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Production by Macrophages: In Vitro and In Vivo Studies1
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Department of Cellular Biology, Faculty of Biology, Complutense University, Madrid, Spain;
Department of Medical Biochemistry and Molecular Biology, University of Seville School of Medicine and Virgen Macarena Hospital, Seville, Spain; and
Department of Biological Sciences, Rutgers University, Newark, NJ 07102
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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is an important mediator of shock and
organ failure complicating Gram-negative sepsis. VIP has been shown to
attenuate the deleterious consequences of this pathologic phenomenon.
In this study we have investigated the effects of VIP and the
structurally related neuropeptide pituitary adenylate
cyclase-activating polypeptide (PACAP38) on the production of TNF-
by endotoxin-activated murine peritoneal macrophages. Both
neuropeptides rapidly and specifically inhibit the LPS-stimulated
production of TNF-, exerting their action through the binding to
VPAC1 receptor and the subsequent activation of the adenylate cyclase
system. VIP and PACAP regulate the production of TNF- at a
transcriptional level. In vitro results were correlated with an
inhibition of both TNF- expression and release in endotoxemic mice
in vivo. The immunomodulatory role of VIP in vivo is supported by the
up-regulation of VIP release in serum and peritoneal fluid by LPS and
proinflammatory cytokines such as TNF-, IL-1ß, and IL-6. These
findings support the idea that under toxicity conditions associated
with high LPS doses, VIP and PACAP could act as protective mediators
that regulate the excessive release of TNF- to reduce inflammation
or shock. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. TNF- is a
monocyte-macrophage-derived cytokine that acts as an important mediator
in the defense mechanism of the host in response to bacterial
colonization or invasion and causes immunopathologic disorders when
secreted in excess 1 . The toxicity caused by Gram-negative bacteria
has been ascribed to LPS, an outer membrane component of bacteria. LPS
represents one of the most potent inducers of TNF- and in high
concentrations causes tissue injury, fever, disseminated vascular
coagulation, and septic shock, often resulting in death 1 . Vasoactive intestinal peptide (VIP)3 and pituitary adenylate cyclase-activating polypeptide (PACAP) are, respectively, 28- and 38-amino acid peptides that have 65% homology and elicit a broad spectrum of biological functions, including actions on natural and acquired immunity 2 . Both VIP and PACAP interact with a family of three VIP/PACAP receptors: VIP1 receptor (VPAC1), which is the classical receptor, shows similar affinities for VIP and PACAP and stimulates almost exclusively the adenylate cyclase system. VPAC2 has similar affinities for both peptides, activates the adenylate cyclase system, and seems to be coupled to a calcium chloride channel in transfected cells. The third type of receptor is the PACAP-preferring receptor (PAC1) that recognizes PACAP much more potently than VIP and activates both adenylate cyclase and phospholipase C. Macrophages have been described to possess VPAC1 3, 4 , and we have recently reported PAC1 receptor gene expression in rat peritoneal macrophages 5 .
Actions on natural and acquired immunity of VIP and PACAP seem to be
mediated through effects on the production and release of cytokines.
Thus, VIP stimulates IL-5 release from schistosome granulomas and
splenic T lymphocytes, and inhibits the expression and secretion of
IL-2, IL-4, and IL-10 in stimulated peripheral T cell and thymocytes
cultures 6 . Moreover, we have recently studied the effects of VIP and
PACAP on the production of the inflammatory cytokine IL-6 7 . We
showed that both neuropeptides exhibited a dual effect on IL-6
production by peritoneal macrophages. Whereas VIP and PACAP inhibited
the release of IL-6 from macrophages stimulated with high LPS doses,
the two neuropeptides enhanced IL-6 secretion in macrophages
unstimulated and stimulated by low LPS doses. To clarify the roles
played by VIP and PACAP in vivo and in vitro in inflammation, we
addressed the following objectives in this study: 1) to determine the
in vitro and in vivo effects of both peptides on LPS-induced TNF-
production and release by macrophages, 2) to investigate the specific
receptor as well as the intracellular signal pathways involved, and 3)
to elucidate the possible existence of a negative feedback mechanism in
the regulation of proinflammatory cytokine production by endogenous
VIP/PACAP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice, 6–10 wk of age (weighing 19–20 g), were purchased from Iffa Credo (France). Mice were kept in the animal house for at least 1 wk before use in a temperature-controlled room with a 12-h light, 12-h dark cycle and were allowed free access to standard laboratory chow and water.
