Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing. Molecular basis for the synergy between interferon-gamma and lipopolysaccharide.

R.B Lorsbach,W.J Murphy,C.J Lowenstein,S.H Snyder,S.W Russell

doi:10.1016/s0021-9258(18)53940-5

R.B Lorsbach, W.J Murphy + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(18)53940-5

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Abstract

Macrophages can become activated to kill both tumor cells and a variety of microbes. Results here show that synthesis of nitric oxide (NO), a mediator of many macrophage cytotoxic functions, was greatly increased when cells of the mouse macrophage cell line RAW 264.7 were costimulated with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), compared to LPS alone. This increase paralleled increases in cytotoxicity. Northern analysis showed that increased production of NO was preceded by markedly enhanced expression of mRNA for the inducible form of macrophage NO synthase (mac-NOS), which catalyzes the synthesis of NO. Cycloheximide inhibited the induction of mac-NOS mRNA by IFN-gamma and LPS, indicating that expression of this mRNA required de novo protein synthesis. Elevated expression of mac-NOS mRNA was not due to an increase in its stability. Rather, the combination of IFN-gamma and LPS induced a much higher rate of transcription of the mac-NOS gene than did stimulation with LPS alone. These results provide one explanation of why priming and triggering stimuli, such as IFN-gamma and LPS, respectively, synergistically activate macrophages and may be applicable to explaining how IFN-gamma augments NO-dependent microbicidal activity in macrophages as well.

Highlights

Genetics, and Immunology, University of Kansas Medical Center, Kansas City, Kansas 66160-7184 and the Departments of **Cardiologyand $$Neuroscience, Johns HopkinsSchool of Medicine, Baltimore, Maryland 21205
Results here show that synthesis of nitric oxide (NO), a mediatorofmany macrophagecytotoxic functions, was greatlyincreased when cells of the mousemacrophage cell line RAW 264.7 were costimulated with bacterial lipopolysaccharide (LPS) and interferon-y (IFN-y), compared to LPS alone
Northern analysis showed that increased production of NO was preceded by markedly enhanced expression of mRNA for the inducible form of macrophage NO synthase, which catalyzes the synthesis of NO

Summary

Nitric Oxide SynthaseEGxepnression in Macrophages

Stratagene (La Jolla, CA); DNase I and phenol, GIBCO/BRL; form- saline with a rubber policeman. Reagent (0.5% sulfanilamide, 0.05% N-(1-naphthy1)ethylenediamine were added, and each sample was digested with 100 pgof dihydrochloride in2.5% HaPo') in 96-well tissue culture plates for proteinase K for 30 min a t 42 "C.Samples were extracted with 10 min at room temperature in the dark. Northern Analysis-RAW 264.7 cells (3 X lo6)were seeded into 100- neutralized by the addition of 1 M HEPES, and nuclear RNAwas mm tissue culture dishes and incubated overnight to allow for adher- ethanol-precipitated inthe presence of ammonium acetate. The plates were dehyde-3-phosphate dehydrogenase [24]) were applied to nitrocelluwashed with an equal volume of 0.1 M sodium acetate,pH 5.2, lose membranes with a dot-blotting manifold and immobilized by containing 10 mM EDTA, and thewash was combined with the SDS/ baking at 80 "C for 2 h. CDNA probes were radiolabeled with [32P]dCTP(3000 Ci/mmol; Du Pont-New England Nuclear) by random primer labeling

RESULTS

LPS nn

DISCUSSION

Because of the unavailability of an antibody specific for