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Production by the Atrial Natriuretic Peptide in Murine Macrophages1
*
Institute of Pharmacy, Center of Drug Research, Pharmaceutical Biology, University of Munich, Munich, Germany; and
Biochemical Pharmacology, University of Konstanz, Konstanz, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. TNF- production in murine bone marrow-derived macrophages
was induced by LPS, and TNF- secretion (±ANP) was determined by
L929 bioassay. ANP dose dependently
(10-8–10-6 M)
inhibited TNF- release by up to 95%. The effect was mediated via
the guanylate cyclase-coupled A receptor, as was shown by employing
dibutyryl-cGMP, the cGMP-inhibitory compound Ly-83583, and the A
receptor antagonist HS-142-1. A specific ligand of the natriuretic
peptide "clearance" receptor inhibited TNF- production only at
10-7 and 10-8 M, but
not at 10-6 M. The B receptor ligand C-type
natriuretic peptide showed no TNF--inhibitory effect. To investigate
the underlying mechanism of ANP-mediated TNF- inhibition, Northern
blot was performed. ANP-treated macrophages displayed decreased
TNF--mRNA levels. Besides the known inhibition of NF-
B
activation, in this study we demonstrated that ANP also attenuates the
activation of the proinflammatory transcription factor AP-1 (gel shift
assay). ANP did not alter subunit composition of AP-1 complexes, as was
shown by supershift assays applying anti-c-jun
and anti-c-fos Abs. To get information on the
ANP effect for human inflammatory processes, we investigated cytokine
production in human LPS-activated blood. ANP significantly attenuated
production of TNF- and IL-1ß without affecting production of IL-10
and IL-1ra. In summary, ANP was shown to attenuate TNF- production
of LPS-activated macrophages via cGMP. The inhibition is suggested to
involve transcriptional processes that are the result of reduced
activation of responsible transcription factors. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The functions of the NP are not restricted to the regulation of volume
homeostasis. ANP and its receptors were demonstrated to be expressed in
diverse tissues besides the cardiovascular and renal system
(11). Our previous work drew attention to a new aspect in
the biological profile of ANP, i.e., its interference with the immune
system (12). ANP and its receptors were shown to be
expressed in thymus (13, 14) as well as in macrophages
(8, 9, 15). Concerning functional consequences, ANP was
demonstrated to inhibit thymocyte proliferation (14) and
thymopoiesis (16). Furthermore, ANP stimulates
phagocytosis and production of reactive oxygen species in macrophages
(17) and inhibits inducible NO synthase (iNOS) in
LPS-activated macrophages (15, 18). This effect was
demonstrated to be mediated via a destabilization of iNOS mRNA and by
reduced activation of NF-B initiated by ANP (19). ANP
was also shown to attenuate ischemia-reperfusion injury of the liver
via its guanylate cyclase-coupled A receptor (20).
Ischemia-reperfusion injury is considered as a state of inflammation
due to the activation of macrophages and the secretion of TNF-
(21). Interestingly, concerning the mechanism underlying
the protective effect exerted by ANP on this kind of cell damage, the
inhibition of NF-B is suggested to be involved (20).
The knowledge about the influence of ANP on immune functions led us to
hypothesize that ANP influences LPS-induced TNF- production in
macrophages. This central proinflammatory cytokine is regulated
transcriptionally, whereby the two transcription factors NF-B and
AP-1 are involved (22). AP-1 is a family of related
transcription factors, which frequently consist of either
c-jun/c-fos heterodimers or
c-jun/c-jun homodimers (23). Changes
in AP-1 activity in response to extracellular signals are regulated
both on the level of transcription of jun and fos
genes and by posttranslational modification of preexisting AP-1
(c-jun/c-jun). The NF-B complex in its
inactive form is located in the cytosol and consists of two DNA binding
subunits, p50 and p65, which are associated with an inhibitory
protein, I-B (inhibitory protein that dissociates from NF-B).
Different stimuli, such as bacterial LPS, lead to a phosphorylation and
degradation of I-B upon which NF-B can translocate into the
nucleus. There, NF-B binds to specific promotor sequences of
proinflammatory genes, such as TNF-, and initiates their
transcription (24).
The macrophage cytokine TNF-, together with other inflammatory
mediators (25), plays a key role in many pathophysiologic
conditions, such as rheumatoid arthritis (26),
atherosclerosis (27), or septic shock (28).
