(Received for publication, October 20, 1995)
From the
Macrophage activation is central to the progression of multiple
diseases via the release of inflammatory mediators such as cytokines
and nitric oxide. Despite the recognized overlap in the regulatory
mechanisms involved in mediator production, little information exists
regarding receptor-initiated signaling pathways that coordinately
control multiple end points, such as tumor necrosis factor-
(TNF-
) and nitric oxide production. In this study, the expression
of inducible nitric oxide synthase (iNOS) in macrophages is shown to be
regulated by calcium and by a purinoreceptor signaling system. The
P
purinoreceptor partial agonist, 2-methylthio-ATP
(2-MeS-ATP), inhibits the expression of iNOS induced by
lipopolysaccharide (LPS) plus interferon-
(IFN-
) in primary
macrophages. Additionally, 2-MeS-ATP attenuates the expression of iNOS
in macrophages isolated from CD-1 mice challenged with LPS, and it
inhibits LPS-induced TNF-
and interleukin-1
(IL-1
)
release, thereby preventing endotoxic death. Thus, purinoreceptors and
calcium are likely to be critical for macrophage activation and the
production of inflammatory mediators stimulated by LPS.
The control of macrophage overproduction of inflammatory
mediators such as TNF-, (
)IL-1
, and nitric oxide
(NO) should greatly facilitate the treatment of septic shock,
rheumatoid arthritis, cerebral malaria, and autoimmune diabetes (1, 2, 3, 4, 5) . In
macrophages, these mediators are regulated primarily at the level of
mRNA expression via the involvement of transcription factors such as
NF-
B (6, 7, 8) and via components that
control message stability(9, 10) . Given the overlap
in the regulatory mechanisms of these genes, it is possible that
several receptor-mediated signal transduction pathways exist that can
modulate the production of selected mediators in response to an
inflammatory stimulus.
Several lines of evidence indicate that
purinoreceptors may control these regulatory
pathways(11, 12, 13, 14) . For
example, we and others have shown that extracellular adenine
nucleotides can regulate the circulating levels of TNF- and
IL-1(12, 14) . Specifically, the administration of the
ATP analog 2-MeS-ATP to LPS-challenged mice reduces serum levels of
TNF-
and IL-1
without affecting the induction of serum levels
of IL-6, suggesting that 2-MeS-ATP has an immunomodulatory effect on
cytokine production or release(12) . This analog also greatly
increases the likelihood of survival (
100%) of these mice after an
injection of a lethal dose of LPS (12) . The effects of
2-MeS-ATP in mice are paralleled by its ability to inhibit a RAW 264.7
macrophage membrane GTPase that is stimulated in the presence of
LPS(11, 12) . Moreover, previous pharmacologic and
kinetic data suggest that the LPS-stimulable GTPase in these membranes
is a G-protein associated with a member of the P
purinoreceptor class(11, 12, 15) . This
receptor class preferentially binds ATP and ADP but not AMP or
adenosine, and many of the P
purinoreceptors share homology
with G-protein-coupled, seven-transmembrane domain
receptors(16) . Macrophages are known to express P
purinoreceptors (which preferentially bind AMP and adenosine but
not ATP or ADP) as well as several members of the P
purinoreceptors, namely those from the P
,
P
, and P
subclasses(17, 18) .
We demonstrate here that
the LPS-stimulable GTPase in RAW 264.7 macrophage membranes is
controlled by P purinoreceptor agonists. Earlier studies
have shown that this receptor class can mobilize calcium(19) ,
an important intracellular second messenger. Because nitric oxide is an
important inflammatory mediator and because extracellular adenine
nucleotides (e.g. 2-MeS-ATP) affect macrophage activation, we
have examined the actions of P
purinoreceptors and calcium
on macrophage iNOS expression. We report that calcium can promote
LPS-stimulated nitric oxide production and that adenine nucleotides
(2-MeS-ATP) can inhibit LPS-induced NO generation by preventing
accumulation of iNOS mRNA; these observations represent two previously
unidentified pathways regulating iNOS expression.
Several agonists of the P receptor class (e.g. ATP, ADP, 5`-adenylylimidodiphosphate (AMPPNP), and
adenosine 5`-O-(3-thiotriphosphate) (ATP
S)) synergize
with LPS to stimulate a macrophage membrane GTPase
activity(12, 15) . Further characterization of this
GTPase shows that its regulation is controlled by a P
-like
purinoreceptor. As shown in Table 1, LPS preparations stimulate
the macrophage GTPase in the presence of ATP or ADP. In contrast, UTP
and 2`- and 3`-O-(4-benzoylbenzoyl)adenosine 5`-triphosphate
(Bz-ATP) at several concentrations are unable to stimulate basal GTPase
activity or synergize with LPS ( Table 1and data not shown).
