J. Fast, Robert. C. Lynch, and. Richard. W. Leu. The Samuel. Roberts. Noble ...... S., Fiore,. N., and. Williamson,. B. An endotoxin-induced serum factor that.
Nitric oxide production by tumor targets in response to TNF: paradoxical correlation with susceptibility to TN F-mediated cytotoxicity without direct involvement in the cytotoxic mechanism David The
J. Fast,
Samuel
Robert
Roberts
C. Lynch,
Noble
Foundation,
and Inc.,
Richard
Abstract: cytotoxic termined
Tumor for some whether
sensitive production a cell line
tumor targets was related to TNF-stimulated of NO by the tumor cell itself. We found that that was sensitive to TNF-mediated cytotoxic-
necrosis factor (TNF) is selectively tumor cells in vivo and in vitro. We deTNF-mediated cytotoxicity for TNF-
ity produced NO in response to TNF as measured by the accumulation of nitrite in the supernatants of TNFstimulated cells. Production of NO in response to TNF was inhibited by the competitive substrate inhibitor, NG monomethyl-L-arginine (NMMA). The kinetics of NO production in response to TNF indicated that most of the NO was produced during the first 24 h and peaked after 48 h of culture and that TNF-stimulated NO production was dose dependent. TNF-resistant cell lines produced less NO than a TNF-sensitive cell line, and the amount nitrite produced correlated with the relative sensitivity each cell line to TNF-mediated cytotoxicity. In addition,
of of
interferon-’y
augmented
NO generated exogenously sodium nitroprusside was
the
by culture cytotoxic for
amount
of
in the presence both sensitive
of and
resistant cells in a dose-dependent manner. We were unable, however, to demonstrate directly a role for NO in TNF-mediated cytotoxicity as NMMAand arginine-free media provided little protection from TNF-mediated cytotoxicity. We tentatively conclude that the ability of adherent murine tumor cells to produce nitric oxide in response to TNF correlates directly with their level of sensitivity to TNF-mediated cytotoxicity, although NO thus produced appears toxic mechanism. Key
Words:
not
J.
nitric
to be Leukoc.
oxide
directly Biol.
tumor
involved in 52: 255-261;
necrosis
Section,
Ardmore,
direct
the cyto1992.
has
susceptibility
a great by
icity
is
TNF,
not
oxygen
been
the
mutase
of [10]
involved in
anaerobic
ity. Other decreased
metabolic consumption
inhibition
of
metabolic
to
suggests
and
superoxide
TNF-mediated
electron
that
[9]
conditions
effects induced of oxygen and
mitochondrial
effects cytotox-
manganous
resistance
number
TNF-mediated Evidence
as
mitochondrial
result
receptor
the
of
understood.
are
overexpression
between
mechanism
completely
radicals
made
TNF [7, 8]. deal is know about
to
Although induced
discytotoxic-
by synthesis
TNF include of ATP [11],
transport
[11],
inhibition
of DNA topoisomerases [12, 13], and dissolution of microfilaments [14]. Despite such a multiplicity of effects, it is not known how these phenomena are related to one another or what second signals are generated in TNF-stimulated cells that
lead
to
TNF
types
cytotoxicity.
has
also of
cidated. brain
Two
Nitric
tion
[16,
20]
toxic
effector
[25],
and
lates
with
[22,
by
molecule the
and
ent
the
results
stimulated that were that response
of
interferon-’y
thus
[19],
cells mechanism
iron in
acid
the
activities
depending in
this
sensitive produced
but
(IFN-’y), to
did
were
on
the
appear
27]
of two
cell
the
target
very
differ-
of
origin. that
do
so
in the
conditions
play
TNF cells also NO
presence they
cytotoxicity.
to
ironsystem
murine tumor cytotoxicity. We did not produce to
which
21]. cytocorre-
of
demonstrate
TNF-mediated
not
killing
transport
23,
induced
under
a
mycobacteria
displays
paper
of [19,
of
[15,
the
vasodila-
inactivation
molecule
presented
to TNF
to
is
electron
cycle
effector
and
[22-24],
and
elu-
stimulation
respectively
The
cell
been
macrophages
tumor of
of
have
leading
NO production by adherent sensitive to TNF-mediated cells that are resistant to TNF
in
became
NO
endothelium
for
citric
a single
biological The
for
oxidative
a variety
through
activated
enzymes
Thus
in
the
[26].