ANP was previously shown to be an autocrine regulator of iNOS
(19), an enzyme strongly induced in inflammatory processes
(29) such as septic shock (30). The
observation that ANP mediates macrophage activation is particularly
interesting because ANP concentrations are highly elevated in septic
shock (31); moreover, LPS-exposed macrophages were shown
to produce increased ANP (9). Thus, due to its effects on
key events of macrophage activation, such as the activation of NF-B,
ANP may represent a promising autocrine substance modulating TNF-
production. Therefore, the aims of this study were 1) to investigate
whether ANP influences the production of this central proinflammatory
cytokine, 2) to consequently determine the type of receptor mediating a
potential ANP effect, 3) to elucidate whether the C-type NP inhibits
TNF- production, and 4) to obtain information on the underlying
mechanism of ANP-mediated TNF- inhibition.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rat ANP 99–126 was purchased from Calbiochem/Novabiochem (Bad
Soden, Germany), and CNP and cANF were purchased from Saxon
Biochemicals (Hannover, Germany). HS-142-1 was a gift from Dr. Y.
Matsuda (Tokyo Research Laboratories, Tokyo, Japan). TNF- cDNA was
obtained from Dr. K. Decker (University of Freiburg, Freiburg,
Germany); antiserum against the macrophage Ag F4/80 was from Serotec
(Wiesbaden, Germany); cell culture medium (RPMI 1640), FCS,
penicillin/streptomycine, and TRIzol were from Life Technologies
(Karlsruhe, Germany) and Biochrom (Berlin, Germany);
[-32P]UTP (800 Ci/mmol) and
[
-32P]ATP (3000 Ci/mmol) were from Amersham
(Braunschweig, Germany); dexamethasone solution was ordered from
Centravet (Bad Bentheim, Germany); heparin (Liquemin) was from Hoffmann
LaRoche (Grenzach-Whylen, Germany); and AP-1 and AP-2 binding
oligonucleotides, SP6 polymerase, and T4 polynucleotide kinase were
obtained from Boehringer Ingelheim Bioproducts (Heidelberg, Germany).
Bradford protein assay was from Bio-Rad (Munich, Germany). All other
materials were purchased from either Sigma (Deisenhofen, Germany) or
ICN Biomedicals (Eschwege, Germany).
Cell culture
Mouse bone marrow macrophages (BMM) were prepared as described previously (8), seeded at a density of 2 x 105 cells/ml in 24-well tissue plates, and grown for 5 days (5% CO2, 37°C) in RPMI 1640 medium supplemented with 20% L-929 cell-conditioned medium, 10% heat-inactivated FCS, and penicillin (100 U/ml)/streptomycin (100 µg/ml). L-929 cell-conditioned medium was removed at least 12 h before experiments. BMM were found >95% pure as judged by FACS analysis (FACScan; Becton Dickinson, Heidelberg, Germany) using an antiserum against the macrophage Ag F4/80 (32).
TNF- bioassay
BMM (24-well plates, 200 µl) were untreated or treated with
LPS (Escherichia coli, serotype 055:B5, 1
µg/ml). The effects of the following substances on TNF- production
were determined: ANP
(10-9–10-6
M), CNP (10-7 and
10-6 M), cANF
(10-8–10-6
M), dexamethasone (10-5
M), dibutyryl-cGMP
(10-5–10-3
M), HS-142-1 (100 and 10 µg/ml), and Ly-83583
(10-7 and
10-6 M). As previously
shown, none of the substances in the used concentrations possesses
cytotoxic activity on BMM (15). Substances were added to
the cells simultaneously with LPS. After 4 h TNF- activity was
measured in the supernatant by an L929 cytotoxicity bioassay. This
assay is based upon quantitation of the cytotoxic activity of TNF-
on L929 cells in the presence of actinomycin D (26). The
mitochondrial reduction of MTT to formazan was determined as an
indicator of L929 cell viability (33). Briefly, L929 cells
were seeded at a density of 2 x 104
cells/well into a 96-well microtiter plate. After incubation for
24 h at 37°C in a humidified atmosphere with 5%
CO2, the medium in the wells was replaced with
fresh medium containing actinomycin D at a final concentration of 1
µg/ml. After 1 h of preincubation with actinomycin D, serial
dilutions of the various supernatants from BMM (see above) were added.
For quantification of TNF- production, a standard curve was prepared
by the addition of recombinant human TNF- (0.75–50 pM; Sigma) to
the cells. The plates were then incubated for an additional 24 h
at 37°C. After removing the supernatant, cells were incubated with
MTT (0.5 mg/ml) for 1 h at 37°C and solubilized in DMSO before
spectrophotometric measurement of produced formazan at 550 nm (SLT
Labinstruments, Heidelberg, Germany). The following control experiments
were conducted. First, samples expected to contain TNF- were
neutralized with TNF- antiserum (Sepharose A-purified goat
polyclonal antiserum against mouse TNF-) and assayed in parallel.