These compounds are ligands for the P
and P
receptor classes, respectively; therefore these results argue against
P
and P
receptor involvement. The role of
P
purinoreceptors is also excluded, because adenosine
(1-1,000 µM), in the presence or absence of LPS did
not stimulate membrane GTPase activity. However, the P
receptor partial agonist 2-MeS-ATP dose-dependently blocked the
responses to LPS in the presence of ATP or ADP (Table 1, (12) , and data not shown). In sum, these results suggest that
the LPS-stimulated GTPase activity in RAW 264.7 membranes is influenced
by P
-like purinoreceptors.
Ligand-occupied P and other purinoreceptors can modulate G-protein-dependent
calcium mobilization(17) . Because ADP most potently stimulated
the LPS-responsive GTPase in RAW 264.7 cell membranes and has little
activity on P
and P
purinoreceptors, we used
this agonist to assess P
-like purinoreceptor-induced
calcium mobilization in Fura-2-loaded RAW 264.7 macrophages. Indeed,
the ratio of Fura-2 fluorescence excited at 340 and 380 nm increased
from a basal level of 2.230 ± 0.022 to 2.970 ± 0.160
after RAW cell treatment with 100 µM ADP, a result
indicative of increased levels of free calcium due to ADP stimulation
of these cells. Of note, previous studies have shown that increased
calcium levels can influence various LPS-mediated signal transduction
events in this and other systems, including the stimulation of a GTPase
in RAW 264.7 membranes, (
)the phosphorylation of
mitogen-activated protein kinases and the translocation of NF-
B (24, 25) . Based on these observations, we
hypothesized that calcium fluxes are part of the mechanism by which
purinoreceptors regulate the production of inflammatory mediators (e.g. TNF-
, IL-1
, NO). Therefore, the role of
calcium on LPS- and/or adenine nucleotide-sensitive end points was
assessed, including its effect on NO production. RAW 264.7 cells do not
appear to contain a calcium-dependent isoform of NO synthase ( Fig. 1and data not shown); however, the role of calcium in
LPS-stimulated expression of iNOS has not been directly investigated.
This is particularly relevant because LPS is known to contain large
levels of Ca
and has been reported to mobilize
Ca
in macrophages(26, 27) .
Figure 1:
RAW 264.7
cells pretreated with calcium ionophore show increased nitric oxide
production in response to LPS or LPS and IFN-. Triplicate cultures
containing 1
10
RAW 264.7 cells each were incubated
with and without A23187 (1 µM) for 4 h, stimulated for 15
min with medium, LPS (1 µg/ml), IFN-
(20 units/ml), or both,
washed, and incubated for an additional 20 h in medium alone. Nitrite
content of culture supernatant samples was assessed using the Griess
reagent(22) . The means and S.E. values are presented for
7-12 experiments, depending on the stimulatory agent
used.
To
examine the relationship between calcium and LPS or IFN-
stimulation of nitric oxide production, initial experiments were
performed showing that a 20-h coincubation of RAW cells with the
calcium ionophore A23187 (0.1-10 µM) and LPS had no
effect on the stimulation of nitrite production induced by LPS
treatment alone. However, when primed with A23187 for 4 h followed by a
brief LPS exposure (15 min), RAW 264.7 cells produce 4-fold higher
levels of nitrite 20 h later, relative to that seen in untreated cells (Fig. 1). Unprimed RAW cells stimulated with LPS did not produce
nitrite nor did macrophages treated with ionophore alone (Fig. 1). Furthermore, RAW cells treated first with LPS for 15
min followed by 4 h of ionophore exposure also did not produce nitrite
(data not shown). These observations are consistent with data showing
that simultaneous treatment of RAW cells with LPS and 5 mM EGTA or 30 µM La
, respectively,
produced 70 and 95% inhibition of the amount nitrite measured 18 h
later. Similar studies by others show that either simultaneous
treatment with LPS and Cd
or pretreatment with
dihydropyridine-type calcium channel blockers followed by LPS
stimulation of macrophages can inhibit LPS-induced nitrite production
by these cells(23, 28) .
We also examined the role
of calcium in IFN- plus LPS-induced nitrite formation by RAW cells
using the same experimental design (Fig. 1). Although 15 min of
stimulation with LPS plus IFN-
is sufficient to induce the
production of nitrite by RAW cells, the level of nitrite produced is
enhanced 2-fold when the cells are primed with A23187. As is the case
for LPS induction, ionophore treatment after the LPS plus IFN-
stimulus does not produce additional nitrite (data not shown).
