JOURNAL
OF SURGICAL
Alterations
RESEARCH
52, 395-400 (1992)
in Macrophage Free Radical and Tumor Necrosis Factor Production by a Potassium Channel Activator H. W. POGREBNIAK,
M.D.,
Thorucic Oncology Section, Surgery Branch, National Presented
at the Annual
Meeting
of the Association
W. MATTHEWS, Cancer Institute, for Academic
The potassium channel activator nicorandil, under evaluation for antianginal management, has been shown to decrease neutrophil respiratory burst. Since our laboratory has demonstrated that reactive oxygen species (ROS) increase tumor necrosis factor (TNF) production, we hypothesized that nicorandil might decrease TNF production from a lipopolysaccharide (LPS) challenge via reduction of respiratory burst. Macrophage viability and TNF production were determined after an 1%hr exposure to 5.0 pg/ml LPS and varying concentrations of nicorandil. Nicorandil was not toxic to macrophages below 12 mM (94 + 3% viability versus control) and decreased ROS and TNF production. Intracellular superoxide production decreased from 164 + 24 OD,,, to 99 + 6 OD&,, with 10 mlMnicorandil and extracellular superoxide decreased from 3 108 +- 111 to 1760 + 2 10 nM. Hydrogen peroxide production was also decreased by 10 mM nicorandil. TNF production in response to 5 bg/ml LPS decreased from 6.8 + 0.6 to 2.7 f 0.4 rig/ml with 10 mM nicorandil. Northern and slot blot analyses demonstrate that nicorandil acts at a post-transcriptional site. These data imply that nicorandil decreases macrophage TNF production from an LPS challenge, possibly through a reduction in respiratory burst. Such compounds may prove useful in the treatment of conditions thought to be associated with free radical-lymphokine interactions such as ischemia-reperfusion injury, oxygen toxicity, adult respiratory distress syndrome, and septic 0 1992 Academic Press, Inc. shock.
INTRODUCTION
Tumor necrosis factor (TNF) is a pluripotent cytokine elaborated primarily by activated macrophages. Although first described as an endotoxin-induced serum factor which caused the necrosis of meth-A sarcomas in uiuo [l], this 17-kDa polypeptide has subsequently been demonstrated to be an important inflammatory mediator. Furthermore, treatment of a variety of cell lines with TNF can induce the release of reactive oxygen species (ROS) [2-61. ROS are generated in uiuo when phagocytic
B.S., AND H. I. PASS, M.D.
National
Institutes
Surgery, Colorado
of Health, Bethesda, Maryland
Springs, Colorado,
November
20892
20-23, 1991
cells undergo respiratory burst, and our laboratory has demonstrated that mild oxidant stress from reactive oxygen species (ROS) can induce macrophages to elaborate significant amounts of TNF in combination with other activating factors [7]. Therefore, ROS may act in an autocrine or paracrine fashion and promote the amplification of TNF production, thereby potentiating oxidant-induced tissue injury. This TNF-mediated tissue injury [6,8] has been implicated in ischemia-reperfusion [9], oxygen toxicity [lo], allograft rejection [ll, 121, septic shock [13,14], and the adult respiratory distress syndrome [9]. Nicorandil is a potassium channel activator that has been shown to decrease neutrophil respiratory burst [Xi]. Since nicorandil inhibits the release of ROS and since ROS stimulate the elaboration of TNF, we hypothesized that nicorandil may decrease TNF production through this reduction of respiratory burst. We, therefore, chose to investigate the influence of nicorandil upon macrophage ROS release as well as the effect of this compound on TNF production. METHODS
Generation of Thioglycollute-Elicited Macrophages
Murine
Three-day thioglycollate-elicited macrophages from lo- to 12-week-old female C57B1/6 mice [animal production colonies of the National Institutes of Health (NIH), Bethesda, MD] were washed twice and then suspended in complete media consisting of phenol red-free DMEM supplemented with 10% heat-inactivated, prime (endotoxin level