The addition of anti-TNF- Ab (1%) completely abolished the
TNF- activity in supernatants of LPS-activated macrophages as well
as of recombinant TNF-, indicating that the assay was highly
specific for TNF-. Second, to further confirm specificity of the
L929 bioassay, some of the experiments were confirmed by a
radioimmunoassay specific for mouse TNF-. Third, to exclude a
potential direct effect of test substances on L929 viability, the
substances were added to actinomycin D-treated L929 cells for 24
h, and formazan production was determined as described above. None of
the substances showed significant alterations of L929 viability.
Detection of TNF- mRNA
BMM were stimulated with or without LPS (1 µg/ml) in the
presence or absence of ANP
(10-6 M) or dexamethasone
(10-5 M) for 4 h
(24-well plates). RNA was prepared using TRIzol reagent and was pooled
from six wells. Northern blot analysis with total RNA (15 µg) was
performed as described previously (8). Membranes were
hybridized to 32P-labeled cRNA probes (2 x
106 cpm/ml). The cDNA probe was a SmaI
linearized rat TNF- cDNA fragment subcloned in a pSPT18 vector (from
Dr. Decker). The TNF- probe was labeled with
[-32P]UTP (50 µCi, Amersham) and Sp6 RNA
polymerase. Signal intensities were evaluated by densitometric
analysis (EASY plus system; Herolab, Wiesloch, Germany). To
control for the amounts of intact mRNA, membranes were rehybridized
with a 32P-labeled ß-actin probe (2 x
106 cpm/ml) described in Ref. 34 .
Signal intensities were evaluated by densitometry, whereby signals for
TNF- were corrected by ß-actin signals. Ratios of LPS-treated
cells were set 100%.
Preparation of nuclear extracts
BMM were grown in 24-well plates and stimulated with LPS (1 µg/ml) in the presence or absence of ANP (10-6 M) for 30 min. Nuclear extracts were prepared as described in Ref. 35 . Briefly, cells were washed with PBS, resuspended in 400 µl hypotonic buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF), and allowed to swell on ice for 15 min. Nonidet P-40 (10%, 25 µl) was added before 10 s of vigorous vortexing and centrifugation at 12,000 x g for 30 s. The supernatant was removed, and the nuclear pellet was extracted with 50 µl of hypertonic buffer B (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF) by shaking at 4°C for 15 min. The extract was centrifuged at 12,000 x g, and the supernatant was frozen at -70°C. The protein concentration was determined by the Lowry method (36).
EMSA
A 22-mer double-stranded oligonucleotide probe containing a
consensus binding sequence for AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3')
was 5' end-labeled with [-32P]ATP (10 µCi)
using T4 polynucleotide kinase. Ten micrograms of nuclear protein was
incubated (20 min at room temperature) in a 15-µl reaction volume
containing 10 mM Tris-HCl (pH 7.5), 5 x 104
cpm radiolabeled oligonucleotide probe, 2 µg poly(dIdC), 4%
glycerol, 1 mM MgCl2, 0.5 mM EDTA, 50 mM NaCl,
and 0.5 mM DTT. Nucleoprotein-oligonucleotide complexes were resolved
by electrophoresis (4.5% nondenaturing polyacrylamide gel; 100 V). The
gel was autoradiographed with an intensifying screen at -70°C
overnight. Specificity of the DNA-protein complex was confirmed by
competition with a 100-fold excess of unlabeled AP-1 and AP-2 (5'-GAT
CGA ACT GAC CGC CCG CGG CCC GT-3') binding sequences, respectively.
For supershift analysis, 1 µg of Abs against
c-jun or c-fos (Santa Cruz Biotechnology,
Heidelberg, Germany) was added to the reaction mixtures 10 min before
the addition of radiolabeled probe. Preimmune rabbit IgG served as a
control.
Cytokine secretion of human whole blood
Cytokine secretion from human whole blood was determined as described previously (37). Briefly, heparinized blood was drawn from healthy volunteers and diluted 1:5 with cell culture medium RPMI 1640 supplemented with penicillin-streptomycin. Further heparin (2 IU/ml final concentration) was added to prevent blood coagulation as a result of dilution. LPS from Salmonella abortus equi was added simultaneously in a volume of 10 µl. After 24 h of incubation at 37°C and 5% CO2, the vials were shaken gently, cells were sedimented by centrifugation, and the supernatants were frozen in aliquots at -80°C until cytokine measurement.