release
the
NO
neurotransmission,
Leishmania
27]
stimulate
activity
produced
sulfur-containing
cells.
to produce
functions
cyclase and
oxide
to
primary
guanylate
Nitric
shown
oxide produced in cGMP production
generates
soluble
been
L-arginine
[15-18].
show
factor
Oklahoma
correlation
and
metabolism
NO produced in response to TNF by both sensitive and resistant cells and correspondingly enhanced the susceptibility of resistant cells to TNF cytotoxicity. Both sensitive and resistant cells were sensitive to NO, however, in that recombinant
W. Leu
Immunology
Nitric
a significant
also oxide
role
in
INTRODUCTION Tumor
nally
necrosis
described
factor-a
for
is
its ability
a multifunctional
to kill
cytokine
transplantable
origi-
tumors
in
vivo [1]. TNF has also been shown to play a role in inflammation [2, 3], shock [4], and the pathogenesis of such diseases as malaria [5]. Such a variety of biological effects reflects observations that TNF receptors have been demonstrated on all nucleated ing of TNF to specific
cells that have been receptors is necessary,
tory,
in
for
cytotoxicity
TNF-sensitive
tested but
tumor
[6]. Bindnot obligacells,
as
no
Journal
Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; NMMA, NCmonomethylLarginine; OD, optical density; PBS, phosphate-buffered saline; rIFN-’y, recombinant interferon-v; SDS, sodium dodecyl sulfate; SNP, sodium nitroprusside; TNF, tumor necrosis factor. Reprint requests: David J. Fast, The Samuel Roberts Noble Foundation, Inc., Biomedical Division, Immunology Section, Post Office Box 2180, Ardmore, OK 73402. Received February 13, 1992; accepted April 24, 1992.
of
Leukocyte
Biology
Volume
52,
September
1992
255
TNF-mediated
cytotoxicity,
to exogenously by TNF-stimulated
generated tumor
for their sensitivity role of NO in the
although
the
NO. Thus the targets provides
to cytotoxicity, process remains
cells
were
sensitive
production a useful
nm
(0D500)
was
matic plate the formula
of NO marker
determined
reader.
with
Percent
although the functional to be determined.
OD600
% Cytotoxicity AND
murine
Gibco BRL gle’s medium (Washington,
all reagents were purchased from (St. Louis, MO). Recombinant from Genzyme (Boston, MA) or
(Gaithersburg, (DMEM) DC), fetal
MD), Dulbecco’s and RPMI 1640 calf serum (FCS)
Sterile Systems (Logan, from Gibco Laboratories monomethyl-L-arginine Diego, CA).
Cell
UT),
RPMI (Grand (NMMA)
1640 Island, from
modified Eafrom Mediatech from Hyclone Select-Amine NY), and Calbiochem
Kit A1G (San
The L929, BALB/3T12-3, were obtained from (Rockville, MD). All plemented Cells were to confluence.
and C3H/MCA American Type cells were maintained
with nonessential harvested by mild
amino trypsin
clone 16 cell lines Culture Collection in DMEM sup-
acids and 10% digestion when
FCS. grown
from
sample x
untreated
100
control
Because
NO
nitrite
production
produced
by
in TNF-sensitive
tumoricidal
tumor
macrophages
in
response to TNF is a potent cytotoxic effector molecule for tumor targets, we tested the hypothesis that TNF may stimulate NO production in tumor cells sensitive to the cytotoxic effects of TNF. In effect, TNF-stimulated production of NO by the TNF-sensitive cell might explain the mechanism of TNF-mediated cytotoxicity. To test this possibility, L929 cells were incubated in the presence or absence of murine rTNF of the culture and assayed
period, for the
a stable end product of NO production. 1 demonstrate that L929 cells stimulated creased their production of nitrite fourfold of nitrite produced by untreated control Figure 1 are data in which NMMA, arginine analogue previously shown to tion [23], totally inhibited TNF-stimulated
cell-free presence
supernaof nitrite,
The
data in Figure with TNF inabove the amount cells. Also shown in a methylated Linhibit NO producproduction of nitrite.
