(Received for publication, May 30, 1996, and in revised form, November 1, 1996)
From the Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892
Nitric oxide (NO) increases tumor necrosis factor (TNF) synthesis in human peripheral blood mononuclear cells by a cGMP-independent mechanism. NO has been shown to inhibit adenylate cyclase in cell membranes. Since cAMP down-regulates TNF transcription, we examined the possibility that NO enhances TNF synthesis by decreasing cAMP. U937 cells were induced to differentiate using phorbol myristate acetate (100 nM for 48 h) and then were incubated for 24 h with sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP). These NO donors increased TNF production (7.0- and 15.6-fold, respectively, at 500 µM) in a dose-dependent manner (p = 0.002). However, SNP and SNAP did not elevate cGMP levels in U937 cell cultures, and the cGMP analog, 8-bromo-cGMP, had no effect on TNF production. In contrast, SNP (p = 0.001) and SNAP (p = 0.009) decreased intracellular cAMP levels by up to 51.5% over 24 h and, in the presence of a phosphodiesterase inhibitor, blunted isoproterenol-stimulated increases in cAMP by 21.8% (p = 0.004) and 27.6% (p = 0.008), respectively. H89, an inhibitor of cAMP-dependent protein kinase, dose dependently increased TNF production in phorbol myristate acetate-differentiated U937 cells in the absence (6.5-fold at 30 µM; p = 0.035), but not in the presence (p = 0.77) of SNAP. Conversely, the cAMP analog dibutyryl cAMP (Bt2cAMP) blocked SNAP-induced TNF production (p = 0.001). SNP and SNAP (500 µM) increased relative TNF mRNA levels by 57.5% (p = 0.045) and 66.2% (p = 0.001), respectively. This effect was prevented by Bt2cAMP. These results indicate that NO up-regulates TNF production by decreasing intracellular cAMP.
Nitric oxide (NO)1 is a free-radical gas produced by many cell types (1-4). NO has a diverse repertoire of important functions (5-9) including neurotransmission (5, 10), vasodilatation (11), antiplatelet activity, and immune modulation (12-14). Most of these effects are mediated through a unique cGMP signaling pathway. NO covalently attacks the heme moiety of soluble guanylate cyclase, activating the enzyme, and thereby elevating intracellular cGMP concentrations (15-17). This increase in cGMP subsequently activates certain protein kinases, which phosphorylate target proteins involved in regulation of cell function (17-19). Although the role of cGMP as a NO second messenger is undisputed, some findings have led to speculation about the existence of cGMP-independent signal transduction pathways for NO.
First, NO is a free radical with the ability to react with a variety of
enzymes besides soluble guanylate cyclase. NO has been shown to
catalyze the covalent binding of NAD to glyceraldehyde-3-phosphate dehydrogenase (20), oxidize iron-containing proteins such as aconitase
or ribonucleotide reductase (21-23), and nitrosylate tyrosine and
cysteine residues in a variety of proteins (24-26). Second, some
effects of NO cannot be reproduced with cell permeable cGMP analogs.
For example, the synthesis of tumor necrosis factor (TNF
), a
proinflammatory cytokine implicated in tissue injury and shock (27), is
increased in human peripheral blood mononuclear cells (28) and
lipopolysaccharide-stimulated neutrophil preparations (29) by exogenous
NO. Although NO increases cGMP concentrations in these cells, cGMP
analogs have no effect on TNF
production (28, 29). Collectively,
these investigations suggest that NO might use cGMP-independent
signaling pathways for some of its cellular functions.
Recently, adenylate cyclase has been added to the list of enzymes that
can be modified by NO (30). Treatment of cell membranes with NO
decreases cAMP production by inhibiting calmodulin activation of type I
adenylate cyclase, presumably through thiol nitrosylation at the
calmodulin-binding site (30, 31). Notably, increases in cAMP in
leukocytes activate cAMP-dependent protein kinase (PKA). This kinase phosphorylates transcription factors that bind to the
cAMP-response element on the TNF promoter, thereby inhibiting TNF
mRNA transcription (32-35). The effect of NO on type I adenylate cyclase suggests that NO might up-regulate TNF
synthesis in human monocytes by decreasing cAMP concentrations.