Following pretreatment with a different calcium ionophore, ionomycin (1
µM), we also observed a 2-fold enhancement of the amount
of nitrite produced from LPS plus IFN-
-stimulated RAW cells
relative to similar controls. Ionophore (A23187) pretreatment followed
by an IFN-
stimulation for 15 min does not induce nitrite
formation by RAW cells indicating that ionophore pretreatment cannot
mimic the effects of LPS on iNOS expression (Fig. 1). Together,
these experiments and the data in Fig. 1demonstrate a
calcium-dependent phase of nitrite production by RAW cells.
Additionally, we have observed a lack of stimulation of nitrite
production by ionophore alone (Fig. 1), a 9-h lag in
ionophore-enhanced nitrite formation after LPS stimulation (data not
shown), and the ability of cycloheximide (2.5 µg/ml) to exert
>95% inhibition of ionophore-pretreated, LPS-induced nitrite
generation. These results implicate a calcium-sensitive factor as an
important regulatory molecule for the expression of iNOS by RAW cells.
To assess whether LPS-induced nitric oxide production is regulated
by purinoreceptors, we performed initial experiments with RAW cells
looking at the ability of adenine nucleotides to stimulate nitrite
production in the presence and absence of LPS. These experiments
revealed that various adenine nucleotides synergize with low levels of
LPS to generate nitrite. These data have now been confirmed by Tonetti et al.(29) , and even though UTP was shown to weakly
synergize with LPS in terms of nitrite production, their findings
extend this observation to suggest that the P
purinoreceptor class on RAW cells is likely to be responsible for this
effect. In the present study, we demonstrate that the P
purinoreceptor partial agonist 2-MeS-ATP also influences
LPS-stimulated nitrite production in RAW cells and in primary mouse
macrophages. As shown in Fig. 2A, concentrations of
2-MeS-ATP of 100 µM and above inhibit LPS-induced nitrite
formation in RAW cells by 60% at 0.1 µg of LPS/ml. At higher
concentrations of LPS, 2-MeS-ATP was less effective at inhibiting RAW
cell production of nitrite. However, in mouse peritoneal macrophages,
2-MeS-ATP inhibits LPS plus IFN-
-induced nitrite and total nitric
oxide production (nitrite plus nitrate) in a concentration-dependent
fashion, i.e. inhibition was detectable at 100
µM, and complete inhibition was observed at a
concentration of 1 mM 2-MeS-ATP (Fig. 2B). In
comparison with the transformed RAW 264.7 cells, the observation that
primary macrophages are more responsive to 2-MeS-ATP inhibition of
LPS-induced nitric oxide production is consistent with our in vitro and in vivo studies examining the antagonistic effects of
2-MeS-ATP on LPS-stimulated TNF-
production(11, 12) . The inhibitory effects of
2-MeS-ATP on nitric oxide production by primary macrophages appears to
be specific for LPS signaling events, as 2-MeS-ATP does not inhibit
nitrite formation induced by treatment of macrophages with TNF-
and IFN-
(Fig. 2C). The fact that nitrite
production by TNF-
requires IFN-
and is not affected by IL-1
(data not shown), together with the observation that 2-MeS-ATP inhibits
LPS plus IFN-
-stimulated but not TNF-
plus IFN-
-induced
nitrite generation, suggest that LPS stimulates nitrite production
independently of TNF-
and IL-1 generation. Additionally, in a
system unrelated to nitric oxide production, 2-MeS-ATP treatment does
not affect LPS-induced procoagulant activity in macrophages, (
)suggesting that the inhibitory effects of this adenine
nucleotide on LPS-stimulated TNF-
, IL-1, and nitric oxide
production are not via a cytotoxic mechanism.
Figure 2:
2-MeS-ATP inhibits LPS-induced, but not
TNF--stimulated, nitric oxide production in peritoneal
macrophages. A, 5
10
RAW 264.7 cells were
treated with 0.1 µg of LPS/ml, 1 µg of LPS/ml, or 10 µg of
LPS/ml and the indicated concentrations of 2-methylthio-ATP. B, 5
10
resident PEC from CD-1 mice were
treated with 1 µg of LPS/ml, 150 units of IFN-
/ml, and the
indicated concentrations of 2-MeS-ATP. C, 5
10
CD-1 PEC were treated with 10 ng of TNF-
/ml, 150 units of
IFN-
/ml and the indicated concentrations of 2-MeS-ATP. The open symbols refer to the levels of nitric oxide metabolites
found in supernatants of cells treated with medium alone. Culture
supernatants were analyzed for nitrite (triangles) or for
nitrite plus nitrate (squares) after 18-24 h of
incubation at 37 °C. Results are the mean ± S.E. of
triplicate measurements from three individual
experiments.
