Inhibition of Inducible Nitric-Oxide Synthase Protects Human T
Cells from Hypoxia-Induced Apoptosis
Juliann G. Kiang, Sandeep Krishnan, Xinyue Lu, and Yansong Li
Armed Forces Radiobiology Research Institute and Departments of Radiation Biology, Pharmacology, and Medicine,
Uniformed Services University of the Health Sciences, Bethesda, Maryland (J.G.K.); Department of Internal Medicine,
Washington Hospital Center, Washington, DC (S.K.); and Department of Cellular Injury, Walter Reed Army Institute of
Research, Silver Spring, Maryland (X.L., Y.L.)
Received August 27, 2007; accepted December 13, 2007
Sodium cyanide-induced chemical hypoxia triggers a series of
biochemical alterations leading to apoptosis in many cell types,
including T cells. It is known that chemical hypoxia promotes
inducible nitric-oxide synthase (iNOS) gene transcription by acti-
vating its transcription factors. To determine whether iNOS and
NO production are responsible for chemical hypoxia-induced
apoptosis, we exposed human Jurkat T cells to sodium cyanide in
the presence or absence of iNOS inhibitors. We found that iNOS
expression is necessary for hypoxia-induced lipid peroxidation
and leukotriene B4generation. The inhibition of iNOS limited T-cell
apoptosis by decreasing the activity of caspase-3 without affect-
ing the expression of Fas/Apo-1/CD95 on the surface membrane
of T cells. These data suggest iNOS-mediated NO produced
endogenously in the T cell alters overall T-cell function and results
in apoptosis. Proper control of iNOS expressed in the T cell may
represent a useful approach to immunomodulation.
The low oxygen cellular environment characteristic of mal-
adies such as stroke, heart attack, anemia, ischemia, and
hemorrhage has been shown to lead to cell injury both in vivo
and in vitro. Hypoxia promotes NO production that leads to
polymorphonuclear neutrophil (PMN) infiltration of tissues
and leukotriene B4(LTB4) generation (Stojadinovic et al.,
1995). It has been reported that hypoxia stimulates the in-
flammatory response by up-regulating the early response
gene, inducible nitric-oxide synthase (iNOS), which leads to
the rapid overproduction of NO. Excessive amounts of NO
seem to cause damage when it combines with superoxide to
form peroxynitrite (ONOO?), a powerful oxidant that can be
cleaved into highly reactive free radicals such as OH?and
NO per se, that leads to cellular damage. In fact, NO at
appropriate levels is critical for normal tissue homeostasis.
For example, maintenance of vascular integrity requires NO
(Drexler, 1999), but abnormally high levels lead to vascular
leakage (Ward et al., 2000).
?(Yasmin et al., 1997). It is the overproduction of NO, not
Deletion of the iNOS gene in mice or treatment of mice
with L-N6-(1-iminoethyl)-lysine (L-NIL), a selective inhibitor
of iNOs, prevents hypoxia-induced injury (Hierholzer et al.,
1998). Other agents such as 5-androstenediol and geldana-
mycin (Kiang et al., 2007a,b) that inhibit iNOS and therefore
the overproduction of NO have also been shown to limit
hypoxia-induced tissue damage. Treatment with N?-nitro-L-
arginine (LNNA), an irreversible inhibitor of constitutive
nitric oxide synthase (cNOS) and reversible iNOS inhibitor,
results in significant reduction of local tissue damage, PMN
infiltration, and LTB4 generation (Chabrier et al., 1999a,b).
Stojadinovic et al. (1995) observed that hypoxia resulting
from ischemia increases generation of PGE2 and LTB4, and
PGE2 and LTB4 are proinflammatory mediators that can
lead to multiple organ dysfunction and failure (Kiang and
Tsen, 2006). Nonetheless, the relation between iNOS and
LTB4 as well as PGE2 was not clear.
Our laboratory has previously investigated various stress
response genes involved in hypoxia, including iNOS, 70-kDa
inducible heat shock protein (HSP-70i), hypoxia-inducible
factor (HIF)-1?, Bcl-2, and p53. In human intestinal epithe-
lial T84 cells and human Jurkat T cells, NaCN-induced hyp-
oxia increases iNOS and HSP-70 mRNA; whereas p53 is
induced only in T84 cells and Bcl-2 only in Jurkat T cells
This work was supported by Armed Forces Radiobiology Research Institute
RAB2CF and Department of Defense RAMII STO C (both to J.G.K.).