Abs and reagents
Synthetic VIP, PACAP38, PACAP27, VIP1–12, and
VIP10–28 were purchased from Novabiochem (Laufelfingen,
Switzerland). The VPAC1 antagonist
[Ac-His1,D-Phe2,K15,R16,L27]VIP3–7-GRF8–27
and the VPAC1 agonist
[K15,R16,L27]VIP1–7-GRF8–27
were donated by Dr. Robberecht (Université Libre de Bruxelles,
Brussels, Belgium). The VIP antagonist
[N-Ac-Tyr1,D-Phe2]-GRF1–29
amide was obtained from Cambridge Research BioChemicals (Wilmington,
DE), and PACAP6–38, secretin, glucagon, and biotinylated
VIP were obtained from Peninsula Laboratories (Belmont, CA).
Oligonucleotides were synthesized by Pharmacia Biotech (Uppsala,
Sweden). The blocking reagent, digoxigenin oligonucleotide tailing kit,
anti-digoxigenin-AP (Fab), and the chemiluminescent substrate for
alkaline phosphatase (CSPD) were obtained from Boehringer
Mannheim (Mannheim, Germany). Murine recombinant (mr) IL-1ß,
mrTNF-, mrIL-6, and capture and biotinylated monoclonal
anti-murine TNF- Abs were purchased from PharMingen (San Diego,
CA). LPS (from Escherichia coli 0111:B4, E. coli
0127:B8, and S. enteridis), FITC-0111:B4 LPS,
calphostin C, 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid
(ABTS substrate), N-laurylsarcosyl, malic acid, forskolin,
PMA, SDS, EDTA, EGTA, and avidin-peroxidase were purchased from Sigma
(St. Louis, MO), and
N-[2-(p-bromocinnamyl-amino)ethyl]-5-iso-quinolinesulfonamide
(H-89) was obtained from ICN Pharmaceuticals (Costa Mesa, CA). Anti-VIP
mAb was a generous gift from Dr. Wong (University of California School
of Medicine, Los Angeles, CA).
Preparation of macrophages and cell cultures
Thioglycolate-elicited macrophages were prepared as reported
previously 8 . Briefly, 2 ml of 4% Brewer’s thioglycolate medium
(Difco, Detroit, MI) was injected i.p. into mice, and peritoneal
exudate cells were collected on day 4 by flushing peritoneal cavity
with ice-cold RPMI 1640 medium (Life Technologies, Grand Island, NY).
Peritoneal exudate cells containing lymphocytes and macrophages were
washed twice and resuspended in ice-cold RPMI 1640 medium supplemented
with 2% heat-inactivated FCS (Life Technologies) containing 0.01 M
HEPES buffer, 1 mM pyruvate, 0.1 M nonessential amino acids, 2 mM
L-glutamine, 50 µM 2-ME, 100 U/ml penicillin, and 100
µg/ml streptomycin (RPMI 1640 complete medium). Cells were plated in
24-well tissue culture plates (Corning Glass, Corning, NY) at 5 x
105 cells/well in a final volume of 1 ml. The cells were
incubated at 37°C for 2 h so they would adhere to plastic, and
nonadherent cells were removed by repeated washing with RPMI 1640
medium. At least 96% of the adherent cells were macrophages as judged
by morphologic and phagocytic criteria and by flow cytometry using
MAC-3 Ab (clone M3/84, CD107b). Macrophage monolayers were incubated in
RPMI 1640 complete medium and stimulated with different concentrations
of LPS (from 10 pg/ml to 10 µg/ml) in the presence or the absence of
VIP or PACAP38 (from 10-13 to 10-7 M) at
37°C in a humidified incubator with 5% CO2. In some
experiments, macrophages were stimulated with IL-1ß (100 U/ml).
Cell-free supernatants were harvested at the designated time points and
kept frozen (-20°C) until assayed for TNF- production by ELISA.
When indicated, to determine the effect of calphostin C, H-89,
forskolin, PMA, and the VIP and PACAP antagonists, cells were incubated
with a concentration range of these agents as indicated in the text and
simultaneously incubated with VIP/PACAP and LPS as described above.
Cytokine determination: ELISA assay for TNF-
The amount of TNF- present in supernatants was determined
using a murine TNF- capture ELISA assay essentially as described
previously 9 . Briefly, a capture monoclonal anti-murine TNF-
Ab (clone MP6-XT22) was used to coat microtiter plates (Corning ELISA
plates) at 2 µg/ml at 4°C for 16 h. After washing and blocking
with PBS containing 3% BSA, culture supernatants were added to each
well for 12 h at 4°C. Unbound material was washed off, and a
biotinylated rabbit anti-mouse TNF- mAb was added at 2 µg/ml
for 2 h. Bound Ab was detected by addition of avidin-peroxidase
for 30 min followed by addition of the ABTS substrate solution.
