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Does Not Inhibit IL-6 or TNF-
Responses of Macrophages to Lipopolysaccharide In Vitro or In Vivo
Departments of
*
Endocrinology and Chemical Biology,
Laboratory Animal Resources, and
Molecular Endocrinology, Merck Research Laboratories, Rahway, NJ 07065
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(PPAR) agonists as
anti-inflammatory agents in cell-based assays and in a mouse model
of endotoxemia. Human peripheral blood monocytes were treated with LPS
or PMA and a variety of PPAR agonists. Although
15-deoxy-
12,14-prostaglandin J2
(15d-PGJ2) at micromolar concentrations significantly
inhibited the production of TNF-
and IL-6, four other high affinity
PPAR ligands failed to affect cytokine production. Similar results
were obtained when the monocytes were allowed to differentiate in
culture into macrophages that expressed significantly higher levels of
PPAR or when the murine macrophage cell line RAW 264.7 was used.
Furthermore, saturating concentrations of a potent PPAR ligand not
only failed to block cytokine production, but also were unable to block
the inhibitory activity of 15d-PGJ2. Thus, activation of
PPAR does not appear to inhibit the production of cytokines by
either monocytes or macrophages, and the inhibitory effect observed
with 15d-PGJ2 is most likely mediated by a
PPAR-independent mechanism. To examine the anti-inflammatory
activity of PPAR agonists in vivo, db/db mice were
treated with a potent thiazolidinedione that lowered their elevated
blood glucose and triglyceride levels as expected. When
thiazolidinedione-treated mice were challenged with LPS, they displayed
no suppression of cytokine production. Rather, their blood levels of
TNF- and IL-6 were elevated beyond the levels observed in control
db/db mice challenged with LPS. Comparable results were
obtained with the corresponding lean mice. Our data suggest that
compounds capable of activating PPAR in leukocytes will not be
useful for the treatment of acute inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is highly
expressed in adipose tissue, colon, spleen, adrenal gland, and
macrophages. Due to alternative promoter use and RNA splicing this
receptor is present as two isoforms: PPAR1 and PPAR2
(4, 5, 6). The latter has additional amino acids at the amino
terminus and is the isoform primarily expressed in adipocytes; the
former appears to be the major isoform in all other tissues. PPAR
has been shown to play a major regulatory role in adipogenesis and the
expression of adipocyte genes involved in lipid metabolism. Its forced
overexpression in fibroblasts and myocytes causes these cells to
differentiate into adipocytes (7, 8). Recently, it has
been demonstrated that the naturally occurring arachidonic acid
metabolite, 15-deoxy-12,14-prostaglandin
J2 (15d-PGJ2) as well as
thiazolidinedione (TZD) and certain novel non-TZD insulin-sensitizing
agents are ligands and agonists of this receptor (9, 10, 11, 12, 13).
PPAR is expressed at high levels in macrophages (14)
and tissues that demonstrate high levels of lipid catabolism,
especially liver (15). Activation of hepatic PPAR
results in increased expression of enzymes involved in fatty acid
-oxidation and, in rodents, peroxisome proliferation and
hepatocarcinogenesis (as reviewed in Refs. 1, 16). Drugs
that serve as hypolipidemic agents in humans, including numerous
fibrates and WY-14653, are PPAR ligands and agonists
(17, 18, 19, 20). PPAR
is widely expressed in a variety of
tissues, including the brain (21, 22). While it has been
shown that it may play a role in regulating cholesterol metabolism in
an animal model of insulin resistance (M. D. Leibowitz, C. Fievet,
N. Hennuyer, J. Peinado-Onsurbe, J. Duez, J. Berger, C. A.
Cullinan, C. P. Sparrow, J. Baffic, G. D. Berger, C. Santini,
R. W. Marquis, R. Tolman, C. Fruchart, R. G. Smith, D.
E. Moller, and J. Auwerx, manuscript in preparation), the physiological
role of PPAR is yet to be fully delineated.
Recently, several laboratories have examined the effects of PPAR
activation on the inflammatory responses of monocytes and macrophages
(23, 24). One group presented data demonstrating that
PPAR agonists could abrogate IFN- activation of nitric oxide
synthase (iNOS) and gelatinase b expression in murine macrophages
(24). In addition, induction of promoters for
proinflammatory genes that are regulated by the AP-1, STAT and NF-
B
transcription factors was antagonized by activation of PPAR in
transfected cell lines (24). A second group of researchers
showed that PPAR agonists could inhibit production of inflammatory
cytokines by pharmacologically activated human monocytes
(23). It was suggested that these inhibitory effects were
occurring at the transcriptional level, because PPAR agonists
blocked induction of the TNF- and IL-2 promoters in a transfected,
macrophage-like cell line. Taken together, these results suggest that
compounds that activate PPAR may be able to serve as
anti-inflammatory agents.