2-MeS-ATP appears to
inhibit nitric oxide production by primary mouse macrophages at the
level of iNOS gene expression. As shown in Fig. 3A,
2-MeS-ATP does not significantly inhibit the enzymatic activity of
iNOS, examined by incubating LPS plus IFN--pretreated primary
macrophages for 3 h with this adenine nucleotide. In contrast, an iNOS
enzymatic inhibitor, aminoguanidine(30) , inhibits nitrite
formation in this whole cell assay system. Fig. 3, B and C, demonstrates that 2-MeS-ATP attenuates LPS plus
IFN-
-stimulated induction of iNOS protein and mRNA in a
concentration-dependent manner, similar to its effects on nitric oxide
production.
Figure 3:
2-MeS-ATP treatment prevents LPS-induced
iNOS expression in peritoneal macrophages, but does not affect iNOS
enzymatic activity. A, CD-1 mouse resident PEC (5
10
cells/400 µl) were incubated for 18 h with LPS (1
µg/ml) and murine IFN-
(150 units/ml). The cells were washed
three times and then incubated for 3 additional h with the indicated
concentrations of 2-MeS-ATP or aminoguanidine. The open triangle denotes the levels of nitrite found in supernatants of cells
treated with medium alone for the entire 21 h. Results are the average
± S.E. of three individual experiments containing three
replicates/condition. B, PEC were incubated for 5 h in
methionine-deficient medium in the presence of LPS (1 µg/ml),
IFN-
(150 units/ml), and 2-MeS-ATP at the indicated
concentrations. [
S]Methionine was added, and the
PEC were incubated for 13 additional h. The cells were harvested, and
murine iNOS was immunoprecipitated and detected by autoradiography.
Results are representative of three individual experiments. C,
CD-1 PEC (4
10
/2.5 ml of medium) were incubated for
6 h with the indicated concentrations of 2-MeS-ATP and LPS (1
µg/ml) plus murine IFN-
(150 units/ml). Total RNA was isolated
and probed for mouse macrophage iNOS by Northern blot analysis. Results
are representative of three individual experiments. D,
5-6-week-old male CD-1 mice were injected intraperitoneally with
saline ± 800 µg of LPS. A second intraperitoneal injection
was given with saline ± 1.3 mg of 2-MeS-ATP. After 18 h,
peritoneal macrophages were isolated by adherence and incubated for an
additional 18 h in medium with and without 1 mM aminoguanidine. Culture supernatants were measured for nitrite
levels, and cell content of iNOS protein was determined by Western
analysis. Data shown are from a representative
experiment.
2-MeS-ATP also attenuates macrophage expression of iNOS induced by intraperitoneal injections of LPS into CD-1 mice (Fig. 3D). LPS induction in vivo causes a 2.5-fold increase in the amount of nitrite found ex vivo relative to the unstimulated control. 2-MeS-ATP treatment in vivo causes a 50% reduction of nitrite levels and iNOS protein associated with these primary mouse macrophages (Fig. 3D, inset). The inhibition of nitrite production by aminoguanidine treatment of the induced macrophages in vitro shows that nitrite production occurs during the ex vivo incubation. These data indicate that 2-MeS-ATP is also effective at inhibiting the expression of iNOS induced by LPS under in vivo conditions.
To address whether calcium mobilization is part of the mechanism by
which purinoreceptors regulate LPS-induced inflammatory mediator
production, 2-MeS-ATP was evaluated for its ability to affect
calcium-sensitive Fura-2 fluorescence in RAW cells treated with and
without ADP (Fig. 4). Consistent with its well characterized
partial agonist activities in other systems(31) , 2-MeS-ATP
treatment alone mediated a smaller increase in free calcium levels than
did ADP alone, as assessed by the ratio of Fura-2 fluorescence excited
at 340 and 380 nm. However, under these conditions, 2-MeS-ATP was
unable to block the increase in free calcium levels stimulated by ADP
treatment of RAW 264.7 cells; rather, the effect appears to be additive (Fig. 4). These results suggest several possibilities. For
example, the inhibitory effects of 2-MeS-ATP on inflammatory mediator
production may be through another P-like or other
purinoreceptor class that signals via a mechanism independent of
calcium. In this case, ADP stimulation of
P
-purinoreceptors could cause calcium level increases that
enhance iNOS expression. The occupation of another purinoreceptor class
by 2-MeS-ATP could then generate a calcium-independent signal that
overrides calcium-dependent effects initiated by ADP-bound
P
purinoreceptors. In addition, the additive increase in
free calcium by the combination of ADP and 2-MeS-ATP may be cytostatic
or cytotoxic to macrophages. However, several observations argue
against this possibility, including the enhancement of LPS-induced RAW
cell nitric oxide production by calcium ionophore pretreatment, the
lack of inhibition by 2-MeS-ATP of LPS-stimulated procoagulant activity
in RAW cells
and TNF-
plus IFN-
generated nitrite
in peritoneal macrophages, and trypan blue exclusion studies which
indicate that cell viability is unaffected by 2-MeS-ATP. Alternatively,
the additive increase in free calcium levels due to ADP and 2-MeS-ATP
treatments could be selectively antagonistic to the LPS-stimulated
pathway, but the experiments with ionophore pretreatment of RAW cells
suggest this is not likely, since the large calcium flux expected with
this treatment was not inhibitory to LPS-induced nitrite production.
