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* Department of Pharmacology, College of Medicine, National Taiwan University; and
Department of Microbiology and Immunology, National Yang-Ming University School of Medicine, Taipei, Taiwan
Correspondence: W. W. Lin, Ph.D., Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan. E-mail: wwl{at}ha.mc.ntu.edu.tw
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-), and
interleukin-6 (IL-6) release in mouse peritoneal macrophages. To
elevate intracellular Ca2+, we used thapsigargin (TG) and
UTP. Although LPS alone cannot stimulate NO synthesis, co-addition with
TG, which sustainably increased [Ca2+]i,
resulted in NO release. UTP, via acting on P2Y6 receptors,
can stimulate phosphoinositide (PI) turnover and transient
[Ca2+]i increase, however, it did not possess
the NO priming effect. LPS alone triggered the release of
PGE2, TNF-, and IL-6; all of which were potentiated by
the presence of TG, but not of UTP. The stimulatory effect of LPS plus
TG on NO release was inhibited by the presence of Ro 31-8220, Go6976,
KN-93, PD 098059, or SB 203580, and abolished by BAPTA/AM and nuclear
factor 
B (NF-B) inhibitor, PDTC. PGE2, TNF-, and
IL-6 release by LPS alone were attenuated by Ro 31-8220, Go6976, PD
098059, SB 203580, and PDTC. Using L-NAME, soluble TNF-
receptor, IL-6 antibody, NS-398, and indomethacin, we performed
experiments to understand the cross-regulation by the four mediators.
The results revealed that TNF- up-regulated NO, PGE2,
and IL-6 synthesis; PGE2 up-regulated NO, but
down-regulated TNF- synthesis; and PGE2 and IL-6
mutually up-regulated reciprocally. Taken together, murine peritoneal
macrophages required a sustained [Ca2+]i
increase, which proceeds after TG, but not UTP, stimulation, to enhance
LPS-mediated release of inflammatory mediators, particularly for NO
induction. Activation of PKC-, ERK-, and p38 MAPK-dependent signaling
also are essential for LPS action. The positive regulatory interactions
among these mediators might amplify the inflammatory response caused by
endotoxin.
Key Words: LPS • UTP • Ca2+ • protein kinase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-),
and interleukin-6 (IL-6) [1
2
3
4
5
]. The knowledge of the
mechanisms involved in the control of inflammatory mediator synthesis
from the major functional cells, macrophages, is a subject of current
interest because of the multiple physiological and pathological effects
elicited by these mediators.
Activated macrophages produce high levels of NO after induction of
inducible nitric oxide synthase (iNOS), which can catalyze the
formation of NO from the terminal guanidino nitrogen atom of
L-arginine [6
]. NO has been shown to be an
effector in the antimicrobial and tumoricidal activities of murine
macrophages [7
, 8
]. TNF- is among the
earliest activated cytokines in inflammation, and its production by
activated macrophages is crucial for the development of an early
defense against many pathogens [9
] and also appears to
be involved in the pathogenesis of endotoxin shock as well as
autoimmune inflammation [10
, 11
].
PGE2, a potent inflammatory mediator, is an important
negative regulator of TNF- production [12
]. IL-6 is
an important cytokine required for acute-phase protein synthesis,
antibody secretion, B cell growth, T cell activation, and maintenance
of optimal immune function [13
, 14
].
Previous studies have indicated that the induction of iNOS by mouse
peritoneal macrophages is controlled by a two-signal process
[15
, 16
]. First, cells must be primed,
either by in vivo treatment with immunostimulant such as
trehalose dimycolate or in vitro treatment with
interferon-
(IFN-). Both treatments result in a number of
biochemical and functional alterations of the macrophages, thus
rendering them sensitive to triggering agents. Second, exposure of
primed macrophages to a second signal such as LPS results in the
activated macrophages capable of maximal NO production and cytotoxicity
[17
, 18
]. Several reports have shown that
Ca2+ elevating agents, such as Ca2+ ionophore
A23187 and thapsigargin (TG) can replace IFN- as a priming signal
[19
20
21
22
]. However, it is unclear whether natural
physiological processes are mimicked by TG.