assay
Nitric oxide generation was determined as accumulated nitrite by a modification of the method of Ding et al. [28]. Briefly, cells were seeded in 6-well cluster plates at 1 x 106/well and cultured overnight. TNF (10 ng/ml) was added and the cells were cultured an additional 48 h, at which time cell-free supernatants were tested for the presence of nitrite by the Griess reaction using 4 volumes of sample to 1 volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochoride, 2.5% H3PO+). The standard procedure for this assay calls for I volume of sample to 1 volume of Griess reagent. However, we have found that a 4:1 ratio of sample to Griess reagent allows more sensitivity at low concentrations of nitrite, approximately twice that of the standard assay. In addition, standard curves generated using the 4:1 remain linear to higher concentrations of nitrite than those generated by the standard procedure. Nitrite in the samples was extrapolated from a standard curve using sodium nitrite as a standard.
TNF
TNF stimulates targets
for 72 h. At the end tants were harvested
lines
Nitrite
auto-
RESU LTS specified otherwise, Chemical Company TNF was obtained
Sigma
test
MR700 determined
METHODS
Reagents Unless
was
(1-
=
0D600
MATERIALS
a Dynatech
cytotoxicity
Kinetics targets To determine in response
by TNF-stimulated
the kinetics of nitrite to TNF, supernatants
production were harvested
tumor by
L929 cells 24, 48, and of
inno
0.14
#{149} -TNF 0.12
9
T
+TNF
a) 0.10 0.08
tested
for sensitivity to TNF published methods [29). seeded with I x 10 cells and
using modifications Briefly, microtiter cultured overnight.
0.06
In experiments in which arginine-free medium was used, the medium was aspirated and replaced with RPMI 1640-10% FCS ± L-arginine. The cells were cultured for 1 h and recombinant TNF (rTNF) was added to the wells. In some experiments, NMMA (500 riM) was added to the wells and incubated for 1 h at 37#{176}Cprior to assay. Recombinant TNF was then added to the wells and incubated an additional 48 h at 37#{176}C,at which maining viable cells in ethanol buffered
production
72 h after TNF stimulation and assayed for the presence nitrite. The data in Figure 2 demonstrate that a significant amount of nitrite, a fivefold increase above that in untreated controls, was detectable after 24 h of culture. The maximum amount of nitrite, 0.133 mM, was produced by cells cubated for 48 h after the addition of TNF. There was
assay
Cells were of previously wells were
of nitrite
time were
the medium stained with
(30%), formaldehyde saline (PBS). Stained
sodium
dodecyl
256
Journal
sulfate
of Leukocyte
(SDS)
was removed crystal violet
(3%), Dulbecco’s cells were solubilized and
Biology
the
0.04
density
Volume
52,
CONTROL
and re(0.5%)
Fig.
1. TNF-stimulated
were cultured
NO
1992
in
6-well
cluster
NMMA
production
plates
by L929
with
Cells.
L929
cells
(1 x
106)
murine rTNF (10 ng/ml) in the presence or absence of NMMA (500 sM) for 72 h, at which time the supernatants were assayed for the presence of nitrite. Results derived from six cxperiments are expressed as mean mM nitrite produced per 1 x 106 cells ± SD.
at 600
September
I
0.00
phosphatewith 1%
optical
1
0.02
o.18 0.16
also
.I[
#{149} -TNF D+TNF
-
production
by
The data sensitivity
0.10
show the response
0.06
NO 0.04
I
0.02 0.00 24
48
experiments
are
difference natants did not Production
expressed
as mean
mM
nitrite
produced
per
in the amount of nitrite present after assayed 1, 2, 4, and 8 h after stimulation contain detectable levels of nitrite (data of NO at 24 and 48 h was totally
the presence of protein synthesis not
cycloheximide is required
(25 for
NO
in the
on NO production production by
the
by these L929-S
cells in cells in-
supernatant derived from I x 106
of nitrite
is dose
Differences in nitrite and -resistant tumor
cells
was five ±
SD.
h. Superwith TNF not shown). abrogated in 72
/Lg/ml) suggesting production to
that occur
next determined sensitivity to in their ability the data of one
whether rTNF-mediated to produce
in Figure sensitive
any
of the
cell
lines.
