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by LPS and IFN- attenuates the oxidative burst in macrophages
Department of Medicine IV-Experimental Division, University of Erlangen-Nürnberg, Faculty of Medicine, 91054 Erlangen, Germany
1Correspondence: University of Erlangen-Nürnberg, Faculty of Medicine, Loschgestrasse 8, 91054 Erlangen, Germany. E-mail: mfm423{at}rzmail.uni-erlangen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) in vitro. Macrophage activation
was achieved with 12-O-tetradecanoylphorbol 13-acetate (TPA), and the
oxidative burst (i.e., oxygen radical formation) was followed by
oxidation of the redox-sensitive dyes hydroethidine and
dichlorodihydrofluorescein diacetate. Prestimulation of macrophages for
15 h with a combination of LPS/IFN- attenuated oxygen radical
formation in response to TPA. Taking the anti-inflammatory properties
of the peroxisome proliferator-activating receptor (PPAR) into
consideration, we established activation of PPAR in response to
LPS/IFN- by an electrophoretic mobility shift, supershift, and a
reporter gene assay. The reporter contains a triple PPAR-responsive
element (PPRE) in front of a thymidine kinase minimal promoter driving
the luciferase gene. We demonstrated that PPRE decoy oligonucleotides,
supplied in front of LPS/IFN-, allowed a full oxidative burst to
recover upon TPA addition. Furthermore, we suppressed the oxidative
burst by using the PPAR agonists
15-deoxy-
12,14-prostaglandin J2, BRL 49653,
or ciglitazone. No effect was observed with WY 14643, a PPAR
agonist. We conclude that activation of PPARs, most likely PPAR,
promotes macrophage desensitization, thus attenuating the oxidative
burst. This process appears important during development of
sepsis.—von Knethen, A., Brüne, B. Delayed activation of PPAR
by LPS and IFN- attenuates the oxidative burst in macrophages.
Key Words: anti-inflammatory • sepsis • desensitization • respiratory burst
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-), interleukin-1 (IL-1), and interferon- (IFN-) that act
both locally and systemically (1)
. It is believed that
monocytes/macrophages are an important source of these factors, with
the implication that their excessive formation in response to
gram-negative or gram-positive bacteria may lead to symptoms known as
septic shock (2
, 3)
. Endotoxin, a lipopolysaccharide (LPS)
-containing molecule, interacts with the LPS binding protein and
elicits monocyte activation and differentiation via the CD14 membrane
receptor (4
, 5)
as well as toll-like receptors, such as
TLR2 and TLR4 (6
7
8
9)
. This is followed by the formation
and secretion of inflammatory cytokines and the generation of reactive
oxygen (ROS) (2)
and nitrogen species (10)
.
To balance inflammation, antiinflammatory components are an important
determinant to counteract toxic cytokine and/or reactive oxygen
formation. For example, in vitro, IL-10 attenuates TNF-
and IL-1 secretion, thereby attenuating proinflammatory responses of
monocytes/macrophages (11
, 12)
. During the development of
sepsis, monocytes/macrophages exhibit a hyper-inflammatory state for
hours to days, after which their function is shifted to a
hypo-inflammatory state characterized by anergy to stimulation with LPS
in vitro (13)
. These cells no longer exert
their function during the innate immune response.
Recently, the anti-inflammatory properties of the nuclear hormone
receptor family known as peroxisome proliferator-activated receptors
(PPARs) were established, although originally being implicated with
obesity, diabetes, and atherosclerosis (14)
. Three
subtypes—PPAR, PPARß (also known as PPAR
), and PPAR—have
been described (15)
. They all function as ligand-dependent
transcription factors that, upon heterodimerization with the 9-cis
retinoic acid receptor, bind to peroxisome proliferator-activated
receptor response elements (PPRE), thus affecting target gene
expression. However, detailed mechanisms of action are not yet being
defined.