We have recently identified and characterized a number of non-TZDs that
serve as ligands and agonists of PPARs (12). These
compounds have been shown to alter the conformation of the receptors
that they activate and promote their interaction with nuclear receptor
coactivators. In addition, these compounds have been used to
demonstrate that activation of PPAR results in an
insulin-sensitizing effect in vivo. In the present study we employed a
subset of these compounds as well as 15d-PGJ2 and
TZDs to determine whether PPAR activation affects the production of
cytokines by monocytes or macrophages in vitro or in vivo. We found
that, with the exception of 15d-PGJ2, PPAR
agonists were unable to significantly inhibit cytokine production by
primary human monocytes, differentiated human macrophages, or murine
RAW 264.7 macrophage-like cells stimulated by LPS or PMA. Additionally,
AD-5075, a potent TZD, was unable to attenuate LPS-induced TNF- and
IL-6 production in obese diabetic or lean mice after a dosing protocol
that provided significant antidiabetic relief in the former. These
results raise doubts as to whether PPAR can modulate acute
macrophage-dependent inflammatory events.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS from Salmonella minnesota R595 and
Escherichia coli K235 was obtained from List Biologicals
(Campbell, MA). A protein-free preparation of LPS from E.
coli K235 was prepared as previously described (25).
The final preparation was devoid of protein as detected by the
colloidal gold total protein staining method (Bio-Rad, Hercules, CA).
All LPS preparations were prepared as 1 mg/ml stocks in Dulbecco’s PBS
without calcium and magnesium (D-PBS) and were sonicated briefly in a
water bath sonicator before dilution and addition to the cells.
Recombinant human TNF- and recombinant human IL-6 were purchased
from R&D Systems (Minneapolis, MN). FCS was obtained from HyClone
(Logan, UT), and human serum was obtained from BioWhittaker
(Walkersville, MD) or Gemini (Calabasas, CA). PMA, penicillin
G, streptomycin sulfate, DMSO, protease inhibitors, and BSA
(fraction V) were purchased from Sigma (St. Louis, MO). D-PBS, RPMI
1640, and Ham’s F-12 were obtained from Mediatech (Herndon, VA).
15d-PGJ2 was obtained from Biomol (Plymouth
Meeting, PA). AD-5075
(5-[4-[2-(5-methyl-2-phenyl-4-oxazoly)-2-hydroxyethoxy]benzyl]-2,4-thiazolidinedione),
L-165,041
(4-[3-[2-propyl-3-hydroxy-4-acetyl]phenoxy]propyloxyphenoxyaceticacid),
L-796,449
(3-chloro-4-(3-(3-phenyl-7-propylbenzofuran-6-yloxy)propylthio)phenylacetic
acid), and L-165,461
(3-chloro-4-(3-(3-ethyl-7-propylbenzisoxazol-6-yloxy)propylthio)phenylacetic
acid) were provided by Gerard Kieczykowski, Philip Eskola, Joseph F.
Leone, Mark S. Levorse, Peter A. Cicala, Gregory D. Berger, Robert
Marquis, Conrad Santini, Soumya P. Sahoo, and Richard L. Tolman (Merck
Research Laboratories, Rahway, NJ).
Purification of monocytes
Human PBMC were obtained by plasmapheresis (University of Pennsylvania, Philadelphia, PA). The cells were washed in RPMI 1640 medium that was supplemented with L-glutamine, 50 U/ml penicillin G, and 50 µg/ml streptomycin sulfate and purified further using Lymphocyte Separation Medium (ICN, Aurora, OH). After centrifugation at 1500 x g for 30 min at room temperature, the interface containing the mononuclear cells was harvested and washed twice in complete RPMI medium. T lymphocytes were then removed using the SRBC rosetting method (26). The monocyte preparation was further washed three times with ice-cold D-PBS before the cells were used in cell-based assays.
Monocyte culture using Teflon beakers
Culture of mononuclear phagocytes in suspension by incubation on
a Teflon surface to which cells do not adhere has been
described previously (27, 28). Briefly, 1 x
107 monocytes were resuspended in 10 ml of RPMI
1640 medium with L-glutamine and 14% normal human
serum/60-ml Teflon beaker. The loosely capped beakers were
incubated at 37°C in a 5% CO2 atmosphere. Cell
recovery from each beaker was 
90%.
