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  • Title: Activated peritoneal macrophages inhibit the proliferation of rat ascites hepatoma AH-130 cells via the production of tumor necrosis factor-alpha and nitric oxide.
    Author: Maekawa H, Iwabuchi K, Nagaoka I, Watanabe H, Kamano T, Tsurumaru M.
    Journal: Inflamm Res; 2000 Oct; 49(10):541-7. PubMed ID: 11089907.
    Abstract:
    OBJECTIVE: To study the effect of peritoneal macrophages on tumor cell proliferation, we cultured ascites hepatoma AH-130 cells with unstimulated, or lipopolysaccharide (LPS)- or interleukin (IL)-2-stimulated rat peritoneal macrophages, 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)-alpha and nitric oxide (NOx, nitrate + nitrite) in the culture supernatants were measured. Furthermore, anti-TNF-alpha antibody (10 microg/ml) and nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMMA, 100 microM) were added to the coculture, and their effect on AH-130 cell proliferation was examined. RESULTS: When AH-130 cells were co-cultured with unstimulated peritoneal macrophages, proliferation of AH-130 cells was not affected. In contrast, when AH-130 cells were cocultured with peritoneal macrophages in the presence of LPS (0.1-20 microg/ml) or IL-2 (1-200 U/ml), proliferation of AH130 cells was dose-dependently suppressed by LPS or IL-2. Moreover, LPS- or IL-2-stimulation 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 TNF-alpha and NOx production by AH-130 cells incubated without macrophages. Interestingly, anti-TNF-alpha antibody and L-NMMA significantly inhibited the suppression of AH-130 cell proliferation by LPS- or IL-2-stimulated macrophages (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 absence of macrophages. CONCLUSION: Together these observations suggest that when peritoneal macrophages are activated by LPS and IL-2, they suppress the proliferation of ascites hepatoma AH-130 cells via the production of TNF-alpha and nitric oxide.
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