For PPAR, it has been confirmed that specific activators such as the
naturally occurring
15-deoxy-12,14-prostaglandin
J2 (15d-PGJ2) (16
, 17)
or synthetic antidiabetic thiazolidinediones, such as
ciglitazone or BRL 49653, effectively reduced proinflammatory cytokine
as well as reactive nitrogen species (RNS) (nitric oxide, or NO)
production in monocytes/macrophages (18
19
20)
.
Considering the properties of PPAR, we hypothesized a role of
PPAR in the process of LPS/IFN--evoked monocyte/macrophage
desensitization. As a test system, we studied the oxidative burst in
primary human macrophages and macrophage cell lines in response to
12-O-tetradecanoylphorbol 13-acetate (TPA). Activation of PPAR
provoked by LPS/IFN- or synthetic PPAR-agonists attenuated the
oxidative burst, whereas scavenging of PPAR by a decoy approach
reversed macrophage desensitization. We conclude that activation of
PPAR by LPS/IFN- is an important determinant of the
activation/deactivation balance in macrophages.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
antibody were from Alexis (Grünberg, Germany). The polyclonal
anti-PPAR antibody was from Santa Cruz Biotechnology (Heidelberg,
Germany). Recombinant murine and human IFN- were provided by Roche
Diagnostics (Mannheim, Germany). Hydroethidine (HE) and
dichlorodihydrofluorescein diacetate (DCF) were from Molecular Probes
(Leiden, The Netherlands). Culture supplements and fetal calf serum
were ordered from Biochrom (Berlin, Germany).
15d-PGJ2 and ciglitazone were bought from Biomol
(Hamburg, Germany). The luciferase assay kit was obtained from Promega
(Mannheim, Germany) and the ß-galactosidase detection kit was from
Tropix (Mannheim, Germany). Oligonuceotides were ordered from
Eurogentec (Seraing, Belgium). All other chemicals were of the highest
grade of purity and commercially available.
Cell culture
The mouse monocyte/macrophage cell line RAW 264.7 and the
premonocytic human cell line U937 were maintained in RPMI 1640
supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and
10% heat-inactivated fetal calf serum (complete RPMI). All experiments
were performed using complete RPMI.
Cell survival
After drug treatment, cell viability was determined by trypan
blue dye exclusion.
Monocyte isolation and culture
For each experiment, cells were isolated from 50 ml buffy coats
(Blutbank Erlangen, Germany). Blood was diluted 1:2 with
phosphate-buffered saline (PBS) and layered on a Ficoll-Isopaque
gradient (P=1077 g ml-1). The
interphase containing peripheral blood mononuclear cells was obtained
after centrifugation (800 g, 20 min). Cells were recovered,
washed twice in PBS and left to adhere on culture dishes (Primaria
3072, Becton Dickinson, Heidelberg) for 90 min at 37°C. Nonadherent
cells were removed. The medium was changed to fresh RPMI 1640
containing 10% heat-inactivated human AB serum (Sigma, Deisenhofen,
Germany) and antibiotics (100 U/ml penicillin, 100 µg/ml
streptomycin). Monocytes (5x105) were cultured
in a volume of 10 ml per plate in an incubator. Medium was changed
every 2–3 days. After 6 days of culture, monocytes acquired a
macrophage-like phenotype (21)
and were used for the
experiments. Recovery of the cells after 6 days was 70%–80% compared
to the starting monocyte population. Flow cytometry confirmed that the
macrophage-like population was 90–95% pure
(CD14+ vs. CD14-). Cell
viability as judged by trypan blue dye exclusion at the time of the
experiment was 
80%.
Flow cytometry of oxygen radical production (hydroethidine assay)
Cells were cultured under nonadherent conditions in non-cell
culture Petri dish (Greiner, Frickenhausen, Germany). After a
prestimulation regime, 5 x 105 cells were
incubated for 30 min with 1 µM TPA. Thereafter, 3 µM HE was added
and incubations went on for 30 min. Cells were harvested, washed with
PBS, and resuspended in 200 µl PBS. Flow cytometry analysis was
performed using a Coulter Epics XL flow cytometer (Beckman Coulter,
Krefeld, Germany) and HE was measured through a 630 nm long-pass filter
(FL3). Data from 10,000 cells were collected to reach significance.