We investigated this question using U937 cells, a human monocytic cell
line that differentiates into monocyte-macrophage-like cells and
produces TNF when exposed to phorbol myristate acetate (PMA)
(36-38). The specific objectives were as follows: 1) to demonstrate that NO up-regulates TNF
production in PMA-differentiated U937 cells
and test the cGMP-dependence of this effect; 2) to determine whether NO
alters resting or stimulated cAMP concentrations in intact cells; 3) to
investigate the effect of inhibitors or activators of PKA on
NO-stimulated TNF
production in this system; and 4) to determine if
NO-induced changes in TNF
mRNA levels were consistent with a
cAMP mechanism.
PMA, S-nitroso-N-acetylpenicillamine (SNAP), 8-bromo-cGMP, dibutyryl cGMP (Bt2cGMP), dibutyryl cAMP (Bt2cAMP), H89, and isoproterenol were all purchased from Calbiochem (San Diego, CA). Sodium nitroprusside (SNP) was from Elkins-Sinn (Cherry Hill, NJ). Ficoll-Hypaque, prostaglandin E2 (PGE2), and 3-isobutyl-1-methyl-xanthine (IBMX) were all obtained from Sigma. Dextran was from Amersham Life Science Inc. TRI REAGENT-LS used for RNA isolation was from Molecular Research Center, Inc. (Cincinnati, OH).
Human neutrophils were isolated from the venous blood of normal
volunteers by dextran sedimentation and Ficoll-Hypaque density centrifugation as described previously (39). U937 cells obtained from
ATCC (Rockville, MD) were cultured in RPMI 1640 supplemented with 25 mM HEPES, 10% heat-inactivated fetal calf serum,
penicillin (100 units/ml), streptomycin (100 µg/ml), and 2% (w/v)
L-glutamine (all from Biofluids, Inc., Rockville, MD). The
cells were grown at 37 °C in a humidified atmosphere containing 5%
CO2. Differentiation was induced by incubating U937 cells
with PMA (100 nM) for 48 h. The cells were then washed
three times with Hank's balanced salt solution without
Ca2+ and Mg2+ (HBSS) to remove residual
PMA.
Differentiated U937 cells
(5 × 105 cells/ml) were incubated with each of the
following reagents for 24 h: increasing concentrations of SNP or
SNAP (0-500 µM); 8-bromo-cGMP or Bt2cGMP
(0-1000 µM); or Bt2cAMP (0-100
µM) in the presence of varying doses of SNAP (0-500
µM). In another experiment, differentiated U937 cells
were pretreated for 6 h with varying concentrations of H89 (0-30
µM), a cell-permeable PKA inhibitor. The cells (5 × 105 cells/ml) were then washed three times with HBSS and
incubated for an additional 24 h in the absence or presence of
SNAP (500 µM). TNF
release into the medium was
measured using an enzyme-linked immunosorbent assay (R&D Systems,
Minneapolis, MN). All reagents were dissolved into RPMI 1640, and
tested negative for endotoxin using a limulus amoebocyte lysate assay
(BioWhittaker, Inc., Walkersville, MD).
At a concentration of 4 × 106 cells/ml, differentiated U937 cells or freshly isolated human neutrophils were preincubated for 15 min in a shaking water bath at 37 °C with Hank's balanced salt solution with Ca2+ and Mg2+ (HBSS+) containing 1 mM IBMX. After addition of SNP or SNAP (0-1000 µM), these cells were incubated for 0-120 min followed by the addition of iced ethanol (65% (v/v) final concentration). The amount of cGMP present in the samples was quantitated by an enzyme immunoassay according to the manufacturer's protocol (Amersham).
