=0.02), while no significant difference was detected in apoptosis rate in the presence of LPS (P=0.08). However, the differences were remarkable between NO production and apoptosis rate in the presence of iron, LPS and IFN-g (P≤0.05).
Conclusion
These findings indicate the immunostimulatory effect of iron on NO production by IFN-g and LPS.
 

Keywords


1.            Feger F, Ferry-Dunazet H, Matsuda MM, Bordenave J, Dupouy M, Nussler AK, et al. Role of iron in tumor cell protection from the pro-apoptotic effect of nitric oxide. Cancer Res 2001; 61:5289-5294.
2.            Kim KK, Kim PK, Kwon YG, Bai SK, Nam WD, Kim YM. Regulation of apoptosis by nitrosative stress. J Biochem Mol Biol 2002; 35:127-133.
3.            Albina JE, Cui S, Mateo RB, Reichner JS. Nitric oxide-mediated apoptosis in murine peritoneal macrophages. J Immunol 1998; 150:5080-5085.
4.            Lu I, Bonham CA, Chambers FG, Watkins SC, Holfman RA, Simmons RI, et al. Induction of nitric oxide synthase in mouse dendritic cells by IFN-gamma, endotoxin, and interaction with allogenic T cells: nitric oxide production is associated with dentritic cell apoptosis. J Immunol 1998; 157:3577-3586.
5.            Gordon SA, Abou-Jaoude W, Hoffman RA, MacCarthy SA, Kim YM, Zhou X, et al. Nitric oxide induces murine thymocyte apoptosis by oxidative injury and a p53-dependent mechanism. J Leukoc Biol 2001; 70:87-95.
6.            Kaneto H, Fujii J, Seo HG, Suzuki K, Matsuoka T, Nakamura M, et al. Apoptotic cell death triggered by nitric oxide in pancreatic beta-cells. Diabetes 1995; 44:733-738.
7.            Dawson VL, Kizushi VM, Huang PL, Synder SH, Dawson TM. Resistance to neurotoxicity in cortical cultures from neuronal nitric oxide synthase-deficient mice. J Neurosci 1996; 16:2479-2487.
8.            Hirakava M, Oike M, Masuda K, Ito Y. Tumor cell apoptosis by irradiation-induced nitric oxide production in vascular endothelium. Cancer Res 2002; 62:1450-1457.
9.            McCauley SD, Gilchrist M, Befus AD. Nitric oxide: a major determination of mast cell phenotype and function. Mem Inst Oswaldo Cruz 2005; 100:11-14.
10.          Choi BM, Pae HO, Jane SI, Kim YM, Chung HT. Nitric oxide as a pro-apoptotic as well as anti-apoptotic modulator. J Biochem Mol Biol 2002; 35:116-126.
11.          Huang H. Iron overload and its association with cancer risk in humans: evidence for iron as a carcinogenic metal. Mutat Res 2003; 533:153-171.
12.          Hida AI, Kawabata T, Minamiyama Y, Mizote A,Okada S. Saccharated colloidal iron enhances lipopolysaccharide- induced nitric oxide production in vivo. Free Radic Biol Med 2003; 34:1426-1434.
13.          Kharazi S, Zavaran HA, Tiraihi T. The role of overproduction of nitric oxide in apoptosis of BALB/C mice macrophages infected with leishmania major in vitro. IAARI 2003; 2(4):209-214.
14.          Pirdel L, Zavaran HA, Tiraihi T. Role of iron overload in apoptosis of Balb/c mice macrophages infected with leishmania major in vitro. Yakhteh 2007; 9(3):196-201.
15.          Mulero V, Brock JH. Regulation of iron metabolism in murine J774 macrophages: role of nitric oxide-dependent and independent pathways following activation with gamma interferon and lipopolysaccharide. Blood 1999; 94:2383-2389.
16.          Ferret PJ, Soum E, Negre O, Fradelizi D. Auto-protective redox buffering systems in stimulated macrophages. BMC Immunol 2002; 3:1-13.
17.          Nishikawa Y, Mishima M, Nagasawa H, Igarash I, Fujisaki K, Otsuka H, et al. Interferon-gamma-induced apoptosis in host cells infected with Neospora caninum. Parasitology 2001; 123: 25-31.
18.          Meier P, Dayer E, Blanc E, Wauters JP. Early T cell activation correlates with expression of apoptosis markers in patients with end-stage renal disease. J Am Soc Nephrol 2002; 13:204-212.
19.          Luttmann W, Dauer E, Schmidt S, Mark O, Hossfeld M, Matthys H, et al. Effects of interferon- and tumour necrosis factor- on CD95/Fas ligand-mediated apoptosis in human blood eosinophils. Scand J Immunol 2000; 51:54-59.
20.          Kim YM, Chung HT, Kim SS, Han JA, Yoo YM, Kim KM. Nitric oxide protects PC12 cells from serum deprivation-induced apoptosis by cGMP-dependent inhibition of caspase signaling. J Neurosci 1999; 19:6740-6747.
21.          Gonzalez A, Lopez AS, Alegre E, Alcazar JL, Lopez-Moratalla N. Dose nitric oxide plays a role in maternal tolerance towards the foetus? J Physiol Biochem 2004; 60: 227-238.
22.          Philippe MA, Ruddell RG, Ramm GA. Role of iron in hepatic fibrosis: one piece in the puzzle. World J Gastroentrol 2007; 13:4746-4754.
23.          Liu TZ, Lee KT, Chern CL, Cheng JT, Stern A, Tasi LY. Free radical-triggered hepatic injury of experimental obstructive jaunide of rats involves overproduction activation of nuclear factor B. Ann Clin Lab Sci 2001; 31:383-390.
24.          Kawabata T, Ma Y, Yamador I, Okada S. Iron-induced apoptosis in mouse renal proximal tunules after an injection of renal carcinogen, iron-nitrilotriacetate. Carcinogenesis 1997; 18:1389-1394.
25.          Hsieh CH, Jeng SF, Hsieh MW, Chen YC, Rau CS, Lu TH, et al. Statin-induced heme oxygenase-1 increases NF-B activation and oxygen radical production in cultured neuronal cells exposed to lipopolysaccharide. J Toxicol Sci 2008; 102:150-159.