Oxygen radical production measured by the
dichlorodihydrofluorescein assay
Cells were cultured under adherent conditions in 24-well plates
at a density of 1 x 105 cells/well. After a
prestimulation regime, medium was changed to 500 µl of a modified
HBSS solution (22)
(124 mM NaCl, 5.8 mM KCl, 10 mM
dextrose, 20 mM HEPES, 0.3 mM CaCl2, pH 7.4)
containing 1 µM TPA. After 30 min, 500 µl HBSS containing 1 µM
TPA and 40 µM DCF (final concentration 20 µM) was added and
incubated for 30 min. Accumulation of oxidized DCF was immediately
followed fluorometrically (Spectrafluor Fluorescence Reader, Tecan,
Crailsheim, Germany) with excitation at 485 nm and emission at 530 nm.
Nuclear protein extraction
Preparation of crude nuclear extract was basically as described
(23)
. Briefly, after cell activation for the times
indicated, 4 x 106 cells were washed in 1
ml of ice-cold PBS, centrifuged at 1000 g for 5 min,
resuspended in 400 µl ice-cold hypotonic buffer (10 mM HEPES/KOH, 2
mM MgCl2, 0.1 mM EDTA, 10 mM KCl, 1 mM DTT, 0.5
mM PMSF, pH 7.9), left on ice for 10 min, vortexed, and centrifuged at
15,000 g for 30 s. Sedimented nuclei were resuspended
in 50 µl ice-cold saline buffer (50 mM HEPES/KOH, 50 mM KCl, 300 mM
NaCl, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, pH 7.9), left
on ice for 20 min, vortexed, and centrifuged at 15,000 g for
5 min at 4°C. Aliquots of the supernatant containing nuclear proteins
were frozen in liquid nitrogen and stored at -70°C. Protein was
determined using a Bio-Rad II Kit.
Electrophoretic mobility shift assays (EMSA)
An established EMSA method, with slight modifications, was used
(24)
. Nuclear protein (5 µg) was incubated for 20 min at
room temperature with 20 µg bovine serum albumin, 2 µg poly(dI-dC
from Pharmacia (Piscataway, N.J.), 2 µl buffer D (20 mM HEPES/KOH,
20% glycerol, 100 mM KCl, 0.5 mM EDTA, 0.25% Nonidet P-40, 2 mM DTT,
0.5 mM PMSF, pH 7.9), 4 µl buffer F (20% Ficoll-400, 100 mM
HEPES/KOH, 300 mM KCl, 10 mM DTT, 0.5 mM PMSF, pH 7.9), and 20,000 cpm
of a [32P]-labeled oligonucleotide in a final
volume of 20 µl. Supershift antibodies (2 µg) were added as
indicated. DNA–protein complexes were resolved at 180 V for 4 h
in a taurine-buffered, native 6% polyacrylamide gel (4% for
supershifts), dried, and visualized (autoradiography using a Fuji X-ray
film). Oligonucleotide probes were labeled by a filling reaction using
the Klenow fragment (Roche Diagnostics, Mannheim, Germany).
Oligonucleotide (1 pmol) was labeled with 50 µCi of
[-32P]-dCTP (3000 Ci/mmol, Amersham,
Braunschweig, Germany), cold nucleotides (dATP, dTTP, dGTP from Life
Technologies, Inc., Eggenstein, Germany), purified on a CHROMA SPIN-10
column (Clontech, Heidelberg, Germany), and stored at -20°C until
use. Oligonucleotides with the consensus PPRE site (boldface letters)
were used (25)
:
5'-GGT AAA GGT CAA AGG TCA AT-3'
3'-A TTT CCA GTT TCC AGT TAG CCG-5'
PPRE reporter gene assay
The plasmid J3 thymidine kinase (TK) pGL3, containing three
copies of the human apoAII gene promoter PPRE with a J site cloned
upstream of the TK promoter in the pGL3 luciferase expression vector,
was kindly provided by B. Staels (U325/INSERM, Institute Pasteur,
Lille, France) (26)
. Results were verified for a PPRE site
of another gene. Therefore, the Aox-TK plasmid was used, which contains
three copies of the acyl CoA oxidase gene promoter PPRE site cloned
upstream of the TK promoter and a luciferase expression vector. This
plasmid was a generous gift from C. K. Glass (University of
California, La Jolla) (27)
.