Absorbances at 405 nm were taken 30 min after addition of the
substrate. A standard curve was constructed using various dilutions of
murine rTNF- in PBS containing 10% FCS. The amount of TNF- in
the culture supernatants was determined by extrapolation of absorbances
to the standard curve. The ELISA was specific for murine TNF- (did
not cross-react with human or rat TNF- or with other murine
cytokines, such as IL-10, IL-6, IL-2, IL-3, IL-4, IL-5, IL-1,
IL-1ß, and IFN-
). The sensitivity of the assay was 10 pg of
TNF-/ml.
RNA isolation and Northern blot analysis
Northern blot analysis was performed according to standard methods. Peritoneal macrophage monolayers (2 x 106 cells/ml) were stimulated with LPS (10 ng/ml) in the absence or the presence of VIP or PACAP38 (10-8 M) for 3 h at 37°C, and total RNA was isolated using the Ultraspec RNA Isolation System (Biotecx, Houston, TX) according to the manufacturer’s instructions. Briefly, macrophage monolayer (2 x 107 cells) was lysed in Ultraspec RNA reagent, followed by chloroform extraction and isopropanol precipitation. The RNA was resuspended in diethylpyrocarbonate-treated water and quantitated spectrophotometrically at 260/280 nm. Twenty micrograms of total RNA from each sample was electrophoresed on 1.2% agarose-formaldehyde gels, transferred to Nytran membranes (Schleicher & Schuell, Keene, NJ), and cross-linked to the nylon membrane using UV light (Linus, Cultek SL, Spain).
The probe for TNF- was an oligonucleotide
(5'-TTGACCTCAGCGCTGAGTTGGTCCCCCTTCTCAGCTGGAAGACT-3') designed
from the murine TNF- mRNA 10 . The probe for the murine 18S
ribosomal RNA, as a quantity control for RNA, was an oligonucleotide
(5'-CCAATTACAGGGCCTCGAAAGAGTCCTGTA-3') derived from the published
sequence. Oligonucleotides were 3' labeled with digoxigenin-dUTP/dATP
mix using terminal transferase according to manufacturer’s
instructions. After UV cross-linking the blots were washed twice for 5
min each time in diethylpyrocarbonate-water and prehybridized at 68°C
for 3 h in prehybridization buffer containing 5x SSC, 1%
blocking reagent (w/v), 0.1% N-laurylsarcosyl (w/v), and
0.02% SDS (w/v). Hybridization was performed at 60°C for 16 h
in the same prehybridization buffer plus 25 ng/ml of TNF- 3'-labeled
oligonucleotide. Thereafter, the blots were washed twice for 5 min each
time in 2x SSC/0.1% SDS (w/v) at room temperature, followed by
washing twice for 5 min each time to a final stringency of 0.1x
SSC/0.1% SDS (w/v) at 60°C. To detect the hybridization signal, the
blots were blocked for 30–45 min in 0.1 malic acid (pH 7.5), 0.15 M
NaCl, and 1% blocking reagent; further incubated with 1/5000
anti-digoxigenin Ab conjugated with alkaline phosphatase for 30 min
in the same buffer; and developed by chemiluminescence in 0.1 M
Tris-HCl (pH 9.5), 0.1 M NaCl, and 0.25 mM CSPD. The
chemiluminescent signals were obtained using Kodak X-OMAT AR film
(Sigma) at room temperature. The membranes were stripped twice for 30
min each time in 50 mM Tris-HCl (pH 8.0) containing 5%
N,N-dimethyl-formamide (v/v) and 1% SDS (w/v) at
68°C and were rehybridized with the 18S ribosomal RNA-labeled
antisense oligonucleotide at 55°C.
In vivo quantitation of TNF- levels
Female mice (6–10 wk old) were randomized to receive a single
i.p. dose of LPS (25 µg/mouse), LPS plus VIP (5 nmol/mouse), or LPS
plus PACAP38 (5 nmol/mouse). After 2 h, blood was removed through
cardiac puncture, and peritoneal exudate was obtained as described
above. The blood samples were allowed to clot for 1 h at room
temperature, and serum was obtained and kept frozen until TNF- ELISA
analysis. The peritoneal suspension was centrifuged for 5 min at
1800 x g, and cell-free supernatant was harvested and
assayed for TNF- ELISA. The peritoneal cells were subjected to
Northern blot analyses as described above.