Culture of RAW 264.7 cells
RAW 264.7 cells (TIB-71) were obtained from American Type Culture Collection (Manassas, VA). The cells were maintained in Ham’s F-12 medium with L-glutamine supplemented with 10% FCS, 50 U/ml penicillin G, and 50 µg/ml streptomycin sulfate in an atmosphere containing 5% CO2.
Induction of cytokine expression in monocytes and macrophages
Freshly isolated or differentiated monocytes were seeded at a
density of 1 x 105 cells/well in RPMI 1640
medium with L-glutamine and 10% normal human serum into
Costar 96-well tissue culture plates (Corning, Corning, NY). RAW 264.7
cells were seeded at a density of 3 x 104
cells/well in Ham’s F-12 medium containing 10% FCS 15–24 h before
the experiment into 96-well plates. The cells were treated with
compound for 1 h before the addition of either 0.1 ng/ml ReLPS
from S. minnesota R595 or 30 ng/ml PMA. After 4-h or
overnight (18- to 24-h) incubation at 37°C in 5%
CO2 and 95% air, the conditioned cell medium was
harvested. The IL-6 and TNF- concentrations were determined by ELISA
as described below.
Cytokine ELISAs
Cytokines (IL-6 or TNF-) were quantitated using a sandwich
ELISA with commercially available Abs. Briefly, 100 µl/well of 4
µg/ml solutions of mAbs to human IL-6 (MAB206, R&D Systems), mouse
IL-6 (MAB406, R&D Systems), human TNF- (MAB610, R&D Systems), or
mouse TNF- (1221-00, Genzyme, Cambridge, MA) in D-PBS were
immobilized on Dynatech Immulon-4 96-well plates (Dynex Technologies,
Chantilly, VA) by overnight incubation. The plates were blocked for
1 h with a blocking buffer containing 1% BSA, 5% sucrose, and
0.05% NaN3 in D-PBS. The blocked plates were
washed five times with wash solution (Kirkegaard & Perry, Gaithersburg,
MD). The cell medium or plasma samples were appropriately diluted in
ELISA diluent containing 1% BSA and 0.05% NaN3
in D-PBS. The diluted supernatants were added to the wells and
incubated for 2 h. The plates were then washed as described above.
Biotinylated Ab (anti-human IL-6 BAF206, R&D Systems, 25 ng/ml;
anti-mouse IL-6, BAF406, R&D Systems, 200 ng/ml; anti-human
TNF-, BAF210, R&D Systems, 200 ng/ml; anti-mouse TNF-,
80-4895-01, Genzyme, 5 µg/ml) were added to the wells in ELISA
diluent, and the plates were incubated for an additional 2 h.
After washing, a 1/20,000 dilution of HRP-conjugated streptavidin
(Zymed, San Francisco, CA) was added to the wells. The plates were
incubated for 30 min. After washing, 100 µl of tetramethylbenzidine
peroxidase substrate solution (Kirkegaard & Perry) was added to each
well, and the color reaction was stopped by adding 50 µl of 1 M
phosphoric acid. The absorbance at 450 nm was determined using a
SpectraMAX 250 plate reader (Molecular Devices, Sunnyvale, CA).
Cytokines were quantitated relative to a standard curve representing a
range of dilutions of recombinant IL-6 or TNF- (R&D Systems). All
steps were conducted at room temperature.
RT-PCR
Total RNA was isolated using Trizol reagent (Life Technologies,
Gaithersburg, MD). To remove potential contamination by genomic DNA for
downstream procedures, total RNA was first treated with 1 U of DNase I
(Life Technologies). Reverse transcription was then performed using the
RT-for-PCR kit from Clontech Laboratories (Palo Alto, CA). Briefly, 1
µg of total RNA was incubated with 20 pmol of
oligo(dT)18, 20 U of RNase inhibitor, and 200 U
of Moloney murine leukemia virus reverse transcriptase in a buffer
containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, and 3 mM
MgCl2 in a total volume of 20 µl at 42°C for
1 h, followed by an incubation at 94°C for 5 min. Kit-provided
human placental RNA (1 µg) was used as a control. Aliquots (1/25th of
the RT reaction) were subjected to PCR amplification using 2 U of Taq
polymerase (Fisher Biotech, Pittsburgh, PA) and a total of 35 cycles (1
min at 94°C, 1 min at 55°C, and 2 min at 72°C). The following
primers were used: human G3PDH amplimer set 5406 (Clontech), mouse
G3PDH amplimer set 5409 (Clontech), and human PPAR forward primer,
5'-GGAAAGACAACAGACAAATCAC; human PPAR reverse primer,
5'-TGCATTGAACTTCACAGCAAAC; mouse PPAR forward primer,
5'-TCATACATAAAGTCCTTCCC; and mouse PPAR reverse primer,
5'-TGTCTGTCTCTGTCTTCTTG. Plasmids pSG5/hPPAR1 encoding human
PPAR1 (5) and pSG5/mPPAR2 containing mouse PPAR2
cDNA (provided by Dr. Bruce Spiegelman, Dana-Farber Institute, Boston,
MA) were used as positive controls in the PCR reactions. These plasmids
were constructed by subcloning the full-length cDNAs into the mammalian
expression vector pSG5 (Stratagene, La Jolla, CA).