Nucleotides contained in large amounts in cell cytosol can reach the
extracellular compartment from cells at inflammatory sites undergoing
cell damage and lysis. Released nucleotides can elicit multiple
physiological actions via acting at the specific P2 receptors. Thus far
five mammalian P2Y receptors, i.e., P2Y1, P2Y2,
P2Y4, P2Y6, and P2Y11, have been
cloned and characterized as G protein-coupled receptors capable of
activating phosphoinositide-specific phospholipase C (PI-PLC) [see
ref. 23
for review]. Our previous study showed that, in murine J774
macrophages, binding of UTP primarily to P2Y6 receptors
stimulates PI breakdown, followed by the increase in intracellular
calcium level ([Ca2+]i), activation of
calmodulin-dependent protein kinase (CaMK), and thus resulting in the
potentiation of LPS-induced iNOS expression [4
]. These
results indicate the possible physiological role of nucleotides in
amplifying the inflammatory response. In this report, we would like to
further address this possible action of nucleotides in the primary
macrophage system with respect to their effects on NO,
PGE2, IL-6, and TNF- formation, and hope to understand
the significant physiological relevance. For this purpose, we performed
experiments in mouse peritoneal macrophages and examined the effects of
nucleotides as well as TG. We also evaluated the roles of signal
molecules, such as calcium, CaMK, protein kinase C (PKC), extracellular
signal-regulated protein kinase (ERK), and p38 mitogen-activated
protein kinase (MAPK).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,ß-methylene-ATP (,ß-MeATP) were from RBI (Natick, MA).
1-[3-(amidinothio)propyl-1H-indoyl-3-yl]-3-(1-methyl-1H-indoyl-3-yl)-maleimide-methane
sulfate (Ro 31-8220),
12-(-2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-indolo-(2,3-a)pyrro-lo(3,4-c)carbazole
(Go6976), KN-93, SB 203580, and NS-398 were purchased from Calbiochem
(La Jolla, CA). IL-6, TNF-, and IL-6 enzyme-linked immunosorbent
assay (ELISA) kits, IL-6 antibody, and IgG antibody were obtained from
R & D Systems (Minneapolis, MN). The ELISA kit for PGE2 was
obtained from Cayman (Ann Arbor, MI).
myo[3H]inositol (20 Ci/mmol) was purchased
from New England Nuclear (Boston, MA). RNAzol was obtained from Biotecx
Laboratories (Houston, TX). All other chemicals were obtained from
Sigma (St. Louis, MO).
Cell culture
The peritoneal macrophages were prepared as previously described
[24
]. BALB/c mice that have been intraperitoneally
injected with 1.5 mL 3% thioglycollate at 3 days before macrophage
isolation were ether-anesthetized, and the peritoneal cavities were
lavaged with ice-cold 0.9% NaCl to remove the elicited peritoneal
macrophages and cultured at 37°C in RPMI 1640, supplemented with 10%
fetal bovine serum and antibiotics (100 U/mL penicillin and 100 µg/mL
streptomycin). Cells were cultured in 24-well plates (for nitrite,
IL-6, PGE2, and TNF- assays) or in 35-mm dishes [for PI
turnover, [Ca2+]i, and reverse
transcription-polymerase chain reaction (RT-PCR) assays] in a
humidified atmosphere of 95% air/5% CO2.
Nitrite, PGE2, TNF-, and IL-6 measurement
Inflammatory mediators accumulated in the cell culture medium
were measured at the indicated time period after LPS with or without TG
or UTP addition. To assess the effects of several inhibitors, drugs
were added to the cells 20 min before LPS/TG addition. Nitrite, an
indicator of NO synthesis, was measured by adding 100 µL of samples
of culture medium to 100 µL of Griess reagent (1% sulfanilamide and
0.1% naphthylethylenediamide in 5% phosphoric acid), then the optical
density at 550 nm was measured using a microplate reader, and the
nitrite concentration calculated by comparison with the absorbance
produced using standard solutions of sodium nitrite in culture medium.
TNF-, PGE2, and IL-6 production were measured in samples
of cell culture supernatant through the use of ELISA kits. Assays were
performed according to the manufacturer’s instructions.