Determination of VIP levels
VIP production was determined using a competitive ELISA. Total peritoneal cell suspension (5 x 106 cells) was incubated with various stimuli as indicated in Results. After different times, the supernatants (1 ml) were collected and lyophilized. A capture anti-VIP mAb (clone CURE.V55) was used to coat microtiter plates (Corning ELISA plates) at 2.5 µg/ml at 4°C for 16 h. After washing and blocking with PBS containing 3% BSA, lyophilized culture supernatants resuspended in 100 µl PBS/10% FCS containing 0.5 ng/ml biotinylated-VIP were added to each well for 12 h at 4°C. Bound biotinylated VIP was detected by addition of avidin-peroxidase for 30 min followed by addition of the ABTS substrate solution. Absorbances at 405 nm were determined 30 min after addition of the substrate. A standard curve was constructed using various dilutions of VIP in PBS containing 10% FCS. The amount of VIP in the culture supernatants was determined by extrapolation of absorbances to the standard curve. The ELISA was specific for VIP (did not cross-react with secretin, glucagon, PACAP27, PACAP38, VIP1–12, or VIP10–28). The sensitivity of the assay was 20 pg of VIP/ml.
Statistical analysis
All values are expressed as the mean ± SD of the number of experiments performed in duplicate, as indicated in the corresponding tables and figures. Comparisons between groups were made using Student’s t test followed by Scheffe’s F test, with p < 0.05 as the minimum significant level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production
To determine the effects of VIP and PACAP on TNF- production,
macrophages were stimulated with LPS, and supernatants were harvested
at different time points and assayed for TNF- production by ELISA.
Stimulation of macrophages with LPS for 6 h resulted in a
dose-dependent release of TNF- (Fig. 1
A). The addition of VIP or
PACAP to LPS-stimulated cultures resulted in an inhibition of TNF-
production by macrophages (Fig. 1, A and B). The
dose-response curves were similar for VIP and PACAP (Fig. 1B), showing maximal effects at
10-8–10-9 M. During stimulation with 1–10
ng/ml LPS, the inhibitory activity of VIP and PACAP was the most
pronounced, with maximum values of 57%, showing an IC50
(i.e., the concentration of neuropeptide producing 50% of maximal
inhibition) of 80 pM (Table I). The time
curves indicate that the TNF- release was significantly inhibited by
VIP and PACAP as early as 3 h, with the maximum inhibitory effect
after 6 h of culture (Fig. 1C). In addition, to
determine whether the effect of VIP and PACAP was sustained over a
longer period of time, macrophages were stimulated with LPS in the
presence of these neuropeptides during a 24-h incubation period. The
reduction of TNF- release was maintained throughout the 24-h
incubation period, indicating that VIP and PACAP do not delay but,
rather, attenuate TNF- release (Fig. 1C). Neither VIP nor
PACAP merely alters the time course of TNF- production by
macrophages stimulated with LPS.
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production was obtained with
macrophages stimulated with 10 ng/ml LPS at a neuropeptide
concentration of 10-8 M after 6 h of culture, we used
these conditions in the rest of the experiments. The inhibitory effects were not the result of a decrease in the number of peritoneal macrophages, as neither VIP nor PACAP affected cell numbers or viability for stimulated macrophages after 8 h of culture (viabilities ranged from 87–96% with or without peptides).
In addition, to determine whether contaminating lymphocytes might have
contributed to the observed cytokine production, freshly isolated
peritoneal suspensions were stimulated with LPS in the presence or the
absence of VIP or PACAP. Similar TNF- values were obtained in
supernatants collected at 6 h from freshly isolated peritoneal
exudate cultures (50–60% macrophages, 30–40% lymphocytes) and from
monolayer cell preparations containing about 96% macrophages,
suggesting that the role of lymphocytes in this system is marginal
(data not shown).
Inhibition of LPS-stimulated TNF- production by VIP and PACAP is
specific
After having established that VIP and PACAP inhibited TNF-
production by LPS-stimulated macrophages, we investigated whether these
effects could be related to the occupancy of specific receptors.