Immunoblot analysis
Purified human monocytes were cultured in Teflon beakers as
described above. On days 0 and 5 in culture, cells were harvested from
two beakers, and cell lysates were prepared. Cells were washed once
with 10 ml of PBS (Mediatech) with protease inhibitors (0.3 U/ml
aprotinin, 2 mM PMSF, 50 µg/ml benzamidine, 3 mM di-isopropyl
fluorophosphate, and 5 µg/ml each of antipain, leupeptin,
chymostatin, and pepstatin A). Recovered cells were directly lysed in
125 µl of SDS sample buffer for 15 min on ice. The SDS sample buffer
consisted of 10% glycerol, 2% SDS, 0.03% bromophenol blue, 1 mM EDTA
(pH 7.0), and 0.06 M Tris (pH 6.8) with protease inhibitors as
described above. The lysates were briefly sonicated and centrifuged for
5 min at 12,000 x g, and supernatants were collected.
The detergent-compatible protein assay (Bio-Rad) was performed to
determine the protein concentration in each sample. The SDS-PAGE
samples were made under reducing conditions using 250 µg of
protein/well. The SDS-PAGE was run on 10% Tris-glycine gels using
standard buffers (NOVEX, San Diego, CA). Following separation, protein
samples were transferred to nitrocellulose filters (NOVEX) for 1.5
h at 300 mA. Filters were blocked with Superblock (Pierce, Rockford,
IL) overnight at 4°C, washed twice with Tris-buffered saline/0.1%
Tween-20, and incubated with mAb E-8 directed against PPAR (Santa
Cruz Biotechnology, Santa Cruz, CA) at 1 µg/ml for 2 h at room
temperature. The filters then were washed twice as described above and
incubated with HRP-conjugated goat anti-mouse IgG diluted 1/3,000
for 2 h at room temperature. The filters were again washed as
described above, and bound Ab was detected using chemiluminescence
(ECL, Amersham, Arlington Heights, IL).
In vivo studies
Specific pathogen-free, 8- to 9-wk-old, male db/db (C57BL6/J+/+Leprdb) or lean control heterozygous mice (The Jackson Laboratory, Bar Harbor, ME) were housed five per cage in static microisolators and allowed ad libitum access to pelleted chow (Purina 5001, Ralston Purina, Richmond, IN) and water. The animal room was maintained on a 12-h light, 12-h dark cycle. The institutional animal care and use committee of Merck Research Laboratories reviewed and approved all animal use, and all animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources), National Research Council, Washington, DC, 1996).
The animals were treated daily for 5 days by oral gavage (0.2 ml/mouse) with vehicle (0.5% carboxymethyl cellulose) with or without AD-5075 (10 mg/kg). On day 5 of the treatment, vehicle (saline) with or without a protein-free preparation of LPS (50 µg/mouse) from E. coli K235 (26) was injected i.p. (0.1 ml/mouse) 1 h after the final dose of vehicle or AD-5075. Blood samples were collected into lithium heparin Microtainer tubes (Becton-Dickinson, Franklin Lakes, NJ), 90 min (tail nick) and 5 h (terminal, CO2 overdose, cardiocentesis) after LPS or vehicle challenge. Cytokines were quantitated by ELISA as described above. Glucose and triglyceride levels were determined by hexokinase and glycerophosphate oxidase methods, respectively (Hitachi 911, Roche, Indianapolis, IN).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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agonists do not inhibit cytokine production by monocytes or
macrophages
A structurally diverse array of PPAR agonists was selected for
this study to test the hypothesis that inflammatory cytokine production
can be inhibited in monocytic cells by a mechanism involving PPAR
(Fig. 1
). The TZD antidiabetic agent,
AD-5075, is a potent PPAR agonist (13). The non-TZD
insulin-sensitizing agents L-796,449, L-165,461, and L-165,041 serve as
potent, moderate, and weak PPAR agonists, respectively
(12). Finally, the prostanoid
15d-PGJ2 has demonstrated PPAR agonist
activity at micromolar concentrations (11) .