Determination of cAMPDifferentiated U937 cells (5 × 105 cells/ml) were incubated in RPMI 1640 containing increasing doses of SNP or SNAP (0-500 µM) for 24 h. The cells were then scraped and spun for collection of cell pellets. Intracellular cAMP was extracted by lysing the cell pellets in 65% iced ethanol and centrifuging at 2000 × g for 15 min. The resultant supernatants were dried and cAMP was quantitated using an enzyme immunoassay according to the manufacturer's protocol (Amersham).
To investigate the effect of NO on agonist-stimulated cAMP responses,
differentiated U937 cells (5 × 105 cells/ml) were
pretreated for 15 min with HBSS containing 500 µM SNP
or SNAP in the presence of IBMX (1 mM). Adenylate cyclase agonists, isoproterenol (1 µM), or PGE2 (10 µM) were then added and total cAMP was extracted at
several time points (0-15 min) and quantitated as described above.
Differentiated U937
cells (5 × 105 cells/ml) were incubated under the
following conditions for 3.5 h: RPMI 1640 alone or with 8-bromo-cGMP (100 µM), Bt2cAMP (100 µM), SNP (500 µM), SNAP (500 µM), or Bt2cAMP (100 µM) and
SNAP (500 µM). RNA was isolated using TRI REAGENT-LS. The
entire open reading frame of human TNF cDNA, in the PAW711
plasmid, was kindly provided by Dr. Alice M. Wang (40). A 253-base pair
TNF
gene fragment, obtained by digestion with the enzymes
AvaI and HincII (Stratagene, La Jolla, CA), was subcloned into the pGEM3Z vector (Promega, Madison, WI). The recombined pGEM3Z plasmid was subsequently sequenced to confirm the presence of
the TNF
fragment insert. Single-strand antisense mRNA probe labeled with 35S was prepared by in vitro
transcription using SP6 polymerase. RPA was performed with a RPA II kit
(Ambion Inc., Austin, TX) using 50 µg of total RNA according to the
manufacturer's instructions. Results obtained were expressed as
percentages of the concurrently run
-actin controls.
All data are expressed as mean ± S.E. All
p values are two-sided. To analyze dose-response curves in
each experiment, the data was first summarized using the nonparametric
Sen-Theil estimate of regression slope (41). From these dose-response
slope estimates, one sample t tests (against the hypothesis
of H0: slope = 0) were done to determine whether the
observed dose-response was significant. To determine if the TNF
response to doses of SNAP was monotonically decreasing as the
Bt2cAMP dose increased, in addition to using the same
technique as described above, we also used Page's nonparametric test
(42) for ordered alternatives in a 2-way ANOVA layout (the first factor
was the increasing doses of Bt2cAMP, the second factor was
experiment). To evaluate the effects of NO on the isoproterenol-induced cAMP response in PMA-differentiated U937 cells, the area under the
curve was determined for each condition/experiment. Then, nominal
p values were computed for each of the two comparisons of
interest: isoproterenol versus isoproterenol/SNAP, and
isoproterenol versus isoproterenol/SNP. Last, these nominal
p values were adjusted by multiplying each by 2, to take
into account the multiple comparisons (Bonferroni adjustment) (43).
When comparing relative TNF
mRNA levels, we used paired
t tests.
First, we confirmed previous reports (37, 38) that
PMA-differentiated U937 cells produce TNF (Fig. 1).
Next, we demonstrated that exogenous NO donors, SNP or SNAP, increased
TNF
release from PMA-differentiated U937 cells over a 24-h
incubation period in a dose-dependent manner
(p = 0.002 for both). At the highest concentrations
examined (500 µM), SNP and SNAP increased TNF
production 7.0- and 15.6-fold, respectively (Fig. 1).
Effect of NO on Total cGMP in U937 Cell Cultures
Production
of cGMP by PMA-differentiated U937 cells in response to exogenous NO
donors was assessed using human neutrophils as a positive control (Fig.