RNA extraction and RT-PCR analysis of P2Y receptor subtypes
RNA was isolated using RNAzol reagent, and RT-PCR carried out
with 10 µg of total RNA as a template, using an RNA PCR kit (GIBCO),
according to the manufacturer’s instructions. The specific primers
were synthesized as follows: P2Y1 (murine) receptor sense
(661–680), 5’-ACG ACT GTG GCC ATG TTC TG-3’ and antisense
(1051–1070), 5’-ATT TCT TCA CTC TTG GAT TG-3’; P2Y2
(human and murine) receptor sense (31–50), 5’-ACC ATC AAT GGC ACC TGG
GA-3’ and antisense (374–393), 5’-CCG GTG CAC GCT GAT GCA GG-3’;
P2Y4 (human) receptor sense, 5’-CAC CGA TAC CTG GGT ATC
TG-3’ and antisense, 5’-CAG ACA GCA AAG ACA GTC AG-3’;
P2Y6 (human and rat) receptor sense (315–334), 5’-GCT TCC
TCT TCT ATG CCA AC-3’ and antisense (779–798), 5’-GTA GGC TGT CTT
GGT GAT GT-3’; P2Y11 (human) receptor sense (94–113),
5’-CTG GTG GTT GAG TTC CTG GT-3’ and antisense (308–327), 5’-GTT
GCA GGT GAA GAG GAA GC-3’. ß-actin mRNA levels were used as an
internal control. The ß-actin primers used were as follows: sense
(613–632), 5’-GAC TAC CTC ATG AAG ATC CT-3’ and antisense
(1103–1122), 5’-CCA CAT CTG CTG GAA GGT GG-3’. Equal amounts of each
RT product (1 µg) were PCR-amplified using Taq polymerase in 30
cycles consisting of 40 s at 95°C, 40 s at 48°C (for
P2Y1 receptor), 54°C (for P2Y2 receptor),
55°C (for P2Y4, P2Y6 receptors and
ß-actin), or 57°C (for P2Y11 receptor) and 2 min at
72°C. The amplified cDNA was run on 1% agarose gels and visualized
by ethidium bromide.
Measurement of PI turnover
PI hydrolysis was measured by the accumulation of inositol
phosphates (IP) in the presence of 10 mM LiCl, as described previously
[25
]. Confluent cells on 35-mm Petri dishes were labeled
with myo[3H]inositol (2.5 µCi/dish) in the
growth medium for 24 h, washed with physiological saline solution
(118 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM
MgCl2, 1.2 mM KH2PO4, 11 mM
glucose, and 20 mM HEPES, pH 7.4) containing 10 mM LiCl and incubated
at 37°C for 20 min. After this preincubation, the nucleotides were
added and incubation continued for another 30 min. The reaction was
terminated by aspiration of the reaction solution and addition of
ice-cold methanol. The cells were scraped off and the
[3H]IP was isolated with an AG-1X8 column (formate form,
100–200 mesh), eluted with 0.2 N ammonium formate/0.1 N formic acid,
and counted by ß-counter. The agonist-elicited [3H]IP
accumulation was expressed as percentages of the basal
[3H]IP level (percent of control).
Measurement of intracellular calcium
[Ca2+]i
Cells grown on glass slides were loaded with 3 µM Fura-2/AM
and pluronic F-127 (0.02% v/v) in RPMI 1640 at 37°C for 45 min. The
fluorescence was monitored on a PTI M-series spectrofluorometer with
dual-excitation wavelengths of 340 and 380 nm and an emission
wavelength of 510 nm. The intracellular calcium was calculated from the
ratio of the fluorescence at the two excitation wavelengths, by use of
a Kd value of 224 nM for the Fura-2/AM
Ca2+ equilibrium, as described by Grynkiewicz et al.
[26
].
Statistical analysis
In each experiment all samples were set up in duplicate, and the
data shown represent the mean ± SE from several
independent experiments. P < 0.05 was considered
significant by evaluation of the data with analysis of variance (ANOVA)
and/or Dunnett’s test. The error bar was omitted when it was within
the symbol representing the mean value.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,ß-MeATP (a P2X receptor
agonist) and adenosine (a selective P1 receptor agonist) did not cause
IP production at 100 µM (data not shown).