Macrophages were transiently transfected using the DEAE-dextran method
as described previously (28)
. Cell selection was
unnecessary because the synthesis of a macrophage unrelated protein was
analyzed. Briefly, 1 day before transfection cells were seeded in
suspension at a density of 1 x 106
cells/ml. 1 x 107 cells were harvested,
washed twice with PBS, and incubated for 3 h at 37°C in 1 ml
RPMI 1640 supplemented with 50 mM Tris-HCl (pH 7.3), 400 µg
DEAE-dextran, 20 µg luciferase reporter construct (J3 TK pGL3 or
Aox-TK), and 5 µg CMV-ß-galactosidase plasmid as an internal
control. To discard the DNA/DEAE-dextran mixture, cells were washed
twice with PBS, seeded at a density of 1 x
106 cells/ml, and cultured for 24 h.
Afterward cells were stimulated for 15 h with LPS/IFN-/NAME,
15d-PGJ2, or ciglitazone. Cell extracts were
assayed for luciferase and ß-galactosidase activity. For
calculations, luciferase activity was normalized for ß-galactosidase
using the formula: luciferase activity/ß-gal activity.
Decoy approach
Cells were exposed to an oligonucleotide containing a PPRE
consensus site as specified for the EMSA. Cells were seeded at a
density of 1 x 106 cells/well into 6-well
plates. Oligonucleotides (3 µM) were added 24 h prior to cell
stimulation. Cell stimulation was performed as indicated.
Oligonucleotide sequences were identical to those used for EMSA. For
control reasons, oligonucleotides with a mutated PPRE site were used
(mutated sites in boldface letters):
5'-GGT AAA GAA CAA AGA ACA AT-3'
3'-A TTT CTT GTT TCT TGT TAG CCG-5'
Statistical analysis
Each experiment was performed at least three times and
statistical analysis was performed using the two-tailed Student’s
t test. Otherwise representative data are shown.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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prestimulation
). To desensitize macrophages, we preactivated cells for 15 h with
a combination of LPS and IFN-. Since these agents are known
activators of inducible NO synthase, at least in murine cells
(25)
, we routinely used NAME to suppress NO formation and
thereby exclude any possible interference of RNS during ROS
determination.
|
When LPS/IFN-/NAME was preincubated for 15 h, we noticed an
attenuated oxidative burst in response to TPA. Inhibition was complete
and was reproduced in U937 cells (Fig. 1)
. In both cell lines,
stimulation with LPS/IFN-/NAME only slightly reduced basal ROS
formation. We then verified these initial observations by using the
redox-sensitive dye DCF (Fig. 2
). Oxidation of the fluorescent molecule was determined in human primary
macrophages, U937 cells, and RAW 264.7 macrophages in response to TPA.
|
Consistently, TPA-evoked oxidation of DCF was significantly antagonized
with LPS/IFN-/NAME prestimulation. The oxidative burst was strongest
in RAW 264.7 cells, revealed an intermediate response in U937 cells,
and was less pronounced in human macrophages. Inhibition of ROS
formation varied between 50% and 75% in the individual cell systems.
Searching for a possible mechanism to explain macrophage
desensitization, we considered PPARs since activation of PPAR is
negatively associated with NO synthase or cytokine expression
(19
, 20
, 25)
. During initial experiments, we asked whether
PPAR agonists would attenuate the oxidative burst as established for
LPS/IFN-/NAME.