Previous reports showed that VIP and both PACAPs, PACAP38 and PACAP27,
bind with similar affinities to VIP/PACAP receptors on different immune
cells, including macrophages 5, 11, 12 . In contrast, the structurally
related peptides secretin and glucagon bind to the VIP/PACAP receptors
poorly or not at all. Also, it has been established that VIP fragments
1–12 and 10–28 and the PACAP fragment 6–38 do not act as agonists in
the inhibition of IL-2, IL-6, and IL-10 7, 13, 14 . We first
investigated the specificity of the inhibitory effect of VIP and PACAP
on LPS-induced TNF- release by comparing the effects of PACAP27,
secretin, glucagon, and the VIP and PACAP fragments. TNF- production
by macrophages was inhibited by VIP, PACAP27, and PACAP38 to similar
levels at all concentrations assayed
(10-7–10-11 M; Fig. 2A), whereas secretin,
glucagon, PACAP6–38, and the two VIP fragments failed to
inhibit TNF- production (Fig. 2A). These results confirm
the specificity of the VIP and PACAP inhibitory activity, and the lack
of effect of the neuropeptide fragments suggests that intact VIP and
PACAP molecules are required for their inhibitory effect.
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production by VIP and PACAP, experiments using
receptor antagonists were performed. We investigated the ability of
PACAP6–38, a PACAP antagonist with specificity for PAC1,
and VPAC2 15 , and of
[Ac-Tyr1,D-Phe2]GRF1–29 amide, a
VPAC1 antagonist 16 , to reverse the effects of VIP and PACAP on
TNF- production by incubating increasing concentrations of these
VIP/PACAP antagonists (10-5–10-8 M) in the
presence of a fixed concentration of VIP or PACAP (10-8
M). The inhibitory effect of VIP and PACAP on TNF- production was
reversed by the VPAC1 antagonist in a dose-dependent manner (Fig. 2
B). The most effective antagonist concentration was
10-5 M, which almost completely abolished the inhibitory
effect of VIP and PACAP (Fig. 2B). A second, recently
developed 17 , VPAC1 antagonist
[Ac-His1,D-Phe2,K15,
R16,L27]VIP3–7-GRF8–27
had a similar effect (Fig. 2C). In contrast, the PAC1
antagonist did not reverse the inhibitory effect of VIP and PACAP (Fig. 2B). Neither the VPAC1 antagonists nor
PACAP6–38 significantly affected cell number or viability
(data not shown) or TNF- levels (Fig. 2). In addition, a newly
described 18 VPAC1 agonist
[K15,R16,L27]VIP1–7-GRF8–27
inhibited in a dose-dependent manner TNF production by macrophages,
similar to VIP and PACAP (Fig. 2D). These results confirm
the specificity of the VIP and PACAP inhibitory activity and suggest
that both neuropeptides exert their actions through binding to VPAC1.
Time course for the inhibitory effect of VIP and PACAP on
LPS-induced TNF- production
In the experiments described above, VIP and PACAP were added to
cells at the same time as LPS. We next investigated the effect of
exposing macrophages to VIP or PACAP before and after LPS stimulation.
We stimulated peritoneal macrophages with LPS (10 ng/ml) and added
10-8 M VIP or PACAP at different times before (24 and
2 h) and after (from 0–4 h) initiation of the cultures.
Supernatants were collected 6 h after the initiation of cultures
and were assayed for TNF- production. The addition of VIP and PACAP
up to 1 h after LPS stimulation resulted in significant levels of
inhibition (57–60%; Fig. 3A). Later additions resulted
in progressively lower degrees of inhibition (Fig. 3A). On
the contrary, preincubation with VIP or PACAP for 24 h did not
inhibit the latter LPS-stimulated TNF- production. However, addition
of VIP or PACAP to the cells 2 h before the addition of LPS
inhibited TNF production as effectively as simultaneous addition (Fig. 3A). Furthermore, the higher concentration
(10-7 M) of both neuropeptides prevented the suppression
of TNF production by a second dose of VIP or PACAP added 24 h
later (Fig. 3B). Preincubation with 10-8 M VIP
or PACAP did not significantly prevent inhibition of TNF production by
a second dose added to the cells with the LPS (Fig. 3B).
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C). Effects of VIP and PACAP on IL-1ß-induced TNF release
Next, to investigate whether the observed inhibition by VIP and
PACAP was specific for endotoxin-induced TNF release or whether these
neuropeptides might also act as inhibitors of TNF- release by
cytokine-activated macrophages, the effects of VIP and PACAP on
IL-1ß-induced TNF- production were determined. VIP and PACAP
attenuated the release of TNF- by macrophages activated with 100
U/ml IL-1ß, but to a greater extent than LPS-induced release (Fig. 4). The maximal VIP/PACAP inhibitory
effect on cytokine-induced TNF- production was exerted at 12 h
(Fig. 4).