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, and IL-6 production (Fig. 2
). Co-incubation with UTP (100 µM) for 24 h did not change
the LPS responses (Fig. 2)
, which is distinct from the P2Y6
receptor-mediated potentiation in J774 cells. In addition to UTP, we
also examined several nucleotide analogs on these events. As shown in
Table 1
, UDP, ATP, 2MeSATP, ,ß-MeATP and adenosine, each at 100 µM,
did not significantly affect LPS-induced mediator release.
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|
,
PGE2, and IL-6 release and primed the NO response upon
simultaneous addition with LPS. The time-dependent potentiation or
priming effects of TG were shown in Figure 3
. In the presence of 30 nM TG, the LPS-induced NO, TNF-,
PGE2, and IL-6 release within 24 h were increased by
4.5-fold (from 5.1 ± 0.4 µM to 23 ± 3.4 µM), 3.7-fold
(from 2014 ± 168 pg/mL to 7452 ± 1370 pg/mL), 5.7-fold
(from 5495 ± 485 pg/mL to 31,322 ± 5156 pg/mL), and
3.9-fold (from 2811 ± 231 pg/mL to 11,103 ± 2108 pg/mL),
respectively.
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, PGE2, and IL-6 release by 48 ± 18, 23 ± 13, and 37 ± 13% (n = 5), respectively
(Table 1) .
Sustained [Ca2+]i increase responsible
for TG potentiation
To explore the ineffectiveness of UTP vs. TG, we determined their
effects on calcium mobilization, which has been reported to be
essential for the priming signal [19
20
21
22
]. Using
Fura-2/AM fluorescence as an index of
[Ca2+]i, as shown in Figure 4
, 30 nM TG caused a slow but sustained increase in
[Ca2+]i for at least 7 min. By contrast, 100
µM UTP elicited a transient increase in the
[Ca2+]i response, reaching its maximum at 1
min, from 122 ± 32 nM to 1008 ± 147 nM (n =
11), and then rapidly decreased. The levels of
[Ca2+]i increase 5 min after the addition of
UTP and TG were 93 ± 15 nM and 305 ± 25 nM, respectively.
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,
PGE2, and IL-6 release by 72 ± 3, 42 ± 10, and
34 ± 4% (n = 3), respectively (data not shown).
The inhibitory effects of BAPTA/AM on TG-elicited potentiation effects
were shown in Figure 5
. Complete abolishment of TG response by BAPTA/AM was observed upon
simultaneous addition of both, while the antagonization ability of
BAPTA/AM time-dependently diminished as it was added at 30 min to
12 h after LPS/TG stimulation. In this context, the priming
effects of TG on NO, TNF-, PGE2, and IL-6 release began
to restore, respectively, at 8-, 8-, 2-, and 12-h delay of BAPTA/AM
addition. These results indicate the requirement of a more sustained
[Ca2+]i to coordinate with LPS for efficient
transcription of iNOS, TNF-, COX-2, and IL-6 genes.
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Role of PKC, CaMK, ERK, p38 MAPK, and NF-B on mediator
release
To explore the underlying mechanism involved in the TG priming
effect, the roles of PKC and NF-B were investigated using
pharmacological approaches. As shown in Figure 6
, the two PKC inhibitors, Ro 31-8220 (1 µM, a selective PKC
inhibitor) [27
] and Go6976 (100 nM, a selective
inhibitor of conventional PKC, -ß, and -) [28
]
not only attenuated LPS-induced TNF-, PGE2, and IL-6
release, but also reduced the priming effect of TG on nitrite and
PGE2 formation. In the presence of PKC inhibitors, the
TNF- and IL-6 potentiation by TG were unaffected.
|
B activation is required for iNOS, COX-2, IL-6, and
TNF- gene expression [3
, 5
,
29
, 30
], the involvement of this
transcription factor in TG effect was studied. As shown in Figure 6
, 30
µM pyrrolidine dithiocarbamate (PDTC), a NF-B inhibitor, abolished
these four inflammatory mediator formation induced by LPS, either alone
or in combination with TG.