2). After 2 h incubation in the presence of IBMX (1 mM), neither SNP nor SNAP elevated total cGMP in U937 cells
(p > 0.1). In contrast, and as expected (28), either
SNP or SNAP increased total cGMP production by human neutrophils in a
dose-dependent manner (p = 0.012 and
p = 0.004, respectively).
To exclude the possibility that cGMP was quickly degraded despite the
presence of a phosphodiesterase (PDE) inhibitor, thereby masking an
increase in cGMP production, total cGMP was also measured at earlier
time points (0, 1, 3, 5, 10, and 15 min) in the presence of IBMX (1 mM) after exposure to SNAP (1 mM). SNAP
increased total cGMP in neutrophils which reached a maximum at 15 min.
However, this NO donor had no effect on cGMP production in
PMA-differentiated U937 cells (Fig. 2, inset). Furthermore,
in separate experiments, SNP or SNAP had no effect on total cGMP in
naive U937 cells, or in U937 cells differentiated with retinoic acid
with or without 1,25-dihydroxyvitamin D3 (data not
shown).
Cell-permeable analogs of cGMP, 8-bromo-cGMP, or
Bt2cGMP were incubated with PMA-differentiated U937 cells
for 24 h. The release of TNF into the medium was not altered by
0-1 mM of either 8-bromo-cGMP (Fig.
3A, p = 0.33) or
Bt2cGMP (Fig. 3B, p = 0.66).
Effect of NO on cAMP Levels in PMA-differentiated U937 Cells
To determine if NO altered cAMP levels, intact
PMA-differentiated U937 cells were incubated in the presence of
increasing concentrations of SNP or SNAP for 24 h. Either SNP or
SNAP decreased intracellular cAMP levels (Fig. 4) in
PMA-differentiated U937 cells in a dose-dependent manner
(p = 0.001 and p = 0.009, respectively). Compared with culture medium alone, SNP or SNAP at the
highest concentrations examined (500 µM) decreased
intracellular cAMP by 37.8 and 51.5%, respectively.
To confirm that NO decreases cAMP levels in PMA-differentiated U937
cells, the effect of NO on agonist-stimulated cAMP responses was
examined in the presence of IBMX (1 mM). As shown in Fig. 5A, SNP or SNAP (500 µM)
blunted isoproterenol-stimulated increases in total cAMP by 21.8%
(p = 0.004) and 27.6% (p = 0.008),
respectively. Furthermore, the inhibitory effects of SNP
(p = 0.045) or SNAP (p = 0.011) were
also demonstrated using PGE2 (10 µM) instead of isoproterenol to activate adenylate cyclase (Fig. 5B).
This experiment was performed in HBSS to avoid activation of
Ca2+/calmodulin-dependent PDE I (44, 45).
Effect of Inhibitors or Activators of PKA on TNF
Finding that NO but not cGMP
increased TNF production, and that NO decreased intracellular cAMP
levels, we next examined the effect of cell-permeable agents that
either inhibit (H89) or activate (Bt2cAMP) PKA on TNF
production. Preincubation of PMA-differentiated U937 cells with H89
increased TNF
production (6.5-fold at 30 µM) in a
dose-dependent manner (Fig. 6,
p = 0.035). The addition of SNAP elevated TNF
production and eliminated the dose effect of H89 (p = 0.77). Conversely, Bt2cAMP blocked SNAP-induced TNF
production (Fig. 7). With increasing concentrations of
Bt2cAMP (0-100 µM), the
dose-dependent effect of SNAP on TNF
production was
abolished (p = 0.001). Cell viability by trypan blue
exclusion was not decreased at the concentrations of
Bt2cAMP employed (0-100 µM, data not
shown).
Effect of NO and Cyclic Nucleotide Analogs on TNF
Relative TNF mRNA
levels were measured using a RPA to investigate the effects of NO,
cGMP, and cAMP on TNF
mRNA transcription (Fig.