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mRNA levels
Having demonstrated that VIP and PACAP had an inhibitory effect on
endotoxin-induced TNF- production, we determined whether this action
occurs at a transcriptional level. We stimulated peritoneal macrophages
with LPS in the presence or the absence of 10-8 M VIP or
PACAP. Total RNA was prepared from 3-h cultures and subjected to
Northern blot analysis. Although no TNF- mRNA was detectable in
unstimulated cells (data not shown), a high level of TNF- mRNA was
present in LPS-stimulated macrophages, and the hybridization signal was
strongly reduced in the presence of either VIP or PACAP (Fig. 5). These results indicate that VIP and
PACAP could inhibit TNF- production at the transcriptional level in
LPS-stimulated macrophage cultures; however, it remains to be
established whether the diminution in steady state TNF- mRNA levels
results from a decrease in either the novo transcriptional rate or
message stabilization, or both.
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production by VIP and PACAP
To study the second messengers involved in the inhibitory activity
of VIP and PACAP on TNF- release, we determined the effects of PMA
(an activator of PKC), calphostin C (a specific inhibitor of PKC) 19 ,
forskolin (an inducer of intracellular cAMP levels) 20 , and H89 (a
potent and specific inhibitor of PKA) 21 on TNF- production by
LPS-stimulated macrophages incubated in the absence or the presence of
VIP or PACAP.
PMA induced TNF- production in a dose-dependent manner, and
VIP/PACAP inhibited the PMA-induced release of TNF- (Fig. 6A). These results suggests
that PKC plays a role in TNF- production by macrophages. Indeed,
calphostin C inhibited in a dose-dependent manner the TNF-
production by LPS-stimulated macrophages (Fig. 6C).
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production in LPS-stimulated macrophages, similar to
VIP/PACAP (Fig. 6B). The treatment with forskolin in
addition to VIP/PACAP resulted in an additive effect only at the
highest forskolin concentration used (10 µM; Fig. 6B). The
fact that the effect of VIP/PACAP was not additive with forskolin
suggests that both neuropeptides and forskolin mediate their actions
through the same intracellular pathway. The involvement of cAMP in the
inhibitory effect of VIP/PACAP on TNF- production is also supported
by the results obtained with the two specific inhibitors, i.e.,
calphostin C and H89. As reported above, calphostin C inhibited TNF-
release by LPS-stimulated macrophages, but did not reverse the
inhibitory effect of VIP/PACAP (Fig. 6C). In contrast,
whereas H89 does not affect TNF- production by LPS-stimulated
macrophages, it reverses the inhibitory effect of VIP/PACAP (Fig. 6D). These results suggest that whereas TNF- production
by LPS-stimulates macrophages involves PKC, the inhibitory effect of
VIP/PACAP is exerted at least partially through increases in
intracellular cAMP.
VIP and PACAP inhibit LPS-induced TNF- production in vivo
An attempt was made to reproduce the in vitro observations in
vivo. To this end, mice were treated with VIP, PACAP, or vehicle
control solution, followed by the administration of LPS (from S.
enteridis; 25 µg/mouse). The three experiments depicted in Fig. 7A show that both
neuropeptides (a single dose of 5 nmol/mouse) decreased the
concentration of TNF- induced by LPS in serum and extracellular
fluid (peritoneal lavage). Next, we used the same experimental protocol
to analyze TNF- mRNA in freshly peritoneal cells by Northern blot
analysis. As shown in Fig. 7B, VIP and PACAP significantly
reduced the expression of TNF- mRNA after 1.5 h of endotoxin
treatment.
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Finally, we investigated the effects of LPS and some
proinflammatory cytokines involved in endotoxic shock, such as IL-1ß,
IL-6, and TNF-, on VIP production by peritoneal cells. As shown in
Fig. 8A, unstimulated
peritoneal cells cultured in vitro release VIP, with maximum release at
24–36 h. Treatment of cultures with LPS, IL-1ß, IL-6, or TNF-
resulted in a significant augmentation of the in vitro VIP production
compared with that in the unstimulated controls. This stimulatory
effect occurred over a period of 12–72 h of exposure to these
endotoxemic mediators, with maximum levels at 24 h (Fig. 8A). At all times, LPS was the strongest activator of VIP
production. The potencies of IL-1ß, IL-6, and TNF- were similar
(Fig. 8A). Anti-mTNF- or anti-mIL-6 Abs partially
reversed the stimulatory effect of LPS on VIP production (Fig. 8B), and cotreatment with both anti-mTNF- and
anti-mIL-6 was clearly additive relative to treatment with either
of these Abs, although the blockage was not total (Fig. 8B).