generated NO is cytotoxic tumor targets
TNF sensitivity is not decreased arginine-free media
dependent
production targets
by
for TN F-sensitive produced targets. resistant
tumor targets were sensitive to NO-mediated cytotoxicity. Cells were cultured with various concentrations of sodium nitroprusside (SNP), which spontaneously generates NO in aqueous solutions, and then assayed for cytotoxicity after 18 h of culture. The data in Figure 6 demonstrate that SNP was cytotoxic for all cell lines tested in a dose-dependent manner. In fact, the resistant cell lines, 3Tl2 and MCA, were more sensitive to SNP-generated NO than was the sensitive cell line, L929. Addition of TNF to these cultures had no additional cytotoxic effect above that of SNP alone (data not shown).
presence
The previous results indicated toxic for tumor targets. We produced by the tumor target
To determine if the production of nitrite in response to TNF was dose dependent, L929 cells were stimulated with various concentrations of rTNF (0.01 to 100 ng/ml) for 48 h and then tested for production of nitrite. The data in Figure 3 demonstrate that nitrite was produced in response to TNF in a dose-dependent manner. The level of nitrite production began to peak at 10 ng/ml, so this concentration was used in the remaining experiments.
poses, tivities
-
Sensitivity of the L929 In contrast, two TNFbecame more sensitive The data in Figure SB
Nitric oxide is a potent cytotoxic effector molecule by tumoricidal macrophages to kill certain tumor Thus we next determined whether sensitive and
72
shown).
Production
production
Exogenously and -resistant
Time (Hr) Fig. 2. Kinetics of NO production. L929 cells were cultured of rTNF (10 ng/ml) for 24, 48, or 72 h. At each time point, removed and assayed for the presence of nitrite. Results
We their vary
and
creased from 0.025 mM in response to TNF alone to 0.04 mM in response to IFN-’y plus TNF, by the 3T12 cells from 0.005 to 0.0012 mM, and by the MCA cells from 0.008 to 0.026 mM. Treatment with IFN--y alone did not stimulate
0.08
(data
effect of IFN-y to TNF. Nitrite
TNF-sensitive
in Figure 5A demonstrate of sensitive and resistant
0.12 a)
z
nitrite
tumor targets. of IFN-’y on
cells to TNF-mediated cytotoxicity. cells was not affected by IFN-y. resistant cell lines, 3T12 and MCA, to TNF when cultured with IFN-’y.
0.14-
.
augment
resistant the effect
by NMMA-
that exogenous next determined in response
and
NO was cytowhether NO to TNF played a
by TNF-sensitive other
cell lines that vary in cytotoxicity would also nitrite. For comparative pur-
4A demonstrate and two resistant
the cell
relative sensilines to TNF-
a)
z E
mediated cytotoxicity. The data in Figure 4B demonstrate the ability of the cell lines to produce NO in response to TNF. The L929 cells, but neither of the resistant cell lines, produced significant amounts of nitrite in response to TNF. Thus, relative
the
ability
sensitivity
to produce of a cell
NO to
correlated TNF-mediated
IFN--1 treatment augments the produced in response to TNF
amount
directly
with
the
cytotoxicity.
of nitrite
Because IFN--y augments the amount of nitrite produced by macrophages [15] and fibroblasts [18], up-regulates TNF receptors [30-32], and increases sensitivity of cells to TNFmediated cytotoxicity [33], we tested whether rIFN-’y would
TNF ng/mI Fig. 3. TNF dose response for NO production. L929 cells were cultured in the presence of TNF at the concentrations indicated. Supernatants were removed after 48 h of culture and assayed for nitrite. Results derived from five experiments
are
expressed
Fast et al.
TNF-stimulated
as
mean
mM
nitric
nitrite
oxide
produced
in tumor
±
SD.
cells
257
100
0.10
.1 C.)
x
80
O.08
60
0.06
0 0
#{149} o +TNF
z
>‘
0
E
40
0.04
20
0.02
0 0.10
1.00
.
B
10.00
--=1
0.00
L929
3T12
MCA
TNF ng/ml Fig.
4.
NO
were
assayed
for
3Tl2,
and
L929,
nitrite.
production
Results
role
in
differs
their
with
sensitivity
MCA
derived
to
cells
were
from
five
TNF-mediated
TNF
sensitivity.
(A)
TNF-mediated cultured
in
the
experiments
3T12,
of
expressed
To
test
and
Results
presence
are
cytotoxicity.
