Activated peritoneal macrophages inhibit the proliferation of rat ascites hepatoma AH-130 cells via the production of tumor necrosis factor-alpha andnitric oxide

Objective: To study the effect of peritoneal macrophages on tumor cell prol iferation, we cultured ascites hepatoma AH-130 cells with unstimulated, or lipopolysaccharide (LPS)- or interleukin (IL)-2-stimulated rat peritoneal m acrophages, and examined the proliferation of AH-130 cells. Materials and Methods: Rat peritoneal macrophages isolated from male Wistar rats were co-cultured with AH-130 cells in the absence or presence of LPS or IL-2. After incubation, proliferation of AH-130 cells was analyzed using flow cytometry. In addition, the levels of tumor necrosis factor (TNF)-alp ha and nitric oxide (NOx, nitrate + nitrite) in the culture supernatants we re measured. Furthermore, anti-TNF-alpha antibody (10 mug/ml) and nitric ox ide synthase inhibitor, N-G-monomethyl-l-arginine (L-NMMA, 100 muM) were ad ded to the coculture, and their effect on AH-130 cell proliferation was exa mined. Results: When AH-130 cells were co-cultured with unstimulated peritoneal ma crophages, proliferation of AH-130 cells was not affected. In contrast, whe n AH-130 cells were cocultured with peritoneal macrophages in the presence of LPS (0.1-20 mug/ml) or IL-2 (1-200 U/ml), proliferation of AH-130 cells was dose-dependently suppressed by LPS or IL-2. Moreover, LPS- or IL-2-stim ulation increased the levels of TNF-alpha and NOx in the supernatants of AH -130 cell and macrophage co-culture, although LPS and IL-2 did not induce T NF-alpha and NOx production by AH-130 cells incubated without macrophages. Interestingly, anti-TNF-alpha antibody and L-NMMA significantly inhibited t he suppression of AH-130 cell proliferation by LPS- or IL-2-stimulated macr ophages (p<0.05). Furthermore, exogenously added recombinant rat TNF-<alpha > (0.26-1300 ng/ml) or NO donor (GSNO, S-nitroso-L-glutathione) (0.1-10 mM) dose-dependently suppressed the proliferation of AH-130 cells in the absen ce of macrophages. Conclusion: Together these observations suggest that when peritoneal macrop hages are activated by LPS and IL-2, they suppress the proliferation of asc ites hepatoma AH-130 cells via the production of TNF-alpha and nitric oxide .