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expression. Freshly isolated human
peripheral blood monocytes contained only relatively small amounts of
PPAR mRNA when assessed by RT-PCR, consistent with previous results
(14, 29). In contrast, G3PDH mRNA was readily
detectable (Fig. 2A).
Cultivation of monocyte preparations in Teflon beakers leads to
differentiation into cells possessing a mature monocyte phenotype
(27, 28). During the maturation process, increased
expression of PPAR was observed (Fig. 2A). The increase
in PPAR mRNA was apparent after only 1 day of culture and was even
greater following culture of the cells for 5 days. Immunoblot analysis
of cell lysates using a commercially available anti-PPAR mAb
failed to demonstrate immunodetectable protein in fresh monocytes (Fig. 2B, lane 3), but resulted in strong staining
after 5 days of differentiation in the Teflon dishes (Fig. 2B, lane 4). Similar results were obtained with
two different anti-PPAR peptide Abs (data not shown).
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agonists on LPS- or PMA-mediated cytokine
induction. Freshly prepared monocytes or those previously
differentiated for 5 days were incubated with each of the inflammatory
agents alone or in the presence of increasing concentrations of PPAR
activators. Following these incubations (4 h for LPS; overnight for
PMA), TNF- and IL-6 concentrations were measured in the cell medium
as described in Materials and Methods.
Unstimulated cells did not produce measurable cytokine (data not
shown), while addition of LPS or PMA caused strong cytokine expression
(Fig. 3). Treatment of freshly isolated
monocytes (Fig. 3, A and B) or 5-day cultured
monocytes (Fig. 3, C and D) with L-165,041,
L-165,461, L-796,449, or AD-5075 at concentrations up to 50 µM did
not inhibit the TNF- or IL-6 synthesis and secretion caused by LPS
(Fig. 3, A and C) or PMA (Fig. 3, B
and D). Of all the compounds tested, only
15d-PGJ2 significantly abrogated cellular
cytokine production, and it did so in freshly isolated monocytes
lacking immunodetectable PPAR as well as in the cultured monocyte
preparations (Fig. 3, A–D). The prostanoid effectively
blocked cytokine secretion in a concentration-dependent manner, with
IC50 values generally between 12.5 and 25 µM.
Similar results were obtained when LPS incubations were allowed to
proceed overnight (not shown). The above results were reliably repeated
when using cell preparations from different healthy donors.
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agonists on cytokine induction in
murine cells, RAW 264.7 cells, a well-established mouse tumor cell line
with a mature macrophage phenotype (30), were used. It has
previously been demonstrated by researchers examining the
anti-inflammatory effects of PPAR agonists that these cells
express PPAR, albeit at low levels (24). We confirmed
the expression of PPAR in our RAW 264.7 culture by RT-PCR (Fig. 4A). Similar to the results we
observed with human monocytes, all the PPAR agonists, with the
exception of 15d-PGJ2, were ineffective in
inhibiting production of TNF- in responses to LPS in RAW 264.7 cells
(Fig. 4B). In contrast, the prostaglandin once again blocked
the induction of cytokine production in a dose-responsive manner.
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agonist that had proven ineffective (as described above) as an
anti-inflammatory agent. Treatment of fresh human monocytes with
15d-PGJ2 resulted in almost identical inhibition
curves for IL-6 production regardless of whether 50 µM AD-5075 was
absent or present during the incubation. The results presented were
obtained after stimulation of the cells with LPS for 4 h (Fig. 5A) or after overnight
incubation with PMA (Fig. 5B), and nearly identical results
were observed when TNF secretion was determined or when overnight
incubations with LPS were used as the stimulus (data not shown).
Similar observations were made when AD-5075 was replaced by the other
PPAR agonist used in this study (not shown).
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serves to inhibit inflammatory responses of monocytic cells
in vitro. In contrast, 15d-PGJ2 appears to
possess anti-inflammatory activity that is most likely mediated by
a PPAR-independent mechanism.
Chronic treatment with a potent PPAR agonist fails to reduce
LPS-induced cytokine production in mice
Animal studies were conducted to determine whether macrophages in
vivo respond to the PPAR agonist, AD-5075, and to allow prolonged
treatment of the cells (5 days). Importantly, these studies allowed a
positive measure of AD5075 efficacy. Obese, diabetic db/db
mice were used in these experiments because their responsiveness to
AD-5075 treatment, previously demonstrated to be mediated through
activation of PPAR (13), can be easily monitored by
measuring decreases in the animals’ elevated blood glucose and
triglyceride levels. The effects of this TZD were also examined in
metabolically normal, lean mice. The animals were treated daily with 10
mg/kg of AD-5075 orally for 5 days. Subsequently, they were injected
i.p. with 50 µg/mouse of LPS to induce acute inflammation, and plasma
levels of TNF- and IL-6 were measured at 90 min and 5 h
postinjection as described in Materials and Methods.