Article, publication date, and citation information can be found at
ABBREVIATIONS: PMN, polymorphonuclear neutrophil; LT, leukotriene; iNOS, inducible nitric-oxide synthase; ONOO?, peroxynitrite; L-NIL,
L-N6-(1-iminoethyl)-lysine; LNNA, N?-nitro-L-arginine; cNOS, constitutive nitric oxide synthase; PG, prostaglandin; HSP, heat shock protein;
HSP-70i, 70-kDa inducible heat shock protein; HIF, hypoxia-inducible factor; siRNA, small interfering RNA; PBS, phosphate-buffered saline; i,
inducible; MDA, malondialdehyde; ANOVA, analysis of variance; CON, control; HX, hypoxia; lipo, Lipofectamine reagent.
U.S. Government work not protected by U.S. copyright
Mol Pharmacol 73:738–747, 2008
Vol. 73, No. 3
Printed in U.S.A.
at ASPET Journals on December 30, 2015
(Kiang et al., 2003). LNNA treatment blocks iNOS, Bcl-2,
and HSP-70 mRNA, but it increases p53 mRNA (Kiang et al.,
2003). We showed in mice that hemorrhage increases iNOS
7 h before HSP-70 and HIF-1? (Kiang et al., 2004), and we
hypothesized that the degree of severity of hypoxia correlates
with the amount of iNOS mRNA expression.
NO has been suggested to mediate apoptosis of T cells and to
regulate peripheral responses. In experimental allergic enceph-
alomyelitis, NO limits inflammatory demyelination by elimi-
death (Zettl et al., 1997). Interestingly, constitutive up-regula-
tion of iNOS in animals that lack glucocorticoid receptor is
responsible for immunosuppression and resistance to the devel-
opment of experimental allergic encephalomyelitis (Marchetti
et al., 2002). In the thymus, NO generated in association with
T-cell receptor stimulation functions to induce deletion of dou-
ble-positive thymocytes, especially when their T-cell receptor is
stimulated (Tai et al., 1997).
Although hypoxia promotes apoptosis in various cell types,
the mechanisms involved are not known. Since hypoxia in-
creases the expression of iNOS, we sought to determine
whether hypoxia mediates apoptosis by increasing the iNOS-
mediated production of NO and lipid peroxidation. We also
asked whether levels of PGE2, LTB4, HSP-70, and HIF-1?
are regulated by iNOS in hypoxic cells. Using sodium cyanide
to induce chemical hypoxia in human Jurkat T cells, we show
that the increased iNOS is responsible for altered NO, LTB4,
lipid peroxidation, caspase-3 activation, and apoptosis.
Fig. 1. Chemical hypoxia up-regulates protein expression of
iNOS, HSP-70i, and HIF-1?. Human Jurkat T cells were
treated with 10 mM NaCN for 1, 2, 3, or 4 h followed by 16-h
recovery. In parallel experiment, cells were treated with 0.1,
0.5, 1, 5, 10, or 50 mM NaCN for 1 h followed by 16-h recovery.
Cell lysates were immunoblotted with antibodies against
iNOS (A and B), HSP-70i (C and D), and HIF-1? (E and F).
Protein was quantitated densitometrically and normalized
with actin. ?, P ? 0.05 versus control groups, determined by
one-way ANOVA and Studentized range test.
iNOS Induces Apoptosis
at ASPET Journals on December 30, 2015
We thank HM1 Neil Agravante and Joan Smith for technical
assistance and Dr. David E. McClain for critiques and discussion.
The opinions and assertions contained in this article are the private
views of the authors and are not to be construed as official or
reflecting the views of the Army, the Department of Defense, The
Armed Forces Radiobiology Research Institute, or the Uniformed
Services University of the Health Sciences.
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Address correspondence to: Dr. Juliann G. Kiang, Armed Forces Radiobi-
ology Research Institute, Bldg. 46, Room 2423, 8901 Wisconsin Ave., Bethesda,
MD 20889-5603. E-mail: email@example.com
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