Desensitization of monocytes/macrophages in response to
PPAR activation
15d-PGJ2, BRL 49653, and
ciglitazone are known PPAR agonists. We exposed RAW 264.7
macrophages to 15d-PGJ2, at a concentration
ranging between 0.1 and 10 µM, and examined HE oxidation in response
to TPA (Fig. 3A
). We noticed that 0.1 µM 15d-PGJ2
reduced the oxidative burst with low efficacy, whereas 1 µM
15d-PGJ2 completely attenuated ROS formation in
response to TPA.
|
Preincubation of macrophages with 10 µM
15d-PGJ2 was as effective as 1 µM in
suppressing the oxidative burst. However, in accordance with published
data, 10 µM 15d-PGJ2 elicited an apoptotic
response in macrophages (data not shown) that was absent at doses below
5 µM 15d-PGJ2. Therefore, a concentration of 1
µM 15d-PGJ2 was used for all subsequent examinations. BRL
49653 was used in a second set of experiments as a PPAR-specific
agonist. Preincubation of macrophages with 10 nM BRL 49653
significantly reduced the oxidative burst induced by TPA (Fig. 3B
). The amounts of 100 nM or 1 µM BRL 49653 were
similarly effective and completely attenuated ROS formation in response
to TPA. To prove the involvement of PPAR, we analyzed the effect of
the PPAR-specific agonist WY 14643. Preincubation of macrophages
with 100 µM WY 14643 left the TPA-induced oxidative burst unaltered
(Fig. 3C
). Control studies revealed that neither PPAR
agonists nor LIN pretreatment significantly altered basal ROS
formation. Taken together, prestimulation of macrophages with specific
PPAR agonists as well as LPS/IFN-/NAME attenuated the TPA-evoked
oxidative burst, whereas the PPAR agonist WY 14643 was not
effective. The statistical evaluation of these flow cytometric results
is summarized in Table 1
.
|
We next examined the effect of 1 µM
15d-PGJ2 and 3 µM ciglitazone on DCF oxidation
in primary human macrophages, U937, and RAW 264.7 cells (Fig. 4
). Activation of PPAR significantly attenuated ROS formation in
response to TPA, in all systems with an equipotent action of
15d-PGJ2 and ciglitazone. The PPAR agonist
ciglitazone revealed no apoptotic response in macrophages at doses
ranging from 0.3 to 6 µM.
|
Reduction of ROS formation was nearly complete in human macrophages,
RAW 264.7, and U937 cells. To demonstrate activation of PPAR in
response to 15d-PGJ2 and ciglitazone, we examined
reporter gene activation in RAW 264.7 macrophages (Fig. 5
) and performed gel shift analysis (Fig. 6A
, B
).
|
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A reporter gene assay confirmed activation of PPAR in response to
15d-PGJ2 and ciglitazone. Two reporter
constructs, each containing three PPRE sites derived from two different
promoters (see Material and Methods) in front of a minimal thymidine
kinase promoter hocked up to the luciferase gene, revealed a three- to
fivefold activation of PPAR in response to agonists. Activation with
15d-PGJ2 and ciglitazone was equally potent. We
conclude that PPAR agonists such as 15d-PGJ2
and ciglitazone provoke PPAR binding to its DNA recognition sites,
which further culminates in trans-activation. This situation
coincides with an attenuated oxidative burst in macrophages.
Activation of PPAR in response to LPS/IFN- and attenuated ROS
formation
We wanted to demonstrate activation of PPAR under conditions of
prestimulation with LPS/IFN-/NAME, a manipulation regime known to
attenuate macrophage activation, i.e., ROS formation. First, we
followed PPAR activation in response to LPS/IFN-/NAME by gel
shift analysis (Fig. 6A
). The response was low under control
situations; activation steadily increased up to 15 h and finally
decreased at 24 h. Maximal activation achieved with
LPS/IFN-/NAME was comparable to the response seen with 1 µM
15d-PGJ2.