Treatment of db/db mice with the PPAR agonist
significantly lowered blood glucose and triglyceride levels (all
p 
0.005; Fig. 6,
J and L) to approximately those observed in lean
mice (Fig. 6, I and K). As previously described
(13), AD-5075 did not lower glucose or triglyceride levels
in the lean mice. Glucose levels were lowered in normal and diabetic
animals by the acute administration of LPS (Fig. 6, I and
J). Such hypoglycemic effects of LPS in db/db and
lean mice are well documented (31) and were, therefore,
expected.
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and IL-6 by activated monocytes and macrophages are two of the
hallmarks of the host response to endotoxin (32, 33, 34).
Following injection of db/db and lean mice with LPS, plasma
levels of these cytokines increased dramatically at both 90 min and
5 h compared with those in mice that did not receive LPS (Fig. 6
, A–H). AD-5075 pretreatment did not diminish plasma cytokine
levels in mice subsequently administered LPS. Rather, chronic treatment
with the potent PPAR agonist resulted in increases in TNF- and
IL-6 plasma levels at both time points after LPS administration. These
increases reached high statistical significance
(p < 0.005) for TNF- at 90 min in both the
lean (Fig. 6A) anddb/db (Fig. 6B) mice
as well as for TNF- (Fig. 6D) and IL-6 (Fig. 6F) at 5 h in db/db mice. Additional studies
examined LPS-induced plasma nitrate and nitrite production resulting
from the in-duction of inducible nitric oxide synthase. The LPS-induced
nitrite/nitrate production was not significantly altered in
db/db mice using the TZD treatment protocol (data not
shown).
These results support the conclusion that activation of PPAR does
not blunt the production of TNF-, IL-6, or iNOS in vivo when the
challenge is LPS.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA and protein
(14, 29, 37). Recent in vitro data suggested that
activation of macrophage PPAR by 15d-PGJ2 as
well as other synthetic PPAR ligands inhibited the expression of
proinflammatory agents, macrophage scavenger receptor A, and inducible
nitric oxide synthase; matrix metalloproteinase-9 and gelatinase
activities were also diminished (23, 24, 29). On the basis
of transfection studies performed in macrophage-like cell lines,
antagonism of the transcription factors AP-1, NF-B, and STAT was
implicated as the mechanism of the observed anti-inflammatory
effects (24). These findings suggested the possibility of
using PPAR agonists in novel treatment protocols for acute and
chronic inflammatory diseases that involve activated macrophages, such
as atherosclerosis and rheumatoid arthritis.
Our experiments were aimed at further exploring the
anti-inflammatory potential of PPAR agonists. We initially chose
in vitro experiments that model a key function of monocytes and
macrophages, the synthesis and secretion of cytokines in response to an
inflammatory stimulus such as bacterial LPS. These cellular responses
strongly depend upon the maximal activation of cytokine expression by
transcription factors such as NF-B and AP-1. We tested the effects
of TZD and non-TZD PPAR agonists of varying potencies in freshly
cultured and differentiated human monocytic cells after stimulation
with either LPS or PMA. Our results demonstrated that regardless of the
state of monocyte differentiation and PPAR expression, both
synthetic TZD and non-TZD PPAR agonists were without effect. Similar
results were obtained with the murine macrophage cell line RAW 264.7.
In addition to the compounds presented here, other TZD PPAR agonists
were also ineffective in suppressing cytokine release (data not shown).
Our results make it unlikely that PPAR agonists would demonstrate
anti-inflammatory effects in vivo through a mechanism involving the
repression of NF-B and/or AP-1.