Finally, the LPS-induced VIP production was corroborated in vivo,
because LPS (25 µg/mouse) increased in a time-dependent manner VIP
levels in the peritoneal suspension and serum, showing a maximum effect
at 12 h (Fig. 8C). Interestingly, basal VIP levels were
significantly higher in thioglycolate-elicited mice than in
unstimulated mice (Fig. 8C). These results indicate that VIP
production in vitro and in vivo is induced by LPS at least partially
through the release of the proinflammatory cytokines TNF-, IL-1ß,
and IL-6.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a proinflammatory
cytokine, by endotoxin-activated macrophages. Our findings show that
VIP and PACAP inhibit in a similar way and in a time- and
dose-dependent manner the LPS-stimulated production of TNF- by
murine macrophages.
The time course indicates that, similar to IL-6 and IL-10 7, 14 , the
effect of VIP and PACAP on TNF- production occurs rapidly, as 5- to
15-min incubations with either neuropeptide are sufficient to achieve a
maximum effect. In addition, the inhibition has a refractory period of
2–3 h, after which the inhibitory activity of VIP or PACAP is reduced
significantly, suggesting that VIP/PACAP block an early event in
TNF- generation in macrophages. The failure of VIP and PACAP to
inhibit TNF- production in response to delayed LPS stimulation could
be explained by down-regulation of VIP receptors, which is known to
occur in the presence of VIP 27 .
Similar to the effect on other cytokines, such as IL-2, IL-6, and IL-10
7, 13, 14 , the inhibition of TNF- requires intact VIP/PACAP
molecules. This is in agreement with previous reports showing that
either C- or N-terminal truncations of VIP lead to significant losses
in biological activity 28, 29 . The nature of the macrophage VIP/PACAP
receptors that mediate the inhibition of TNF- production is not
clear. Peritoneal macrophages were previously shown to express PAC1 and
VPAC1 mRNA, and both high affinity and low affinity VIP/PACAP binding
sites were identified on peritoneal macrophages 4, 5, 30 . The
Kd of the high affinity binding sites for VIP
and PACAP38 (0.6–1.44 nM) corresponds to the neuropeptide
concentrations (10-9–10-8 M) that showed the
highest inhibitory effect in our study. To confirm the specificity of
the observed effects and to provide additional evidence for the
receptor-mediated nature of the effects of VIP and PACAP, we
investigated the ability of the PACAP6–38 fragment, an
antagonist with specificity for both the PAC1 and VPAC2 15 , and of
two VPAC1 antagonists 16, 17 to reverse the inhibitory effect of VIP
and PACAP on TNF- production. The lack of effect of
PACAP6–38 eliminates PAC1 and VPAC1 as possible mediators.
In contrast, the two VPAC1 antagonists are fully active in reversing
the inhibitory effect of both VIP and PACAP, suggesting that this
receptor is a viable candidate. This was confirmed by the fact that a
VPAC1 agonist 18 showed a similar effect as VIP/PACAP by inhibiting
TNF- production.
The VPAC1 is coupled primarily to the adenylate cyclase system in
several immune cells, including peritoneal macrophages 30, 31, 32 . To
fully understand the mechanism of action of VIP and PACAP, it is
important to clarify which transduction pathways are involved in the
inhibition of TNF- production by macrophages. It has been shown that
TNF- production is inhibited by agents that increase intracellular
cAMP levels and is stimulated by activation of the PKC pathway 1, 33, 34, 35 . In the present study, using selective activators and
inhibitors of PKA and PKC, we demonstrated that VIP and PACAP inhibit
TNF- production by macrophages through PKA activation and elevation
of cAMP levels following binding to VIP1R.
In a recent study we reported that in contrast to the broad inhibitory
effects of VIP and PACAP on LPS-induced IL-6 release, both
neuropeptides were ineffective in reducing production of IL-6 by
macrophages stimulated with either IL-1ß or TNF-; on the contrary,
we found that VIP and PACAP increased IL-6 levels 7 . However, in the
present study we have shown that both neuropeptides inhibited in a
dose-dependent manner IL-1ß-induced TNF- production. Similar
results have been previously described in human monocytes for another
anti-inflammatory substance, the endogenous nucleoside adenosine
36 . This discrepancy between the inhibitory effects of VIP/PACAP and
adenosine on IL-1ß-induced TNF- release on one side and the
potentiating effect on the release of IL-6 on the other side, further
argues against a common inhibitory mechanism for both cytokines.
