Iron down-regulates macrophage anti-tumour activity by blocking nitric oxide production

Abstract

Although the inhibitory effect of iron on macrophage production of tumoricidal free radical nitric oxide (NO) has been reported, its possible influence on macrophage anti-tumour activity has not been established. In the present study, FeSO4 markedly reduced IFN-gamma + LPS-induced NO synthesis in mouse and rat macrophages. The effect of iron coincided with the loss of macrophage cytotoxic activity against NO-sensitive C6 rat astrocytoma and L929 mouse fibrosarcoma cell lines, as measured by MTT assay for cellular respiration and the crystal violet test for cell viability. Tumour cell survival did not improve further in the presence of FeSO4 if macrophage NO release and cytotoxicity were already blocked by aminoguanidine. In accordance with the results obtained with exogenous iron, cell membrane permeable iron chelator o-phenanthroline enhanced both macrophage NO release and anti-tumour activity. Iron also down-regulated NO production and increased the viability of L929 fibrosarcoma cells stimulated with IFN-gamma + LPS in the absence of macrophages. However, neither NO release nor cell viability was affected by iron addition to cultures of the C6 astrocytoma cell line. Iron was unable to prevent L929 and C6 cell death induced by the NO releasing chemicals SNP and SIN-1, indicating that iron-mediated inhibition of NO synthesis, rather than interference with its cytotoxic action, was responsible for the protection of tumour cells. Collectively, these results indicate that iron might protect tumour cells by reducing both macrophage and tumour cell-derived NO release.

Fig. 1

Effect of iron on IFN-γ + LPS-induced NO production in macrophages. (a) Mouse macrophages or (b) rat macrophages were stimulated with IFN-γ + LPS, in the presence or absence of different concentrations of FeSO₄. (c) Mouse macrophages were stimulated with IFN + LPS, in the presence or absence of FeSO₄ and/or o-phenanthroline (OPH; 0·2 mm). (a–c) Nitrite concentration was determined after 48 h, and macrophage viability was assessed at the same time by MTT test (control viability of 100% refers to macrophage cultures without FeSO₄); *P < 0·05 refers to cell cultures without FeSO₄ (a,b) or o-phenanthroline (c).

Fig. 2

Iron down-regulates macrophage anti-tumour activity. (a–d) Co-cultures of murine (a,b,e,f) or rat macrophages (c–e) with C6 (a,c,e) or L929 (b,d,f) cells were stimulated with IFN-γ + LPS, in the presence or absence of different concentrations of FeSO₄. (g) Rat macrophages were incubated with C6 cells and stimulated with IFN-γ + LPS, in the presence or absence of CuSO₄. (h) Co-cultures of murine macrophages and L929 cells were stimulated with IFN-γ + LPS, in the presence or absence of FeSO₄ (0·2 mm) and o-phenanthroline (OPH; 0·2 mm). (a–h) After 48 h of incubation, nitrite concentration (a–d,g,h) was measured, and cell viability was determined by MTT (a–d,g,h) or crystal violet (e,f) assay. Control value for NO production (100%) represents macrophage NO release in the absence of FeSO₄. Control value for the cell viability (100%, with standard deviations less than 10% in a–d,g,h) refers to viability of tumour cells incubated in the absence of macrophages; *P < 0·05 refers to cell cultures without FeSO₄ (a–f) or o-phenanthroline (h).

Fig. 3

Iron protects tumour cells by down-regulating macrophage and tumour cell NO release. (a,b) Murine macrophages were incubated with C6 (a) or L929 (b) cells and stimulated with IFN-γ + LPS in the presence or absence of aminoguanidine (AG; 2 mm) and FeSO₄ (0·2 mm ive (b)). (c,d) Confluent L929 (c) or C6 (d) cells were stimulated with IFN-γ + LPS, in the presence or absence of various concentrations of FeSO₄. (a–d) Nitrite concentration and cell viability were determined after 48 h. Control value (100%) for NO production represents macrophage or tumour cell NO release in the absence of AG or FeSO₄. Control value for the cell viability (100 ± 5·2%) refers to viability of tumour cells incubated in the absence of macrophages or IFN-γ + LPS (c,d); *P < 0·05 refers to cultures without FeSO₄.

Fig. 4

Iron does not affect tumoricidal action of exogenous NO. C6 cells were incubated with various doses of NO donors SNP (a,c) and SIN-1 (b,d), in the presence or absence of different concentrations of FeSO₄. Nitrite concentration (a,b) and cellular respiration (c,d) were determined after 48 h of incubation.