Cytokines were measured by sandwich ELISA. IL-1ß and TNF- Ab pairs
were from Endogen (Eching, Germany), and IL-10 Ab pairs were from R&D
Systems (Wiesbaden, Germany). rIL-1ß (Endogen), recombinant TNF-
(Bender, Vienna, Austria), and recombinant IL-10 (R&D Systems) were
used as standards. Streptavidin-conjugated peroxidase (Dianova,
Hamburg, Germany) and the chromogen tetramethylbenzidine were used for
detection of the immune complexes.
Statistical analysis
All experiments were performed at least three times. Data are expressed ± SEM. Values with p < 0.01 were considered significantly different compared with 100% (LPS-treated cells only) by one sample t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production in BMM
Murine bone marrow-derived macrophages were stimulated with LPS (1
µg/ml) for 4 h to evoke TNF- synthesis. TNF- in the
supernatant was measured by determining the cytotoxic activity on L929
cells by employing recombinant TNF- as a standard. Coincubation of
BMM with ANP
(10-8–10-6
M) and LPS (1 µg/ml) resulted in a dose-dependent, significant
reduction of TNF- production up to 95% (Fig. 1
). Dexamethasone, a known inhibitor of
TNF- production (38), completely abolished TNF-
secretion (Fig. 1) and served as a control. ANP
(10-6 M) in the absence of
LPS did not alter the basal TNF- secretion and did not interfere
with the L929 bioassay (data not shown).
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synthesis
To determine which NP receptor mediates the inhibitory effect of
ANP on TNF- synthesis, the following experiments were performed with
LPS-stimulated cells. As shown in Fig. 2A, the stable analogue of
cGMP, dibutyryl-cGMP, at a concentration of
10-3 and
10-4 M significantly
reduced TNF- secretion by up to 70%. Furthermore, an antagonist of
the guanylate cyclase-coupled NPR-A, HS-142-1 (39), dose
dependently (10 and 100 µg/ml) abrogated the TNF--reducing effect
of ANP (10-6 M) in BMM
(Fig. 2B). Ly-83583
(10-7 and
10-6 M), a compound known
to inhibit cGMP production (40), partially abolished the
reduction of TNF- secretion by ANP
(10-6 M) (Fig. 2C). The specific NPR-C ligand cANF did not elicit a
significant decrease in TNF- secretion at a concentration of
10-6 M (Fig. 2D). However, cANF significantly reduced TNF- production
in concentrations of 10-7
and 10-8 M. ANP,
dibutyryl-cGMP, HS-142-1, Ly-83583, and cANF were tested for their
effect on TNF- synthesis in unstimulated cells and did not exhibit
any effect (data not shown).
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synthesis of BMM
BMM were previously shown to express all types of NP receptors
(15) and therefore may represent targets for all types of
NP. To elucidate whether the TNF--inhibitory effect is specific for
the binding of ANP to the NPR-A, we examined the ligand for the other
guanylate cyclase-coupled receptor (NPR-B), CNP. As shown in Fig. 3, CNP even at a concentration of
10-6 M did not affect
TNF- formation of LPS-activated BMM (Fig. 3). CNP had no effect on
basal TNF- production and did not interfere with the TNF-
bioassay (data not shown).
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mRNA levels
To investigate the mechanism of regulation of TNF- production
by ANP, Northern blot analysis was performed to determine whether ANP
inhibits TNF- mRNA accumulation when added simultaneously with LPS.
BMM were activated with LPS (1 µg/ml) in the presence or absence of
ANP (10-6 M), and mRNA was
isolated. In unstimulated cells, no TNF- mRNA was detectable (Fig. 4). ANP
(10-6 M) caused a marked
reduction of LPS-induced TNF- mRNA steady-state levels.
Dexamethasone (10-5 M), a known inhibitor of
TNF- induction (38), completely blocked TNF- mRNA
accumulation. Rehybridization with a ß-actin probe served as a
control for the amounts of intact RNA.
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Because ANP is known to interfere with transcriptional processes
(19, 41), we determined its effect on binding activity of
AP-1, a transcription factor known to be important for TNF-
induction (22). AP-1 binding activity of nuclear extracts
was assessed by EMSA after stimulation of cells with LPS (1 µg/ml)
for 30 min. Formation of the specific DNA probe-AP-1 complex was
markedly reduced when nuclear extracts of cells coincubated with ANP
(10-6 M) were employed
(Fig. 5). Binding specificity was
determined by addition of a 100-fold excess of unlabeled AP-1 or AP-2
binding sequence. Excess of unlabeled AP-1 binding sequence abolished
binding reaction to the labeled sequence, whereas AP-2 binding sequence
showed no significant influence on binding reaction (Fig. 5).