To provide unequivocal prove for the involvement of PPAR in DNA
binding, we performed a supershift analysis (Fig. 6B
).
Activation achieved with 15d-PGJ2 or
LPS/IFN-/NAME was supershifted with a PPAR2 antibody, whereas an
unrelated PPAR antibody left the response unaltered. In a second set
of experiments, we used the luciferase reporter assay to demonstrate
transcriptional activation of PPAR after the addition of
LPS/IFN-/NAME (Fig. 6C
). In cells transfected with either
of two different luciferase reporter constructs containing PPAR
binding sites in their promoter, the addition of LPS/IFN-/NAME
elicited a three- to fivefold increase of luciferase activity. Having
established the effect of LPS/IFN-/NAME in PPAR activation, we
demonstrated the role of PPAR in attenuating the oxidative burst.
Decoy oligonucleotides can be used to scavenge and thereby to
inactivate transcription factors (26
, 27)
. Using this
experimental approach, we provide evidence that LPS/IFN-/NAME
attenuated ROS formation via PPAR activation (Fig. 7A
, B
). As shown in Fig. 7A
, oxidation of HE was elicited in response to 1 µM TPA in RAW 264.7
macrophages, whereas prestimulation with LPS/IFN-/NAME abolished ROS
formation. The presence of PPRE decoy oligonucleotides attenuated the
down-modulatory behavior of LPS/IFN-/NAME and allowed it to regain
HE oxidation in response to TPA. A similar effect was observed using
ciglitazone as a specific PPAR agonist. Prestimulation with 3 µM
ciglitazone abolished ROS formation, whereas PPRE decoy
oligonucleotides fully restored the oxidative response.
|
In a next step, we reproduced results obtained with RAW 264.7
macrophages in human primary macrophages (Fig. 7B
). DCF
oxidation occurred after the addition of TPA and was attenuated when
LPS/IFN-/NAME was prestimulated. Addition of PPRE decoy
oligonucleotides restored DCF oxidation despite LPS/IFN-/NAME
prestimulation. In contrast, prestimulation of human macrophages with
LPS/IFN-/NAME that had been exposed to oligonucleotides containing a
mutant PPRE site beforehand were unable to regain the oxidative
response after TPA addition. We conclude that LPS/IFN-/NAME causes
activation of PPAR, which in turn attenuates macrophage activation
as shown here for the oxidative burst.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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provokes a profound reduction in the oxidative burst. Desensitization
was the result of PPAR activation. This was confirmed by gel shift
analysis, a reporter gene assay, and a decoy oligonucleotide approach.
Our notion on the involvement of PPAR was strengthened by the
finding that PPAR agonists such as 15d-PGJ2;
more important, BRL 49653 or ciglitazone reproduced the effect of
LPS/IFN- whereas the PPAR-specific agonist left the TPA-induced
oxidative burst unaltered. Obviously, PPAR activation reduces the
ability of macrophages to produce ROS and thus to act proinflammatory.
Macrophages are key players in the innate immune response
(32)
. Immunological activation of macrophages is achieved
by the Th 1 cytokine IFN- and LPS, the outer membrane constituent of
gram-negative bacteria (10)
. Cell activation results in
the release of various proinflammatory products such as cytokines and
reactive nitrogen as well as oxygen species (10)
. A unique
property of endotoxin is the ability to evoke a state of
hypersensitivity contrasted by low responsiveness, known as endotoxin
tolerance (33
, 34)
. Tolerance development is controlled at
the cellular level and may limit the extend of proinflammatory
responses to protect the host from excessive destruction. This would
require a highly orchestrated compensatory mechanism to control a whole
battery of activation signals, including ROS formation. It was
attractive to speculate on the involvement of PPAR, a recently
described member of the PPAR ligand dependent transcription factor
family implicated in anti-inflammatory signaling (20
, 27)
.