In murine J774 macrophages, we have reported that the
Ca2+/CaMK-dependent pathway is responsible for the
potentiation effect of UTP and TG on LPS-induced iNOS gene expression
[4
]. Here, using KN-93, a selective CaMK inhibitor
[31
], we also found a similar mechanism for TG effect in
peritoneal macrophages. KN-93 at 10 µM markedly decreased the TG
responses on all the four mediator releases, but failed to affect those
of LPS alone (Fig. 6)
. KN-93 inhibited the TG response on TNF-,
PGE2, IL-6, and NO release by 69 ± 8, 63 ± 14,
73 ± 4, and 74 ± 2% (n = 3), respectively.
These results suggest the [Ca2+]i increase
and subsequent CaMK activation mediated TG effect.
Because accumulating reports have indicated the involvement of ERK and
p38 MAPK in iNOS, TNF-, COX-2, and IL-6 gene expression in response
to LPS or cytokines [4
, 32
33
34
35
36
37
38
], we also
like to address this issue in peritoneal macrophages. As shown in
Figure 7
, PD 098059 (30 µM, a selective MEK inhibitor)
[39
] and SB 203580 (3 µM, a selective p38 MAPK
inhibitor) [40
] dramatically attenuated the responses of
LPS/TG, particularly in terms of TNF- and PGE2 release.
|
receptor
(sTNFR), IL-6 specific antibody, NS-398 (a COX-2 inhibitor), and
indomethacin (a nonselective COX inhibitor) were examined. As shown in
Figure 8
, 300 µM L-NAME only reduced the NO response but did
not affect the others. Upon released TNF- being neutralized by sTNFR
(5 µg/mL), the release of NO, PGE2, and IL-6 in response
to LPS either with or without TG were attenuated. IL-6 antibody at a
concentration completely neutralizing IL-6, 0.1 µg/mL, can reduce
PGE2 release by 47 ± 12% (n = 3) in
response to LPS and by 41 ± 9% (n = 3) in the
presence of TG. IL-6 antibody did not affect LPS/TG-induced NO and
TNF- release, however, both mediator releases were not affected by
IgG control antibody.
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release in response to LPS with or without TG.
To further explore whether TG potentiation is due to the involvement of
contaminated lymphocyte-generated IFN-, which is also reported as a
primer for LPS response [15
], we checked the effects of
anti-IFN- antibody. We found that IFN- antibody at 10 µg/mL did
not affect TG-induced NO potentiation (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Murine macrophage-derived NO, PGE2, TNF-, and IL-6
release have been shown to be dramatically induced by LPS, and the
regulation of these inflammatory mediators in macrophages is
controlled, at least in part, at the level of gene transcription. A
crucial transcription factor that regulates expression of these genes
is NF-B, which binds to the specific promoter sequences, B
binding sites, and induce gene transcription [41
]. With
regard to these well-documented notions, we have gotten support from
our present studies in mouse peritoneal macrophages. With respect to
signaling, particularly of the involvement of several protein kinases,
our results here also confirmed our previous notion that PKC, ERK, and
p38 MAPK are pivotal signaling molecules to transduce LPS action in
macrophages. Despite these confirmed results, we have observed an
additional regulatory pathway mediated by the increase in
[Ca2+]i in integration with others and thus
potentiating mediator release. Moreover, the reciprocal
cross-amplification or inhibition between mediators also implies the
physiological significance in the progress of inflammation.
The regulatory machinery required for iNOS induction seems to be not
exactly the same in different macrophage cell types and quite depends
on the nature of the cells [42
]. At least as compared
with J774 macrophages and peritoneal macrophages, the requirement of
the priming signals is different. In the former, LPS treatment itself
is sufficient to induce abundant NO production, whereas in the latter,
LPS alone has little effect on NO production unless the cells were
primed with either IFN- or TG. As previously indicated, the capacity
to express iNOS gene in mouse peritoneal macrophages is induced by a
multi-step cascade of events. NO synthesis is induced by the sequential
exposure of responsive peritoneal macrophages to priming and trigger
signals such as IFN- and LPS, respectively, for full activation
[15
, 18
, 22
,
42
43
44
]. Furthermore, calcium ionophores and TG are also
playing triggering signals for the induction of NO synthesis in
peritoneal macrophages [45
]. Thus, calcium-elevating
agents such as calcium ionophore and TG can replace either priming or
triggering signals, but themselves cannot contribute to the induction
of NO formation. Although elevation of
[Ca2+]i is one of the mechanisms contributing
to the induction of NO release, the relationship between priming
effects and [Ca2+]i increase is complex, and
the mechanism is still unknown.