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Fig. 5
shows a markedly reduced AP-1 DNA binding activity in ANP +
LPS-treated cells compared with LPS treatment only. Supershift analysis
employing specific Abs against the most important c-jun and
c-fos subunits both resulted in markedly reduced complex
formation. This observation shows that AP-1 DNA binding proteins in
LPS-treated cells are predominantly composed of c-fos and
c-jun (Fig. 6), although no
supershifted ones were observed. No marked alteration of AP-1
composition could be detected in ANP
(10-6 M) cotreated cells
compared with those that received LPS treatment only (Fig. 6). Addition
of preimmune IgG had no effect on binding reaction (data not
shown).
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To investigate whether our shown data could be extended on other
species and cell types, we determined cytokine release in LPS-activated
whole human blood from healthy donors. ANP
(10-7 and
10-6 M) significantly
inhibited production of the proinflammatory cytokines TNF- and
IL-1ß (Fig. 7). ANP showed no effect on
the secretion of IL-1ra and IL-10 (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in macrophages represents an important
pathomechanism in diverse inflammatory processes (26, 27, 28). Therefore, special interest focuses on the regulatory
mechanism of TNF- production and on tools for potential
pharmacological intervention (42). By characterization of
a novel endogenous substance (i.e., ANP) regulating TNF- expression,
this study might therefore be of special interest. The presented work
investigates for the first time mechanisms by which the cardiovascular
hormone ANP selectively influences TNF- production by
macrophages.
The fact that inhibition of TNF- production by ANP is mediated via
the NPR-A was conclusively demonstrated because dibutyryl-cGMP dose
dependently mimicked the ANP effect. Employment of the microbial
polysaccharide HS-142-1, which selectively blocks the guanylate
cyclase-linked NP receptors and cGMP production (39), dose
dependently reversed the ANP effect. Because no soluble guanylate
cyclase has been detected in the macrophages employed
(19), the reversal of the ANP effect by Ly-83583 can be
attributed to an inhibition of cGMP production linked to the
particulate guanylate cyclase-linked NPR-A receptor. In accordance with
our observations, other data report an inhibition of TNF- production
by cyclic nucleotides (43, 44, 45), both cGMP and cAMP.
The effect was observed by the employment of cGMP analogues as well as
of phosphodiesterase inhibitors in murine and human mononuclear cells.
However, in human monocytes, ANP exerted no effect on TNF-
production (46). This observation was explained by the
lack of functional NPR-A on the cells investigated and thus the missing
cGMP answer after ANP treatment (46). On the other hand,
Suga et al. (47) reported a significant increase in cGMP
production in T helper precursor-1-derived human macrophages after ANP
treatment, whereas CNP was without effect. This observation may explain
our finding that CNP did not affect TNF- secretion.
In contrast to our findings, cGMP was also reported to stimulate
TNF- synthesis (48, 49, 50, 51). This discrepancy requires
explanation: the cells in which cGMP displayed a TNF--inducing
effect were either from rat or human source and represented different
macrophage populations, such as Kupffer cells or peritoneal
macrophages. These data together with our results lead to the
suggestion that the effect of cGMP on TNF- synthesis is highly
dependent on species and cell type. To get a first hint toward a
potential relevance of our findings during inflammatory processes in
man, we performed experiments using whole human blood. As previously
shown, human whole blood cytokine release offers the opportunity to
assess primary leukocyte responses in a physiological cell environment
without major preparation artifacts (37). Moreover, data
obtained using this ex vivo (52) system has been shown to
go in parallel with human in vivo data (53). Importantly,
ANP in this cellular system also exerted its TNF- inhibitory action
and additionally attenuated IL-1ß secretion. These interesting
findings suggest an anti-inflammatory potential of ANP in humans
as well.
Surprisingly, the specific NPR-C ligand cANF exhibited an inhibitory
effect on TNF- production only in lower concentrations
(10-7 and
10-8 M) and not in
concentrations as high as
10-6 M. This kind of
action exerted by cANF might probably be linked to the cAMP system
because activation of NPR-C is known to alter adenylate cyclase
activity (10). cAMP was shown to dose dependently enhance
or suppress TNF- release (45, 50, 51). Activation of
NPR-C with cANF may specifically influence cAMP levels and therefore
may reveal effects that are not related to the cGMP-dependent
inhibition of TNF- release exerted by ANP.
Previous data demonstrated that macrophages express mRNA coding for all
three receptor subtypes, i.e., NPR-A, NPR-B, and NPR-C
(15). The fact that macrophages express the NPR-B led us
to hypothesize that they should be target cells for CNP action.