Figure 4:
ADP- and 2-MeS-ATP-induced calcium
mobilization in RAW 264.7 cells. 5 10
RAW cells
were plated on glass coverslips and loaded with Fura-2 as described
under ``Experimental Procedures.'' After obtaining a basal
ratio of Fura-2 fluorescence excited at 340 and 380 nm (2.285 ±
0.090 for the entire experiment), adenine nucleotides were added alone
or simultaneously at the indicated concentrations. Changes in Fura-2
fluorescence were monitored for 9 min followed by stimulation with 5
µM ionomycin as a positive control. Data represent the
mean change in fluorescence and the standard deviation after the basal
340/380 ratio for each individual tracing is subtracted from the peak
and averaged with data from two other coverslips. A representative
experiment is shown.
This study demonstrates that 2-MeS-ATP inhibits LPS-induced iNOS
gene expression in vitro and in vivo. Previous
findings have shown that this nucleotide can dramatically reduce serum
levels of TNF- and IL-1
in LPS-challenged mice, without
affecting LPS-induced IL-6 serum levels in these mice or procoagulant
activity in LPS-stimulated macrophages. The prevention of endotoxic
death suggests that 2-MeS-ATP has an important immunomodulatory effect
on the inflammatory process(12) . Our results, in combination
with observations that other adenine nucleotides can enhance
LPS-stimulated nitrite formation in RAW 264.7 cells (29) and
LPS-induced IL-1 production in mice(14) , suggest that
macrophage purinoreceptors control a signal transduction pathway that
intersects with LPS signaling. Because this study demonstrates that
calcium is important for iNOS expression, one pathway that can be
suggested is that ligand occupation of G-protein-coupled
purinoreceptors results in calcium mobilization, ultimately modulating
LPS-stimulated iNOS expression by affecting one of the multiple
calcium-dependent factors that can be involved in initiating iNOS
transcription. 2-MeS-ATP in this model may serve to bind, but not
activate, a P
-like purinoreceptor, thereby blocking
stimulation of this receptor class by ATP or ADP, but not preventing
the ability of ATP or ADP to signal through other receptor classes. As
noted above, the role of calcium in the inhibitory mechanism of
2-MeS-ATP action remains to be more fully characterized; however, a
role for calcium in iNOS synthesis, and possibly in TNF-
and IL-1
expression(11, 12) , is suggested by these data and
observations by others showing that calcium channel blockers can
prevent sepsis in rats (32) .
Because multiple
receptor-mediated pathways mobilize calcium in macrophages (e.g. complement, Fc, and platelet-activating factor receptors, all
of which are stimulated during sepsis(1, 18) ), the
enhancement of mediator expression via elevated calcium is a mechanism
that is not exclusive to purinoreceptors. However, purinoreceptors play
an important role in the inflammatory response (18) . During
infection and/or stress, large levels of extracellular ATP and ADP are
released locally from cellular damage and from platelet activation, as
well as systemically from the adrenal gland(18, 33) .
Additionally, many cell types have been shown to release ATP through
cell surface proteins containing an ATP binding cassette motif,
resulting in autocrine stimulation of
purinoreceptors(17, 34) . This raises the possibility
that LPS may cause ATP release from stimulated macrophages which could
then activate purinoreceptors. Regardless of the source of adenine
nucleotides, the stimulation of macrophages by ATP, ADP, or other
adenine nucleotides present at the inflammatory site and the functional
inhibition by 2-MeS-ATP with respect to LPS-induced TNF-
, IL-1,
and nitric oxide production strongly indicate that purinoreceptors can
control signal transduction pathways that are critically important to
macrophage activation.