Superoxide Formation and Macrophage Resistance to Nitric Oxide-mediated Apoptosis

doi:10.1074/jbc.272.11.7253

Superoxide Formation and Macrophage Resistance to Nitric Oxide-mediated Apoptosis*

From the Faculty of Medicine, Department of Medicine IV, Experimental Division, University of Erlangen-Nürnberg, 91054 Erlangen, Federal Republic of Germany the
§ Department of Toxicology, National Health Institute, 70211 Kuopio, Finland, and
^¶ Case Western Reserve University, Molecular Cardiovascular Research Center, School of Medicine, Cleveland, Ohio 44106-4958

^‡ To whom correspondence should be addressed:
University of Erlangen-Nürnberg, Faculty of Medicine, Loschgestrasse 8, 91054 Erlangen, Federal Republic of Germany
. Tel.: 49-9131-856311; Fax: 49-9131-859202.

Abstract

RAW 264.7 macrophages, when challenged with a combination of lipopolysaccharide (10 μg/ml) and interferon-γ (100 units/ml), respond with endogenous NO^{^·} formation, which ultimately results in apoptotic cell death. Apoptosis is detected morphologically by chromatin condensation. Concomitantly we noticed the accumulation of the tumor suppressor protein p53. NO^{^·}-derived apoptosis was blocked by the NO^{^·}-synthase inhibitor N^G-monomethyl-L-arginine. Repetitive treatment of RAW 264.7 macrophages with lipopolysaccharide/interferon-γ, followed by subculturing viable cells, allowed us to select resistant macrophages which we called RES. RES cells still produced comparable amounts of nitrite/nitrate in response to agonist treatment but showed no apoptotic markers, i.e. chromatin condensation or p53 accumulation. However, RES macrophages undergo apoptosis in the presence of exogenously supplied NO^{^·}, released from the NO-donors S-nitrosoglutathione or spermine-NO. Assessment of cytochrome c reduction established that RES cells released twice the amount of superoxide compared to RAW 264.7 macrophages under both resting and stimulated conditions. We linked increased superoxide production to cellular macrophage resistance by demonstrating decreased apoptosis after simultaneous application of S-nitrosoglutathione or spermine-NO and the redox cycler 2,3-dimethoxy-1,4-naphthoquinone. Our results suggest that macrophage resistance toward NO^{^·}-mediated apoptosis is, at least in part, due to increased superoxide formation. Therefore, the balance between reactive nitrogen and reactive oxygen species regulates RAW 264.7 macrophage apoptosis.

Footnotes

↵* Financial support was received from Deutsche Forschungsgemeinschaft and the European Community. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

NO

nitric oxide

NOS

nitric-oxide synthase

GSNO

S-nitrosoglutathione

LPS

lipopolysaccharide

IFN-γ

interferon-γ

NMMA

N^G-monomethyl-L-arginine

DMNQ

2,3-dimethoxy-1,4-naphthoquinone

TBS

Tris-buffered saline.
- Received December 13, 1995.
- Revision received December 2, 1996.