OF SURGICAL
Alterations
RESEARCH
52, 395-400 (1992)
in Macrophage Free Radical and Tumor Necrosis Factor Production by a Potassium Channel Activator H. W. POGREBNIAK,
M.D.,
Thorucic Oncology Section, Surgery Branch, National Presented
at the Annual
Meeting
of the Association
W. MATTHEWS, Cancer Institute, for Academic
The potassium channel activator nicorandil, under evaluation for antianginal management, has been shown to decrease neutrophil respiratory burst. Since our laboratory has demonstrated that reactive oxygen species (ROS) increase tumor necrosis factor (TNF) production, we hypothesized that nicorandil might decrease TNF production from a lipopolysaccharide (LPS) challenge via reduction of respiratory burst. Macrophage viability and TNF production were determined after an 1%hr exposure to 5.0 pg/ml LPS and varying concentrations of nicorandil. Nicorandil was not toxic to macrophages below 12 mM (94 + 3% viability versus control) and decreased ROS and TNF production. Intracellular superoxide production decreased from 164 + 24 OD,,, to 99 + 6 OD&,, with 10 mlMnicorandil and extracellular superoxide decreased from 3 108 +- 111 to 1760 + 2 10 nM. Hydrogen peroxide production was also decreased by 10 mM nicorandil. TNF production in response to 5 bg/ml LPS decreased from 6.8 + 0.6 to 2.7 f 0.4 rig/ml with 10 mM nicorandil. Northern and slot blot analyses demonstrate that nicorandil acts at a post-transcriptional site. These data imply that nicorandil decreases macrophage TNF production from an LPS challenge, possibly through a reduction in respiratory burst. Such compounds may prove useful in the treatment of conditions thought to be associated with free radical-lymphokine interactions such as ischemia-reperfusion injury, oxygen toxicity, adult respiratory distress syndrome, and septic 0 1992 Academic Press, Inc. shock.
INTRODUCTION
Tumor necrosis factor (TNF) is a pluripotent cytokine elaborated primarily by activated macrophages. Although first described as an endotoxin-induced serum factor which caused the necrosis of meth-A sarcomas in uiuo [l], this 17-kDa polypeptide has subsequently been demonstrated to be an important inflammatory mediator. Furthermore, treatment of a variety of cell lines with TNF can induce the release of reactive oxygen species (ROS) [2-61. ROS are generated in uiuo when phagocytic
B.S., AND H. I. PASS, M.D.
National
Institutes
Surgery, Colorado
of Health, Bethesda, Maryland
Springs, Colorado,
November
20892
20-23, 1991
cells undergo respiratory burst, and our laboratory has demonstrated that mild oxidant stress from reactive oxygen species (ROS) can induce macrophages to elaborate significant amounts of TNF in combination with other activating factors [7]. Therefore, ROS may act in an autocrine or paracrine fashion and promote the amplification of TNF production, thereby potentiating oxidant-induced tissue injury. This TNF-mediated tissue injury [6,8] has been implicated in ischemia-reperfusion [9], oxygen toxicity [lo], allograft rejection [ll, 121, septic shock [13,14], and the adult respiratory distress syndrome [9]. Nicorandil is a potassium channel activator that has been shown to decrease neutrophil respiratory burst [Xi]. Since nicorandil inhibits the release of ROS and since ROS stimulate the elaboration of TNF, we hypothesized that nicorandil may decrease TNF production through this reduction of respiratory burst. We, therefore, chose to investigate the influence of nicorandil upon macrophage ROS release as well as the effect of this compound on TNF production. METHODS
Generation of Thioglycollute-Elicited Macrophages
Murine
Three-day thioglycollate-elicited macrophages from lo- to 12-week-old female C57B1/6 mice [animal production colonies of the National Institutes of Health (NIH), Bethesda, MD] were washed twice and then suspended in complete media consisting of phenol red-free DMEM supplemented with 10% heat-inactivated, prime (endotoxin level