8). SNP or SNAP increased TNF
mRNA levels by
57.3% (p = 0.045) and 66.2% (p = 0.001), respectively. An analog of cAMP, Bt2cAMP, decreased
TNF
mRNA levels (p = 0.002), and prevented the
effect of SNAP (Bt2cAMP versus
Bt2cAMP and SNAP: p = 0.93). In contrast to
the NO donors, 8-bromo-cGMP had no effect on TNF
mRNA levels
(p = 0.52).
We demonstrated that NO increased TNF production in
PMA-differentiated U937 cells by decreasing intracellular cAMP levels, indicating that NO uses cAMP, rather than cGMP as a second messenger for some of its cellular effects. This conclusion is based on these
findings: 1) two structurally dissimilar NO donors increased TNF
production in a dose-dependent manner; 2) both SNP and SNAP increased cGMP concentrations in human neutrophil cultures, but had no
effect on cGMP concentrations in PMA-differentiated U937 cell cultures;
3) cell-permeable analogs of cGMP, 8-bromo-cGMP and
Bt2cGMP, did not alter TNF
production by
PMA-differentiated U937 cells; 4) SNP or SNAP not only decreased
intracellular cAMP in a dose-dependent manner, but also
blunted isoproterenol- and PGE2-stimulated cAMP responses
in PMA-differentiated U937 cells; 5) an inhibitor of PKA, H89,
increased TNF
release in the absence but not in the presence of
SNAP; 6) conversely, an activator of PKA, Bt2cAMP,
abolished the effect of SNAP on TNF
production; and 7) finally, NO
donors and Bt2cAMP but not 8-bromo-cGMP caused changes in
relative TNF
mRNA levels that were consistent with a cAMP
mechanism for the observed effects of NO. Collectively, these
experiments demonstrate that NO-induced up-regulation of TNF
production in this human cell line uses cAMP, not cGMP, as its second
messenger.
Many of the known effects of NO have been attributed to its ability to
generate cGMP through its action on soluble guanylate cyclase (15-18).
However, we were unable to demonstrate that NO donors increase cGMP in
either naive or differentiated U937 cells. It seems unlikely that our
inability to detect NO-stimulated increases in cGMP was due to
degradation of cGMP. U937 cells have extremely low cGMP hydrolytic
activity and do not contain the cGMP-specific PDE isoenzyme (PDE V)
(45, 46). Furthermore, our experiments were conducted in the presence
of a potent, nonselective PDE inhibitor. These data indicate that U937
cells lack NO-sensitive soluble guanylate cyclase. Moreover, membrane
permeable cGMP analogs, 8-bromo-cGMP and Bt2cGMP, were
unable to mimic the effect of NO on TNF production, a finding that
has also been reported in human peripheral blood mononuclear cells and
neutrophil preparations (28, 29). These results further suggest that NO
regulates TNF
production in PMA-differentiated U937 cells by a
cGMP-independent mechanism.
The ability of NO to decrease intracellular cAMP levels and blunt
isoproterenol- and PGE2-stimulated cAMP responses provide direct evidence for our speculation that NO increases TNF production in PMA-differentiated U937 cells by decreasing cAMP levels. Decreases in intracellular cAMP can result either from its reduced synthesis by
adenylate cyclase or from increased catabolism due to increased PDE
activity (47). The result that NO decreased cAMP concentrations in the
presence of IBMX, a nonspecific PDE inhibitor, support the hypothesis
that changes in cAMP levels were due to decreased synthesis, rather
than increased catabolism by PDE. Interestingly, cGMP can either
increase cAMP hydrolysis by activating PDE II (45, 46) or decrease cAMP
hydrolysis by inhibiting PDE III (48, 49). Increased or decreased cAMP
hydrolysis mediated by cGMP is unlikely in our experiments since U937
cells lack PDE II activity (45, 46), a PDE inhibitor was used, and as
shown here, U937 cells do not produce cGMP in response to a NO signal. However, in other cell types that contain NO-sensitive soluble guanylate cyclase, the ability of NO to decrease cAMP production may be
masked by decreased cAMP hydrolysis via cGMP-mediated inhibition of PDE
III activity (48-51).