Recent study has shown that in peritoneal macrophages,
platelet-activating factor even inducing rapid and transient increases
of [Ca2+]i did not elicit the priming effect
with respect to LPS action [45
]. Confirming these
results, in this report, we observed the priming effects of TG, but not
of UTP, on iNOS induction. Both TG and UTP can elicit
[Ca2+]i increase in peritoneal macrophages to
different extents. UTP via acting on P2Y6 receptors induced
a much less sustained increase in [Ca2+]i
than TG, whose response was maintained for at least 7 min as long as we
examined. In this study we also provided evidence to support the
requirement of intracellular Ca2+ in iNOS priming in
peritoneal macrophages. Reduction of [Ca2+]i
by BAPTA/AM at different periods after LPS initiating its triggering
cascade further indicated that the Ca2+-dependent signaling
event lasting at least for 8 h is required for iNOS priming. On
the other hand, the potentiating effect in J774 macrophages only
depends on a short period of [Ca2+]i
elevation, because short-term presence of UTP for 30 min with LPS is
sufficient to achieve the potentiation [4
]. Thus, these
results together suggest that a much more sustained
Ca2+-dependent signaling pathway is necessary to integrate
with LPS signaling for iNOS expression in peritoneal macrophages
compared with J774 cells. Except NO production, the release of the
other three mediators, PGE2, TNF-, and IL-6, also were
potentiated by TG and dependent on [Ca2+]i
level, respectively, for 2, 8, and 12 h. Certainly, except
intracellular Ca2+, other regulators with different
significance involving the control of gene expression in various cell
types need to be further explored.
Despite intensive research, the nature of the messengers that transduce
TG priming signal in macrophages is still unclear. Our previous study
has shown that in J774 macrophages, UTP and TG can potentiate the
induced activation of NF-B via a CaMK-dependent pathway
[4
]. In agreement with this notion, the priming effects
of TG on several mediator releases in peritoneal macrophages were also
shown to be inhibited by KN-93, indicating the CaMK activation as being
a key step. The interesting aspect of Ca2+ signal,
particularly of the frequency, duration, amplitude, and spatial
distribution aspect, in its capacity to encode cellular responses has
been previously demonstrated in several cell types. It means that these
variables of Ca2+ signal were proposed as a means by which
Ca2+ could achieve specificity in signaling. In this
respect, lymphocytes, for example, require a prolonged Ca2+
stimulus for activation of immune response genes but respond to brief
Ca2+ stimuli in other ways [46
]. A large
transient Ca2+ rise in B lymphocyte activates JNK, whereas
NF-ATc translocation to the nucleus is induced by a low sustained
Ca2+ plateau [47
]. In activated T
lymphocytes, sustained high intracellular Ca2+ is required
for both full activation of calcineurin and JNK, which respectively
maintains NF-ATc in the nucleus and activates AP-1
[48
49
50
]. Accordingly, high levels of Ca2+
are required to activate NF-B in both B cells [51
]
and microglial cells [52
].
Previous studies have shown that the priming effect of elevating
[Ca2+]i on iNOS induction did not involve PKC
activation in peritoneal macrophages [53
] and J774
macrophages [4
]. In this study we also found that the
two PKC inhibitors Ro 31-8220 and Go6976, although they inhibited the
responses of LPS on TNF-, IL-6, and PGE2 release, failed
to affect the priming responses on TNF- and IL-6. On the contrary,
the priming effect on PGE2 release was attenuated by PKC
inhibition. In addition to PKC, ERK and p38 MAPK are two important
kinase molecules essential for LPS signaling on mediator release, as we
can observe that all the four mediator releases caused by LPS alone or
in the presence of TG were dramatically reduced by MEK inhibitor, PD
098059 and p38 MAPK inhibitor, SB 203580.
