Growth and viability of macrophages continuously stimulated to produce nitric oxide

Abstract

Deregulated production of nitric oxide (NO) has been implicated in the development of certain human diseases, including cancer. We sought to assess the damaging potential of NO produced under long-term conditions through the development of a suitable model cell culture system. In this study, we report that when murine macrophage-like RAW264.7 cells were exposed continuously to bacterial lipopolysaccharide (LPS) or mouse recombinant interferon-␥ (IFN-␥) over periods of 21-23 days, they continued to grow, but with doubling times 2 to 4 times, respectively, longer than the doubling time of unstimulated cells. Stimulated cells produced NO at rates of 30 to 70 nmol per million cells per day throughout the stimulation period. Within 24 hr after removal of stimulant, cells resumed exponential growth. Simultaneous exposure to LPS and IFN-␥ resulted in decreased cell number, which persisted for 2 days after removal of the stimulants. Exponential growth was attained only after an additional 4 days. Addition of N-methyl-L-arginine (NMA), an NO synthase inhibitor, to the medium inhibited NO production by 90% of all stimulated cells, partially reduced doubling time of cells stimulated with LPS or IFN-␥, and partially increased viability and growth rates in those exposed to both LPS and IFN-␥. However, when incubated with LPS and IFN-␥ at low densities both in the presence and in the absence of NMA, cells grew at a rate slower than that of unstimulated cells, with no cell death, and they resumed exponential growth 24 hr after removal of stimulants. Results from cell density experiments suggest that macrophages are protected from intracellularly generated NO; much of the NO damaging activity occurred outside of the producer cells. Collectively, results presented in this study suggest that the type of cellular toxicity observed in macrophages is markedly inf luenced by rate of exposure to NO: at low rates of exposure, cells exhibit slower growth; at higher rates, cells begin to die; at even higher rates, cells undergo growth arrest or die. The ability of RAW264.7 cells to produce NO over many cell generations makes the cell line a useful system for the study of other aspects of cellular damage, including genotoxicity, resulting from exposure to NO under long-term conditions.

Mutagenesis associated with nitric oxide production in macrophages

“…Mutagenesis was studied in cells continuously stimulated with IFN-␥ to produce NO over many generations (6) and also in those stimulated simultaneously with IFN-␥ and LPS under conditions that induced maximal NO production. Increases in MF associated with continuous stimulation are summarized in Fig.…”

“…Hygromycin B-resistant RAW264.7 macrophages were cultured at 37°C in DMEM supplemented with 10% heat-inactivated calf serum, 100 units͞ml penicillin, 100 g͞ml streptomycin, 1 mM L-glutamine (all from BioWhittaker), and 100 g͞ml hygromycin B (Boehringer Mannheim), as described (6). To establish a population with a low background MF, six populations, each consisting of Ϸ10 8 cells, were produced by expansion of initial cultures of Ϸ200 cells.…”

“…We also reported recently that growth and viability of stimulated mouse macrophage-like RAW264.7 cells are strongly influenced by NO (6). Under certain stimulation conditions, cells continued to divide and to produce NO over many generations, a capability making them a useful experimental system for characterization of toxicity resulting from long-term exposure to NO.…”

“…Cells treated with IFN-␥ in the absence of NMA were later used for isolation of individual hprt mutants (see below). Cumulative NO production was determined by measurement of nitrite plus nitrate content of the medium throughout the stimulation period (6). A second experiment of similar design was conducted, in which cells were stimulated for only 14 days.…”

“…To determine mutagenicity associated with continuous, long-term NO production, a total of 1.5 ϫ 10 8 cells were incubated for 23 days in six 150-mm plates with 30 ml of medium per plate containing 100 units͞ml IFN-␥ (Genzyme) plus 10 units͞ml polymyxin B (PMB) (Sigma), in the presence or absence of 2 mM N-monomethyl-L-arginine (NMA) (ChemBiochem Research, Salt Lake City, UT), as described (6). Untreated cells were cultured in parallel as negative controls.…”