Although PPAR is expressed at high levels in adipocytes and affects
adipocyte gene expression and differentiation, it is also found at
lower levels in several other cells (19)
. Besides
antidiabetic thiazolidinediones, fatty acids or their metabolites and
components of oxidized low density lipoproteins have been described as
PPAR agonists (35)
. We now provide evidence that the
classic macrophage stimulators LPS/IFN- promote PPAR activation.
This is established by gel shift analysis, a supershift response in the
presence of PPAR but not PPAR antibodies, and a reporter gene
assay coupled to luciferase activity. Whereas reporter gene assays do
not discriminate between PPAR isoforms, our supershift experiment
points to PPAR. We assume that LPS/IFN- induce a PPAR response
via production of activating ligands, which awaits further
characterization. Possible candidates are
15d-PGJ2, a derivative of prostaglandin
D2 synthesized by cyclooxygenase-2
(36)
, 13-hydroxy octadecadienoic acid, or
15-hydroxyeicodatetraenoic acid, which are products of the 12/15
lipoxygenase pathway. These compounds have recently been implicated in
PPAR activation in macrophages (37)
. Along this line,
it appears that combined expression of cytosolic phospholipase
A2, cyclooxygenase-2, and prostaglandin D
synthase produce sufficient endogenous cyclopentenone prostaglandins to
act anti-inflammatory (38)
. Since in macrophages the
expression and activation of cyclooxygenase-2 is a standard response to
LPS/IFN- (39)
, we envision a subsequent PPAR
signaling pathway to limit further cell activation, thereby preventing
a self-perpetuating autodestructive loop. Desensitization is evident at
the level of ROS formation, a heretofore unrecognized feature of
PPAR action.
Our results support the anti-inflammatory properties of PPAR and
appear in line with the action of PPAR in attenuating TNF-
formation or iNOS induction (20
, 40)
. For the latter
studies, synthetic PPAR agonists have been used to inhibit TNF-
and iNOS gene expression in part by antagonizing transcription factors
such as AP-1, STAT, and NF-
B (27
, 39)
. Since
cyclopentenone prostaglandins, which are proposed PPAR agonists,
directly block IB kinase (IKKß), thereby exerting
anti-inflammatory activity, the specificity of prostaglandin
metabolites was questioned (40)
. Other studies also
challenge a direct relationship between the anti-inflammatory action of
15d-PGJ2 and PPAR activation, based on the
observation that only 15d-PGJ2, but not other
PPAR agonists, blocked cytokine formation (42)
.
Variations among studies may reside in the period of time that PPAR
agonists are present. Prestimulation with LPS/IFN- required at least
12 h in order to block the oxidative burst. Short preincubation
periods in the range of hours may activate preexisting PPAR, whereas
longer lasting incubation periods also allow transcriptional
up-regulation of PPAR and thus provoke stronger/diverse responses.
These experimental drawbacks prompted us not only to use chemically
diverse PPAR agonist in attenuating the oxidative burst, but also to
apply a decoy approach in more rigorously demonstrating the requirement
of PPAR for LPS/IFN- action. As a further working hypothesis on
molecular actions of PPAR in attenuating ROS formation, we will
examine whether gene activation and/or inhibition by PPAR is
required or whether PPAR complexes with protein partners, thereby
blocking activation of the NAD(P)H-oxidase.
During our studies, we noticed a proapoptotic action of
15d-PGJ2 at concentrations above 5 µM. This
agrees somewhat with the study of Chinetti and co-workers, who reported
activation of PPAR to be association with programmed cell death
(41)
. At variance, activation of PPAR is not a
proapoptotic signal in general for the following reasons: 1)
PPAR activation and apoptotic signaling can be separated at lower
agonist concentrations; 2) ciglitazone caused PPAR
activation but failed to initiate apoptosis; and 3)
LPS/IFN-/NAME stimulation was not proapoptotic either. Obviously, a
general proapoptotic role for PPAR in macrophages can be excluded
and, moreover, can clearly be separated from its action in tolerance
development.