In this study we also showed the cross-interaction on the mediator
synthesis between endogenous NO, PGE2, TNF-, and IL-6.
In accordance with a previous report observed in either macrophages or
other cell types, PGE2 represents a positive feedback
mediator in NO [54
, 55
] and IL-6 synthesis
[56
], but as a negative regulator on TNF- release
[12
]. TNF- itself also plays a positive modulation on
NO, PGE2, and IL-6 synthesis [37
,
57
, 58
], whereas IL-6 can enhance
PGE2 synthesis only.
Taken together, these results suggest that not only PKC, ERK, and p38
MAPK are all required for LPS action in peritoneal macrophages, but
also the sustained increase in intracellular calcium elicits a priming
event. The positive regulatory interactions among NO, PGE2,
TNF-, and IL-6 imply the amplified inflammatory response caused by
endotoxin.
|
ACKNOWLEDGEMENTS |
|---|
Received April 12, 2000; revised August 9, 2000; accepted September 25, 2000.
|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and TNF- in mouse macrophages J. Immunol. 162,415-522This article has been cited by other articles:
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D. Poeckel, L. Tausch, S. George, J. Jauch, and O. Werz 3-O-Acetyl-11-keto-boswellic Acid Decreases Basal Intracellular Ca2+ Levels and Inhibits Agonist-Induced Ca2+ Mobilization and Mitogen-Activated Protein Kinase Activation in Human Monocytic Cells J. Pharmacol. Exp. Ther., January 1, 2006; 316(1): 224 - 232. [Abstract] [Full Text] [PDF]
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 M. Yadav, S. K. Roach, and J. S. Schorey Increased Mitogen-Activated Protein Kinase Activity and TNF-{alpha} Production Associated with Mycobacterium smegmatis- but Not Mycobacterium avium-Infected Macrophages Requires Prolonged Stimulation of the Calmodulin/Calmodulin Kinase and Cyclic AMP/Protein Kinase A Pathways J. Immunol., May 1, 2004; 172(9): 5588 - 5597. [Abstract] [Full Text] [PDF]
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 H.-M. Wu, H.-C. Wen, and W.-W. Lin Proteasome Inhibitors Stimulate Interleukin-8 Expression via Ras and Apoptosis Signal-Regulating Kinase-dependent Extracellular Signal-Related Kinase and c-Jun N-Terminal Kinase Activation Am. J. Respir. Cell Mol. Biol., August 1, 2002; 27(2): 234 - 243. [Abstract] [Full Text] [PDF]
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 C.-J. Tsi, Y. Chao, C.-W. Chen, and W. W. Lin Aurintricarboxylic Acid Protects against Cell Death Caused by Lipopolysaccharide in Macrophages by Decreasing Inducible Nitric-Oxide Synthase Induction via Ikappa B Kinase, Extracellular Signal-Regulated Kinase, and p38 Mitogen-Activated Protein Kinase Inhibition Mol. Pharmacol., July 1, 2002; 62(1): 90 - 101. [Abstract] [Full Text] [PDF]
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 B.-C. Chen, W.-T. Wu, F.-M. Ho, and W.-W. Lin Inhibition of Interleukin-1beta -induced NF-kappa B Activation by Calcium/Calmodulin-dependent Protein Kinase Kinase Occurs through Akt Activation Associated with Interleukin-1 Receptor-associated Kinase Phosphorylation and Uncoupling of MyD88 J. Biol. Chem., June 28, 2002; 277(27): 24169 - 24179. [Abstract] [Full Text] [PDF]
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Y.-W. Hsu, K.-H. Chi, W.-C. Huang, and W.-W. Lin Ceramide Inhibits Lipopolysaccharide-Mediated Nitric Oxide Synthase and Cyclooxygenase-2 Induction in Macrophages: Effects on Protein Kinases and Transcription Factors J. Immunol., May 1, 2001; 166(9): 5388 - 5397. [Abstract] [Full Text] [PDF]
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