L929,
cytosoxicity.
rTNF
(10
as mean
this
MCA
cells
derived
from
ng/ml)
mM
for
nitrite
were
plated
eight
experiments
48
h,
at
in
which
produced
per
microtiter are
time
the
wells
at
expressed
a density as
supernatants
I x 106 cells
±
of
mean
%
were
assayed
I x
l0
cells/well
cytotoxicity for
the
and
SD. (B)
±
presence
of
SD.
cells with NMMA before assay for sensitivity to TNFmediated cytotoxicity in the presence of IFN-’y also had no effect, suggesting that the mechanism of IFN-y-enhanced TNF sensitivity is not due to increased production of NO (data not shown).
possibility,
TNF cytotoxicity assays were performed in which the production of NO was blocked with either NMMAor arginine-free media. The data in Figure 7 demonstrate that NMMAand arginine-free media had no effect on the sensitivity of these cells to TNF-mediated cytotoxicity. Although not shown here, NO production by TNF-stimulated cells was abrogated by NMMA-free (Fig. 1) and arginine-free media. We also tested the NO scavengers hemoglobin and methylene blue [2] and found them to have no effect on any cell line in terms of their sensitivity to TNF-mediated cytotoxicity (data not shown). Pretreatment of L929-S, 3T12, and MCA
DISCUSSION TNF
possesses
the ability resistant sensitive
a variety
of biological
to kill tumor to the cytotoxic cells are readily
effects,
one
of which
is
cells. Most tumor cells, however, are effects of TNF. In addition, TNFconverted to a resistant phenotype
a) >.
z
0
x 0
E
0
>‘
0
0.625
0.156
2.5
L929-S
10
3T12
MC-1
IFNF] ng/mI Fig.
5.
cells/well, as for
258
mean
Effect
of IFN-y
treated %
with
on IFN--y
cytotoxicity
48 h, at which
Journal
TNF-mediated (50
± SD.
time
the
(B)
supernatants
of Leukocyte
cytotoxicity
U/ml),
and
L929, were
Biology
and
assayed
3T12,
for
and
assayed
their
MCA for
Volume
NO production. (A) L929, sensitivity to TNF-mediated cells
nitrite.
52,
were
Results
cultured derived
September
in from
1992
3T12, and cytotoxicity.
the
presence
five
experiments
MCA cells were Results derived
of IFN--y are
(50 expressed
U/mI),
plated from TNF
as mean
in microtiter six experiments (10
mM
ng/ml), nitrite
wells at 1 x I0 are expressed
or per
IFN--y
1 x
106
and cells
TNF ±
SD.
by long-term presented resistance
exposure to low levels of TNF [34]. The results here may provide a potential explanation for TNF in some cells. We have demonstrated that the sen-
sitivity of a cell line to TNF-mediated cytotoxicity correlated directly with the ability of that cell line to produce NO in response to TNF. In addition, treatment with IFN--y converted resistant cells to TNF-sensitive while concomitantly reconstituting their ability to produce NO. In a similar system, Amber et al. demonstrated that IFN--y and TNF stimulated cytotoxicity in the murine EMT-6 mammary adenocarcinoma cell line by an NO-dependent mechanism [35, 36]. However, we were unable to demonstrate that NO plays a direct role in TNF-mediated cytotoxicity, even though these cells were shown to be sensitive to killing by exogenous NO. The NO synthase inhibitor NMMA and arginine-free media inhibited NO production by TNF-stimulated tumor targets but had no significant effect on TNF-mediated cytotoxicity. Hemoglobin and methylene blue also had no protective effect on TNF-mediated cytotoxicity. Nonetheless, our results suggest that resistance to TNF may occur because cells down-regulate their ability to produce NO in response to TNF. The mechanism(s) for TNF resistance is unknown. Other investigators have demonstrated that some TNF-resistant cells produce TNF themselves [37, 38]. This phenomenon is analogous to the induction of TNF-resistant cells in vitro by chronic exposure to rTNF [34]. It is not known whether endogenous production of TNF by resistant cells simply down-regulates TNF receptors, as has been shown in other systems [39], or is due to some other mechanism such as uncoupling of the cytoplasmic domain of the TNF receptor with an associated kinase or other second signal-generating enzyme. In this regard, Higuchi et al. have demonstrated that cGMP is involved in the mechanism of TNF-mediated cytotoxicity [40]. Their results would corroborate ours in that cGMP is often produced in response to NO [16, 19, 21]. A possible mechanism to explain how IFN-y makes cells more sensitive to TNF-mediated cytotoxicity may be related to their increased NO production. Resistant cells incubated
100 #{149}
80
L929
#{149} McA
‘60
0
x 0
0
0
0.156
0.625
2.5
[TNFJ ng/ml Fig.