Our previous experiments regarding VIP modulation of cytokine
expression indicated that the inhibitory effects of VIP on
IL-2/IL-6/IL-10 and IL-4 production result from different molecular
mechanisms, i.e., transcriptional vs post-transcriptional regulation,
respectively 6, 7 . The present study indicates that, similar to IL-2,
IL-6, and IL-10 and in contrast to IL-4, the inhibitory effect of VIP
and PACAP on TNF- production occurs through a transcriptional
mechanism. In this sense we have recently reported that VIP and PACAP
inhibit TNF- transcriptional activation in LPS-stimulated Raw 264.7
cells by affecting both NF-
B nuclear translocation and the
composition of the cAMP response element binding complex (CREB/c-Jun)
37 .
The in vitro inhibitory effect of VIP/PACAP on LPS-induced TNF-
production correlates with the in vivo inhibition of both TNF-
expression and release in endotoxemic mice. VIP and PACAP have been
described as components of the lymphoid microenvironment, including the
peritoneal immune population. Indeed, we have previously reported that
VIP is produced by peritoneal lymphocytes 4 , and Gaytan et al. found
PACAP immunoreactivity in lymphocytes from different lymphoid organs
38 . In the present study we have demonstrated that LPS stimulation
induced in vivo and in vitro VIP production and release by peritoneal
cells (possibly lymphocytes). In addition, several proinflammatory
cytokines, such as IL-1ß, IL-6, and TNF-, stimulated VIP synthesis
in the peritoneal microenvironment. The fact that the LPS-induced VIP
production is partially reversed by the addition of Abs against these
proinflammatory cytokines suggests that the stimulatory action of LPS
on VIP release is mediated at least partially through the LPS-induced
synthesis of IL-1ß, IL-6, and TNF- by peritoneal macrophages. In
this sense, several lines of evidence suggest that the production and
expression of VIP and VIP binding sites are regulated by inflammatory
mediators, such as IL-1, IL-6, TNF-, antigenic stimulation, and
glucocorticoids (39-44). These findings suggest the existence of
a negative feedback, in which mediators produced by inflammatory cells
following endotoxin stimulation stimulate VIP and VIP-R expression, and
the subsequent VIP/VIP-R interactions negatively regulate the local
inflammatory response. Therefore, the in vivo effect of VIP and PACAP
on TNF- production, the expression and production of both
neuropeptides, and their receptors in peritoneal immune cells together
with the concept of a VIP- and PACAP-mediated general immunoregulatory
feedback mechanism suggest that these neuropeptides might be endogenous
modulators of inflammatory cytokine production by activated immune
cells.
There is abundant evidence that TNF- is an important mediator of
shock and organ failure complicating Gram-negative sepsis 1 . Could
the administration of VIP/PACAP early in sepsis, before the development
of hypotension, be useful? In this sense, elevated VIP levels were
reported in patients with Gram-negative septic shock and in some
endotoxic animal models 45, 46, 47, 48, 49 . We have recently demonstrated that
VIP and PACAP protect mice from lethal endotoxemia through the
inhibition of TNF- and IL-6 production 50 . In addition, an
anti-shock effect of PACAP on experimental endotoxin shock in dogs
has been previously reported 51 . We propose that during a normal
immune response, the timely production and/or release of VIP and
possibly PACAP within the lymphoid organs following antigenic
stimulation serve to down-regulate the ongoing immune response, mostly
through the modulation of cytokine production. During septic shock,
however, due to severe septicemia leading to an overstimulation of the
immune system, the effect of checkpoint molecules such as IL-10, IL-13,
VIP, and PACAP is overwhelmed by the proinflammatory network. Based on
the present study demonstrating that VIP and PACAP inhibit TNF-
production and on the protective effect of these anti-inflammatory
neuropeptides in lethal endotoxic shock, the exogenous administration
of VIP or PACAP could offer an alternative to existing treatments for
septic shock syndrome.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mario Delgado, Department of Biological Sciences, Rutgers University, 101 Warren St., Newark, NJ 07102. E-mail address:
3 Abbreviations used in this paper: VIP, vasoactive intestinal peptide; PACAP, pituitary adenylate cyclase-activating polypeptide; GRF, growth hormone-releasing factor; mr, murine recombinant; PKC, protein kinase C; PKA, protein kinase A; ABTS, 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid.
Received for publication July 6, 1998. Accepted for publication November 9, 1998.
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from macrophages: enhancement and suppression are dose-dependently regulated by prostaglandin E2 and cyclic nucleotides. J. Immunol. 141:2388.[Abstract]
transcriptional activation by regulating NF-B and CREB/c-Jun. J. Biol. Chem. 273:31427. and tumor necrosis factor- differentially regulate enkephalin, vasoactive intestinal peptide, neurotensin, and substance P biosynthesis in chromaffin cells. Endocrinology 130:2252. and IL-6.
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