Our notion that activation of PPAR in macrophages may be of
considerable importance for tolerance development under septic
conditions correlates well with the work of Leininger et al.
(43)
. They observed induction of PPAR to coincide with
or to closely follow an endotoxin challenge and host responses to acute
inflammation in peripheral porcine blood monocytes. It will be
challenging to define conditions in vivo when activation of
PPAR contributes to a low responsiveness of macrophages and to see
whether this is associated with tolerance development toward
LPS.
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ACKNOWLEDGMENTS |
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Received for publication April 3, 2000.
Revision received July 24, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 A. von Knethen, M. Soller, N. Tzieply, A. Weigert, A. M. Johann, C. Jennewein, R. Kohl, and B. Brune PPAR{gamma}1 attenuates cytosol to membrane translocation of PKC{alpha} to desensitize monocytes/macrophages J. Cell Biol., February 26, 2007; 176(5): 681 - 694. [Abstract] [Full Text] [PDF]
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 A. Weigert, A. M. Johann, A. von Knethen, H. Schmidt, G. Geisslinger, and B. Brune Apoptotic cells promote macrophage survival by releasing the antiapoptotic mediator sphingosine-1-phosphate Blood, September 1, 2006; 108(5): 1635 - 1642. [Abstract] [Full Text] [PDF]
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 R. Kohl, S. Preiss, A. von Knethen, and B. Brune Oxidized low-density lipoprotein depletes PKC{alpha} and attenuates reactive oxygen species formation in monocytes/macrophages Cardiovasc Res, August 1, 2006; 71(3): 574 - 585. [Abstract] [Full Text] [PDF]
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 S. Y. Lee, E. J. Kang, G. Y. Hur, K. H. Jung, H. C. Jung, S. Y. Lee, J. H. Kim, C. Shin, K. H. In, K. H. Kang, et al. Peroxisome proliferator-activated receptor-{gamma} inhibits cigarette smoke solution-induced mucin production in human airway epithelial (NCI-H292) cells Am J Physiol Lung Cell Mol Physiol, July 1, 2006; 291(1): L84 - L90. [Abstract] [Full Text] [PDF]
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 C. D. Cool, S. D. Groshong, J. Oakey, and N. F. Voelkel Pulmonary Hypertension: Cellular and Molecular Mechanisms Chest, December 1, 2005; 128(6_suppl): 565S - 571S. [Full Text] [PDF]
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A. von Knethen, A. Tautenhahn, H. Link, D. Lindemann, and B. Brune Activation-Induced Depletion of Protein Kinase C{alpha} Provokes Desensitization of Monocytes/Macrophages in Sepsis J. Immunol., April 15, 2005; 174(8): 4960 - 4965. [Abstract] [Full Text] [PDF]
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 S.-Y. Lim, J.-H. Jang, H.-K. Na, S. C. Lu, I. Rahman, and Y.-J. Surh 15-Deoxy-{Delta}12,14-Prostaglandin J2 Protects against Nitrosative PC12 Cell Death through Up-regulation of Intracellular Glutathione Synthesis J. Biol. Chem., October 29, 2004; 279(44): 46263 - 46270. [Abstract] [Full Text] [PDF]
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 Hongkuan Fan and J. A. Cook Review: Molecular mechanisms of endotoxin tolerance Innate Immunity, April 1, 2004; 10(2): 71 - 84. [Abstract] [PDF]
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B. Zingarelli, M. Sheehan, P. W. Hake, M. O'Connor, A. Denenberg, and J. A. Cook Peroxisome Proliferator Activator Receptor-{gamma} Ligands, 15-Deoxy-{Delta}12,14-Prostaglandin J2 and Ciglitazone, Reduce Systemic Inflammation in Polymicrobial Sepsis by Modulation of Signal Transduction Pathways J. Immunol., December 15, 2003; 171(12): 6827 - 6837. [Abstract] [Full Text] [PDF]
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0.05 vs. TPA only). For details,
see Materials and Methods.