As already noted, NO has been shown to inhibit
calmodulin-dependent adenylate cyclase activity in isolated
cell membranes by oxidizing cysteine residues at the calmodulin-binding
site (30). Other studies have shown that calcium ionophores potentiate cAMP responses in human peripheral blood mononuclear cells and neutrophils stimulated with isoproterenol and PGE2, and
this potentiation was inhibited by calmodulin inhibitors (52-54).
These observations indicate that the calmodulindependent
adenylate cyclase subtype that is inhibited by NO is present in human
leukocytes. Although this suggests a possible mechanism for NO
modulation of intracellular cAMP levels, other possibilities exist.
Substitution of cysteine residues for other amino acids in the
2-adrenergic receptor markedly shifts the dose-response
curve to the right for isoproterenol-stimulated increases in
intracellular cAMP concentrations (55). This suggests that NO could
reduce agonist-stimulated cAMP responses by decreasing receptor
affinity through the nitrosylation of key cysteine-containing domains.
Inhibitors and activators of PKA were used to further explore the
possibility that NO was using a cAMP-dependent signaling pathway. H89, a specific cell-permeable inhibitor of PKA (56), dose-dependently increased TNF release. This finding
demonstrates in PMA-differentiated U937 cells that basal cAMP levels
and the resulting degree of PKA activation are inhibitory of TNF
synthesis. Interestingly, H89, at the doses tested, did not further
up-regulate SNAP-induced TNF
production, suggesting that PKA was
maximally inactivated by the decrease in cAMP levels caused by NO. In
contrast, Bt2cAMP only slightly suppressed basal TNF
release, but completely abolished SNAP-induced increases in TNF
production. This cAMP analog can permeate cell membranes and is
resistant to hydrolysis by PDE (57), enabling it to persist in cell
cultures and mimic prolonged elevations of intracellular cAMP. Toxicity
caused by the butyrate moiety of the Bt2cAMP molecule was
unlikely to be responsible for this effect, since the concentrations of
Bt2cAMP used were relatively low and Bt2cGMP
(up to 1 mM), which also contains a butyrate moiety did not
alter TNF
production. Furthermore, cell viability by trypan blue
exclusion was not decreased by Bt2cAMP. Together, these
results reinforced our conclusion that NO increases TNF
production
in PMA-differentiated U937 cells by decreasing cAMP levels.
Similarly, NO donors were also found to increase relative TNF
mRNA levels and Bt2cAMP completely prevented this
effect. An analog of cGMP, 8-bromo-cGMP, had no effect. These results
are consistent with a cAMP mechanism acting at the level of
transcription for the observed effects of NO on TNF
production in
PMA-differentiated U937 cells. Evidence is also available that cAMP can
down-regulate TNF
expression at a post-transcriptional level in
monocytes and macrophages (58-60).
Previously, NO was demonstrated to increase TNF mRNA levels in
HL-60 cells (61), but we found no change in TNF
mRNA levels in
human neutrophil preparations (28). These inconsistent findings may be
ascribed to the different methods employed to measure mRNA levels.
In our present study, we measured TNF
mRNA levels using a RPA,
which may be quantitatively more reliable than the reverse transcription polymerase chain reaction assay used in our previous experiments with neutrophils (28). Furthermore, differentiated U937
cells may contain more copies of TNF
mRNA than neutrophils. Besides the cAMP mechanism, our data do not exclude the possibility of
additional mechanisms for the up-regulation of TNF
production by NO.
For example, NO could activate or induce other transcription factors,
such as NF-
B (29).
In conclusion, the present study indicates that NO increase TNF
production in PMA-differentiated U937 cells by decreasing intracellular
cAMP. To our knowledge, this is the first demonstration in intact cells
that NO signal transduction can use cAMP rather than cGMP to regulate
cell function.