Interestingly, CNP was demonstrated to be highly increased in septic
shock patients (54), leading to the anticipation that CNP
may be involved in respective inflammatory processes, such as TNF-
production. However, the property to inhibit TNF- secretion seems to
be specific for ANP because CNP displayed no activity. This observation
is in concordance with previous observations in macrophages showing a
lack of effect of CNP on iNOS, whereas ANP represents a potent
inhibitor of this proinflammatory enzyme (15). No
difference in stability between ANP and CNP is responsible for this
fact (15). Others also reported that CNP elicits very low
or no biological activity in different cell systems despite the
presence of NPR-B (55, 56, 57). One possible explanation for
this observation might be the heterogeneity of the NPR-B receptor. As
in diverse tissues, two forms of NPR-B receptor could also be detected
in macrophages (15, 58). These two forms of NPR-B differ
from each other only by a 75-bp deletion at the 3' flanking region and
possess practically the same high binding affinity for CNP
(58). However, the shorter form could not induce cGMP
production upon binding by CNP (56). The expression of
both forms of NPR-B transcripts in the macrophages was shown by us
previously (15). Because the ANP effect on TNF-
synthesis is shown to be mediated by cGMP, an insufficient amount of
cGMP produced by CNP may indeed be responsible for the lack of TNF-
inhibition by this peptide.
After demonstrating a specific inhibitory action of ANP on TNF-
production, the molecular mechanism of action should be investigated.
Because TNF- is a transcriptionally regulated cytokine
(22), the effect of ANP on TNF- mRNA had to be
determined. Northern blot analysis revealed markedly reduced levels of
TNF- mRNA initiated by ANP. The levels of TNF- mRNA are mainly
controlled via activation of respective transcription factors
(22, 23). We hypothesize that ANP decreases the
transcription of TNF- mRNA by interfering with two prominent
transcription factors involved in TNF- regulation, NF-B and AP-1
(22). In this study, we demonstrate for the first time
that ANP also inhibits activation of AP-1 in macrophages. This further
pivotal proinflammatory transcription factor is known to be involved in
TNF- gene expression (22, 23). Because different AP-1
complexes are suggested to exhibit different transcriptional activities
(23), we investigated whether ANP alters AP-1 composition.
However, a modulation of subunit composition by ANP could not be
observed.
Few data exist concerning the influence of cGMP and ANP on AP-1
activation, respectively, and data are controversial. In accordance
with our results, Isono et al. (59) reported that ANP
inhibits activation of AP-1 in glomerular mesangial cells. In contrast,
cGMP analogues were demonstrated to activate transcription from
AP-1-responsive promoters in thyroid follicular cells as well as in
fibroblasts (60). These data together with our results
support the notion that the influence of cGMP on AP-1-dependent gene
transcription might represent a highly cell type-specific and
stimulus-dependent event. It is important to note that ANP has recently
been shown to interfere with other cell signaling systems that control
gene transcription, e.g., ANP abrogates endothelin-3-induced
stimulation of egr-1 transcription and basic fibroblast factor
transactivation in astrocytes (41). Furthermore, ANP was
shown to attenuate activation of NF-B in different cell/organ
systems (19, 20). Taken together, increasing evidence
suggests ANP as an important regulator of gene transcription.
Modulation of TNF- synthesis by ANP may have broad implications in
situations such as endotoxic shock (28), where in fact
increased ANP plasma levels have been reported (31). Thus,
our data lead us to suggest ANP as an endogenous regulator of
inflammatory response.
In summary, we could demonstrate a novel mechanism of action for the
cardiovascular hormone. ANP specifically interacts with TNF-
production of LPS-activated macrophages and whole human blood
cells. This effect is mediated via cGMP and may involve transcriptional
inhibition because of attenuated activation of AP-1.
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Acknowledgments |
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cDNA probe. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Alexandra K. Kiemer, Institute of Pharmacy, Center of Drug Research, Pharmaceutical Biology, University of Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.
3 Abbreviations used in this paper: ANP, atrial natriuretic peptide; cANF, C-atrial natriuretic factor; CNP, C-type natriuretic peptide; NP, natriuretic peptide; NPR, natriutetic peptide receptor; IL-1ra, interleukin-1 receptor antagonist; iNOS, inducible NO synthase; BMM, bone marrow macrophage.
Received for publication September 29, 1999. Accepted for publication April 21, 2000.