The observations that PPAR is expressed in monocytic cells and that
the naturally occurring prostaglandin D2
metabolite 15d-PGJ2 is a PPAR agonist have
suggested a potential role for this receptor not only in lipid
metabolism but also in control of inflammation (10, 11, 38). When tested in our monocyte assays,
15d-PGJ2 was, indeed, the only PPAR agonist
that effectively blocked LPS- or PMA-induced cytokine expression. Based
on the full weight of the data, it appears unlikely that
15d-PGJ2 effects are directly mediated through
PPAR activation. The anti-inflammatory potency of
15d-PGJ2 in our experiments and in those
previously published (23, 24) substantially exceeded that
of other more potent TZD and non-TZD PPAR agonists. Importantly, we
demonstrate here that synthetic PPAR ligands were unable to block
the inhibitory activity of 15d-PGJ2 when used at
concentrations that should displace the prostaglandin all but
completely from the receptor. These findings, we believe, support the
contention that 15d-PGJ2 may act through
mechanisms not involving PPAR. Vaidya et al. have rendered a similar
conclusion from studies showing that 15d-PGJ2,
but not AD-5075, inhibited responses of neutrophils to TNF- and
formyl peptides (39). It was hypothesized that the
prostanoid may exert its effects through interaction with an as yet
unknown prostaglandin receptor. In neutrophils, this receptor appears
to be unrelated to the prostaglandin D2 receptor,
because a potent specific agonist of the receptor did not affect
peroxide production. Given the similarity of these observations, it is
conceivable that monocytes and neutrophils share a common signaling
pathway initiated by 15d-PGJ2. Collectively,
these findings emphasize the need to exercise caution when interpreting
results obtained with 15d-PGJ2 and the importance
of studying the actions of a broad spectrum of PPAR agonists before
invoking this receptor as a mediator of critical biological
responses.
The above studies suggest that PPAR does not affect the acute
response of macrophages to a stimulant such as LPS. Macrophages exhibit
a distinct phenomenon, macrophage activation, which entails
differentiation to a state characterized by unique patterns of gene
expression and responsiveness to stimuli. PPAR was first described
as a nuclear receptor that plays a critical role in adipocyte
differentiation (1, 7, 8), and it is thus possible that
PPAR may critically regulate monocyte activation or differentiation.
Because monocyte differentiation is controlled by poorly defined
factors acting locally in tissues, we tested the role of PPAR
agonists in animals by measuring responses of animals to challenges
with LPS, a widely used model of acute inflammation. The use of obese
diabetic mice (db/db) in the study in addition to normal
lean animals allowed us to determine the effectiveness of the TZD
treatment on PPAR activity by measuring the decline in blood glucose
and triglyceride levels at the time of endotoxin administration
(12). In both lean and db/db mice the
chronically administered PPAR agonist did not show effects on IL-6
and TNF- production and, therefore, confirmed the results we had
previously obtained in vitro with cultured monocytes and macrophages.
Instead, the blood levels of TNF- and IL-6 in the mice actually
increased, often significantly, compared with those in vehicle-treated
animals. Using the same treatment protocol, similar results were
obtained with C57BL/6J mice maintained on either a low fat diet or a
high fat, obesity-inducing diet (data not shown). The rise in
LPS-induced cytokine production is consistent with PPAR playing a
role in macrophage activation or differentiation, but with effects
opposite those proposed by prior authors.
Recent studies of a homologous transcription factor, PPAR, are
consistent with our studies of PPAR. Hill et al. reported that
chronic treatment of animals with PPAR agonists (fenofibrate and
Wy-14,643) results in a marked increase in TNF- levels and
significantly lowers 50% lethal doses of LPS in a mouse model of
endotoxemia (40). Although these agents modestly
down-regulated TNF expression in primary macrophages in vitro
(40), important differences between PPAR and PPAR
must be noted. The PPAR agents both increase liver weight and
decrease serum lipoprotein levels. Because hepatic macrophages may
contribute importantly to plasma TNF levels, and because lipoproteins
may strongly neutralize LPS, these two effects may explain the larger
increase in cytokine levels observed by Hill et al.
(40).
Our data cannot rule out the possibility that the activation of PPAR
may prove effective in antagonizing macrophage function in other
settings. Certain stimuli, for example treatment with live or killed
bacteria, are known to raise serum IFN- levels in animals and
produce hypersensitivity to the effects of LPS (41, 42).
It is conceivable that under these experimental conditions PPAR
agonists would prove effective in alleviating inflammation.
Interestingly, mice with a targeted deletion of the IFN- receptor
demonstrate a significant decrease in their disposition to develop
atherosclerotic lesions, suggesting that macrophage activation driven
by IFN- promotes atherosclerosis (43). As mentioned
above, PPAR is expressed in macrophages from atherosclerotic lesions
(24, 29), and recently published data provided evidence
that a PPAR agonist, troglitazone, can effectively reduce
atherosclerosis in animals (44). It is therefore likely
that PPAR agonists may not act globally as regulators of all
inflammatory mediators but, rather, may control only a specific subset
of proinflammatory genes. In keeping with this idea, recent studies
have observed that both PPAR and PPAR agonists failed to block
production of the cytokine IL-8, but at the same time strongly
inhibited MMP-9 secretion from a monocytic cell line, THP-1 (H. Shu, B.