7.
Effect
cytotoxicity. cells/well lowing Results
of L929
and
NMMAcells
assayed
for
pretreatment derived
with from
and
were
arginine-free
plated
their
sensitivity
NMMA-free
six experiments
media
in microtiter (500 are
wells
on
expressed
sM)
TNF-mediated
at a density
to TNF-mediated or
tumor tion.
40
arginine-free
as mean
%
cytotoxicity
mass, In this
thus regard,
protecting activated
other tumor macrophages
U
1
2
4
8
[SNP] mM Fig.
6.
Exogenous
cells
were
presence viability
plated
NO at
is cytotoxic 2
x I0
in
for
tumor
targets.
microtiter
wells
L929, and
3T12,
were
cultured
of SNP at the concentrations indicated for 24 h, at which of the cells was assessed by crystal violet staining. Results four
experiments
are
1 x 10 folmedia.
SD.
±
cells from destruchave been shown
in an autocrine cells would have
to eliminate the TNF-producing effector cells; therefore need not make NO, which might be self-destructive, demonstrated by their sensitivity to exogenous NO. were so, therapies might be devised in which resistant
20
0
of
cytotoxicity
in the presence of IFN-’y and TNF exhibit an augmented response in terms of the amount of NO they produce above the response to TNF alone coincident with their increased sensitivity to TNF-mediated cytotoxicity. We do not know whether this is due to IFN-y-mediated up-regulation of TNF receptors [30-32] or to some other mechanism. Unpublished results from our laboratory suggest that IFN-.y treatment abrogates TNF production by resistant cells, suggesting that binding of endogenously produced TNF is important in TNF resistance (D. Fast and S. Lee, unpublished observation). If NO produced by TNF-sensitive cells in response to TNF
to be sensitive to NO self-damage [41]. In this scenario, TNF-resistant
from
10
is not directly involved in the mechanism of TNF cytotoxicity, the question of its mechanistic role arises. It is possible that NO production by TNF-stimulated tumor targets modulates the function of other cell types, as is the case with endothelial cells that promote vasodilation of vessels by action on smooth muscle target cells [16, 20]. Similarly, NO functions in the brain to enhance neurotransmission [19, 21]. It is also possible that the production of NO in response to TNF provides a defense mechanism for TNF-sensitive cells to eliminate TNF-producing immune effector cells from the
3T12
-0-
>, 0
expressed
as
%
cytotoxicity
±
SD.
and
MCA in
time
the the
derived
manner no need they as If this cells
could be induced to produce NO, leading to their elimination. Since TNF-sensitive tumor targets were shown to be killed by exogenously generated NO, it seems probable that they might be damaged by endogenous production of NO in response to TNF stimulation, as has been reported for activated macrophages [41]. Otherwise, one must speculate that some mechanism exists to protect these cells from NO-
Fast et at’.
TNF-stimulated
nitric
oxide
in
tumor
cells
259
mediated self-destruction. Nitric oxide produced by endothelial cells has been reported to be neutralized by superoxide [16]. Thus it is possible that superoxide produced in response to TNF stimulation could protect tumor targets from NO self-damage. We have observed that catalase and taurine enhanced TNF-mediated cytotoxicity of resistant cell lines lished
that was partially observation).
reversed by NMMA These findings suggest
(D. Fast, unpuba role for oxida-
tive burst products (i.e., H202) in the TNF-resistant phenotype, which appears to be coupled to NO production. The expression of manganous superoxide dismutase in response to TNF [10] may be a mechanism for cells to neutralize excess superoxide produced under these conditions. Studies are in progress to define this potential mechanism further. Taken together, we have provided evidence for the induction of NO by TNF-stimulated tumor targets, which was correlated with their susceptibility to TNF-mediated cell damage. Although NO was apparently not directly associated with target cell damage, it seems evident that its production must provide an important biological function that remains to be elucidated.
authors
thank
Laura
Smith
for expert
preparation
of the
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