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 S. M. Sacre, A. Lo, B. Gregory, R. E. Simmonds, L. Williams, M. Feldmann, F. M. Brennan, and B. M. Foxwell Inhibitors of TLR8 Reduce TNF Production from Human Rheumatoid Synovial Membrane Cultures J. Immunol., December 1, 2008; 181(11): 8002 - 8009. [Abstract] [Full Text] [PDF]
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V. von Bulow, S. Dubben, G. Engelhardt, S. Hebel, B. Plumakers, H. Heine, L. Rink, and H. Haase Zinc-Dependent Suppression of TNF-{alpha} Production Is Mediated by Protein Kinase A-Induced Inhibition of Raf-1, I{kappa}B Kinase beta, and NF-{kappa}B J. Immunol., September 15, 2007; 179(6): 4180 - 4186. [Abstract] [Full Text] [PDF]
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 T. Nakayama, N. Harada, M. Asano, N. Nomura, T. Saito, A. Mishima, and K. Okajima Atrial Natriuretic Peptide Reduces Ischemia/Reperfusion-Induced Spinal Cord Injury in Rats by Enhancing Sensory Neuron Activation J. Pharmacol. Exp. Ther., August 1, 2007; 322(2): 582 - 590. [Abstract] [Full Text] [PDF]
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 T. J. Wang, M. G. Larson, M. J. Keyes, D. Levy, E. J. Benjamin, and R. S. Vasan Association of Plasma Natriuretic Peptide Levels With Metabolic Risk Factors in Ambulatory Individuals Circulation, March 20, 2007; 115(11): 1345 - 1353. [Abstract] [Full Text] [PDF]
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 K. Ladetzki-Baehs, M. Keller, A. K. Kiemer, E. Koch, S. Zahler, A. Wendel, and A. M. Vollmar Atrial Natriuretic Peptide, a Regulator of Nuclear Factor-{kappa}B Activation in Vivo Endocrinology, January 1, 2007; 148(1): 332 - 336. [Abstract] [Full Text] [PDF]
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 L. R. Potter, S. Abbey-Hosch, and D. M. Dickey Natriuretic Peptides, Their Receptors, and Cyclic Guanosine Monophosphate-Dependent Signaling Functions Endocr. Rev., February 1, 2006; 27(1): 47 - 72. [Abstract] [Full Text] [PDF]
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V. von Bulow, L. Rink, and H. Haase Zinc-Mediated Inhibition of Cyclic Nucleotide Phosphodiesterase Activity and Expression Suppresses TNF-{alpha} and IL-1{beta} Production in Monocytes by Elevation of Guanosine 3',5'-Cyclic Monophosphate J. Immunol., October 1, 2005; 175(7): 4697 - 4705. [Abstract] [Full Text] [PDF]
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 D. C. Irwin, M. C. Tissot van Patot, A. Tucker, and R. Bowen Direct ANP inhibition of hypoxia-induced inflammatory pathways in pulmonary microvascular and macrovascular endothelial monolayers Am J Physiol Lung Cell Mol Physiol, May 1, 2005; 288(5): L849 - L859. [Abstract] [Full Text] [PDF]
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 K. Suzuki, T. Suda, T. Naito, K. Ide, K. Chida, and H. Nakamura Impaired Toll-like Receptor 9 Expression in Alveolar Macrophages with No Sensitivity to CpG DNA Am. J. Respir. Crit. Care Med., April 1, 2005; 171(7): 707 - 713. [Abstract] [Full Text] [PDF]
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 A. T. Bender, C. L. Ostenson, E. H. Wang, and J. A. Beavo Selective up-regulation of PDE1B2 upon monocyte-to-macrophage differentiation PNAS, January 11, 2005; 102(2): 497 - 502. [Abstract] [Full Text] [PDF]
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 A. M. Kapoun, F. Liang, G. O'Young, D. L. Damm, D. Quon, R. T. White, K. Munson, A. Lam, G. F. Schreiner, and A. A. Protter B-Type Natriuretic Peptide Exerts Broad Functional Opposition to Transforming Growth Factor-{beta} in Primary Human Cardiac Fibroblasts: Fibrosis, Myofibroblast Conversion, Proliferation, and Inflammation Circ. Res., March 5, 2004; 94(4): 453 - 461. [Abstract] [Full Text] [PDF]
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 N. C. Weber, S. B. Blumenthal, T. Hartung, A. M. Vollmar, and A. K. Kiemer ANP inhibits TNF-{alpha}-induced endothelial MCP-1 expression--involvement of p38 MAPK and MKP-1 J. Leukoc. Biol., November 1, 2003; 74(5): 932 - 941. [Abstract] [Full Text] [PDF]