Wong, G. Zhou, Y. Li, J. P. Berger, J. W. Woods, S. D.
Wright, and T.-Q. Cai, manuscript in preparation).
In summary, we have shown that PPAR agonists other than
15d-PGJ2 do not inhibit cytokine production in in
vitro and in vivo models of acute inflammation. The prostanoid appears
to exert its actions via a PPAR-independent mechanism. The results
from our study raise significant doubts about the potential global
utility of PPAR agonists as anti-inflammatory agents. Rather,
PPAR agonists may function selectively by regulating proinflammatory
genes involved in the development of inflammatory diseases such as
atherosclerosis. This may at least in part explain the protective role
of activators of PPAR in atherosclerosis (44).
|
Acknowledgments |
|---|
2 cDNA. We also thank Thomas Blake (Merck
Research Laboratories) for preparing the human T cell-depleted monocyte
fractions and Daniel Fletcher (Merck Research Laboratories) for
measurements of iNOS activity. |
Footnotes |
|---|
2 Abbreviations used in this paper: PPAR, peroxisome proliferator-activated receptor; TZD, thiazolidinedione; 15d-PGJ2, 15-deoxy-12,14-prostaglandin J2; D-PBS, Dulbecco’s PBS; AD-5075, 5-[4-[2-(5-methyl-2-phenyl-4-oxazoly)-2-hydroxyethoxy]benzyl]-2,4-thiazolidinedione; L-165,041, 4-[3-[2-propyl-3-hydroxy-4-acetyl]phenoxy]propyloxyphenoxy acetic acid; L-796,449, 3-chloro-4-(3-(3-phenyl-7-propylbenzofuran-6-yloxy)propylthio)phenylacetic acid; L-165,461, 3-chloro-4-(3-(3-ethyl-7-propylbenzisoxazol-6-yloxy)propylthio)phenylacetic acid.
Received for publication May 20, 1999. Accepted for publication November 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2: tissue-specific regulator of an adipocyte enhancer. Genes Dev. 8:1224.1 and 2. Biochem. Biophys. Res. Commun. 224:431.[Medline]
(mPPAR) gene: alternative promoter use and different splicing yield two mPPAR isoforms. Proc. Natl. Acad. Sci. USA 92:7921.2, a lipid- activated transcription factor. Cell 79:1147.[Medline]
(PPAR). J. Biol. Chem. 270:12953. and promotes adipocyte differentiation. Cell 83:813.[Medline]
12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR. Cell 83:803.[Medline]
and PPAR ligands produce distinct biological effects. J. Biol. Chem. 274:6718.: binding and activation correlate with antidiabetic actions in db/db mice. Endocrinology 137:4189.[Abstract]
and induces apoptosis of human monocyte-derived macrophages. J. Biol. Chem. 273:25573.-oxidation pathway by a novel family of nuclear hormone receptors. Cell 68:879.[Medline]
and . Proc. Natl. Acad. Sci. USA 94:4312. and . Proc. Natl. Acad. Sci. USA 94:4318. contains a CGG triplet repeat and is prominently expressed in the thalamic nuclei. Biochem. Biophys. Res. Commun. 217:1015.[Medline]
agonists inhibit production of monocyte inflammatory cytokines. Nature 391:82.[Medline]
is a negative regulator of macrophage activation. Nature 391:79.[Medline]
: differentiation-dependent peroxisomal proliferator-activated receptor (PPAR) expression and reduction of MMP-9 activity through PPAR activation in mononuclear phagocytes in vitro. Am. J. Pathol. 153:17. (PPAR) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein. Proc. Natl. Acad. Sci. USA 95:7614.12,14-prostaglandin J2 inhibits the 2-integrin-dependent oxidative burst: involvement of a mechanism distinct from PPAR ligation. J. Immunol. 163:6187. Interferon mediates Propionibacterium acnes-induced hypersensitivity to lipopolysaccharide in mice. Infect. Immun. 60:1994. potentiates atherosclerosis in ApoE knock-out mice. J. Clin. Invest. 99:2752.[Medline]
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) and the presence
() of 50 µM AD-5075. After a 60-min
preincubation period, 0.1 ng/ml LPS (A) or 30 ng/ml PMA
(B) was added. Cell medium was harvested after 4-h (LPS)
or overnight (PMA) incubation at 37°C, and cytokine levels were
determined by ELISA. The results are shown are the mean of each
condition performed in duplicate with SDs.