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R. Morita, N. Ukyo, M. Furuya, T. Uchiyama, and T. Hori Atrial Natriuretic Peptide Polarizes Human Dendritic Cells Toward a Th2-Promoting Phenotype Through Its Receptor Guanylyl Cyclase-Coupled Receptor A J. Immunol., June 15, 2003; 170(12): 5869 - 5875. [Abstract] [Full Text] [PDF]
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A. K. Kiemer, N. Bildner, N. C. Weber, and A. M. Vollmar Characterization of Heme Oxygenase 1 (Heat Shock Protein 32) Induction by Atrial Natriuretic Peptide in Human Endothelial Cells Endocrinology, March 1, 2003; 144(3): 802 - 812. [Abstract] [Full Text] [PDF]
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 K. Kataoka, T. Muta, S. Yamazaki, and K. Takeshige Activation of Macrophages by Linear (1right-arrow3)-beta -D-Glucans. IMPLICATIONS FOR THE RECOGNITION OF FUNGI BY INNATE IMMUNITY J. Biol. Chem., September 20, 2002; 277(39): 36825 - 36831. [Abstract] [Full Text] [PDF]
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A. K. Kiemer, N. C. Weber, R. Furst, N. Bildner, S. Kulhanek-Heinze, and A. M. Vollmar Inhibition of p38 MAPK Activation via Induction of MKP-1: Atrial Natriuretic Peptide Reduces TNF-{alpha}-Induced Actin Polymerization and Endothelial Permeability Circ. Res., May 3, 2002; 90(8): 874 - 881. [Abstract] [Full Text] [PDF]
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A. K. Kiemer, M. D. Lehner, T. Hartung, and A. M. Vollmar Inhibition of Cyclooxygenase-2 by Natriuretic Peptides Endocrinology, March 1, 2002; 143(3): 846 - 852. [Abstract] [Full Text] [PDF]
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 T. Suganami, M. Mukoyama, A. Sugawara, K. Mori, T. Nagae, M. Kasahara, K. Yahata, H. Makino, Y. Fujinaga, Y. Ogawa, et al. Overexpression of Brain Natriuretic Peptide in Mice Ameliorates Immune-Mediated Renal Injury J. Am. Soc. Nephrol., December 1, 2001; 12(12): 2652 - 2663. [Abstract] [Full Text] [PDF]
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 A K Kiemer and A M Vollmar The atrial natriuretic peptide regulates the production of inflammatory mediators in macrophages Ann Rheum Dis, November 1, 2001; 60(90003): iii68 - 70. [Abstract] [Full Text] [PDF]
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 A. K. Kiemer and A. M. Vollmar Induction of L-Arginine Transport Is Inhibited by Atrial Natriuretic Peptide: A Peptide Hormone as a Novel Regulator of Inducible Nitric-Oxide Synthase Substrate Availability Mol. Pharmacol., September 1, 2001; 60(3): 421 - 426. [Abstract] [Full Text] [PDF]
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 W.-G. Forssmann, M. Meyer, and K. Forssmann The renal urodilatin system: clinical implications Cardiovasc Res, August 15, 2001; 51(3): 450 - 462. [Abstract] [Full Text] [PDF]
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 F. El-Gehani, M. Tena-Sempere, H. Ruskoaho, and I. Huhtaniemi Natriuretic Peptides Stimulate Steroidogenesis in the Fetal Rat Testis Biol Reprod, August 1, 2001; 65(2): 595 - 600. [Abstract] [Full Text] [PDF]
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M. J. Coffey, R. Natarajan, P. H. Chumley, B. Coles, P.-R. Thimmalapura, M. Nowell, H. Kuhn, M. J. Lewis, B. A. Freeman, and V. B. O'Donnell Catalytic consumption of nitric oxide by 12/15- lipoxygenase: Inhibition of monocyte soluble guanylate cyclase activation PNAS, June 20, 2001; (2001) 141136098. [Abstract] [Full Text] [PDF]
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M. J. Coffey, R. Natarajan, P. H. Chumley, B. Coles, P.-R. Thimmalapura, M. Nowell, H. Kuhn, M. J. Lewis, B. A. Freeman, and V. B. O'Donnell Catalytic consumption of nitric oxide by 12/15- lipoxygenase: Inhibition of monocyte soluble guanylate cyclase activation PNAS, July 3, 2001; 98(14): 8006 - 8011. [Abstract] [Full Text] [PDF]
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A. K. Kiemer, N. C. Weber, R. Furst, N. Bildner, S. Kulhanek-Heinze, and A. M. Vollmar Inhibition of p38 MAPK Activation via Induction of MKP-1: Atrial Natriuretic Peptide Reduces TNF-{alpha}-Induced Actin Polymerization and Endothelial Permeability Circ. Res., May 3, 2002; 90(8): 874 - 881. [Abstract] [Full Text] [PDF]
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