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12,1412,14 Prostaglandin J21 ,2
*
Cellular and Molecular Immunology Section, Laboratory of Experimental Immunology, and
Laboratory of Molecular Immunoregulation, Division of Basic Science, National Cancer Institute-Frederick Cancer Research Development Center, National Institute of Health, Frederick, MD 21702; and
Department of Molecular Oncology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(PPAR), such as 15-deoxy-
12,14PGJ2
(15d-PGJ2) have been proposed as a new class of
antiinflammatory compounds with possible clinical applications. As
there is some controversy over the inhibitory effects of
15d-PGJ2 on chemokine gene expression, we investigated
whether 15d-PGJ2 itself affected chemokine gene expression
in human monocytes/macrophages and two monocytic cell lines. Here we
demonstrate that the 15d-PGJ2 can induce IL-8 gene
expression. In contrast, monocyte chemoattractant protein-1 gene
expression was suppressed by 15d-PGJ2, while the expression
of RANTES was unaltered. Furthermore, concomitant treatment of
monocytes/macrophages with 15d-PGJ2 (2.5 x
10-6 M) potentiated LPS-induced gene expression of IL-8
mRNA, but suppressed PMA-induction of IL-8 mRNA. In addition, treatment
of U937 and THP-1 cells with 15d-PGJ2 also resulted in
induction of IL-8 gene expression. Further studies demonstrated that
15d-PGJ2 regulated IL-8 gene expression via a
ligand-specific and PPAR-dependent pathway. Our observations
revealed a previous unappreciated function and mechanism of
15d-PGJ2-mediated regulation of cytokine gene expression in
monocytes/macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pharmacological intervention can be applied to the regulation of
cytokine production. Recently, much attention has been focused on the
role of 15-deoxy-12,14
PGJ2
(15d-PGJ2)4
in the regulation of the inflammatory process (4, 5, 6, 7, 8, 9, 10, 11, 12, 13).
15d-PGJ2 is one of the derivatives of the
PGD2 metabolism pathway and is a natural ligand
for peroxisome proliferator-activated receptor (PPAR)
(14, 15, 16, 17). It has been documented that the fluctuation of
15d-PGJ2 is associated with inflammatory process
(4, 5), suggesting it may play an important role in the
regulation of inflammatory reaction in vivo.
Several studies with monocytes/macrophages demonstrated that
15d-PGJ2 could inhibit the expression of genes
coding for IL-1
, TNF-
, cyclooxygenase-2, NO synthase-2 and matrix
metalloproteinases (7, 8). These observations raise the
possibility that 15d-PGJ2 may be a potential
therapeutic compound for treatment of inflammatory diseases. However,
other studies have failed to observe an inhibitory effect of
15d-PGJ2 on induced expression of TNF- and
IL-6 in freshly prepared human monocytes/macrophages (13).
Thus, whether 15d-PGJ2 will be of therapeutic
value as an antiinflammatory agent remains controversial.
Because chemokines are mediators of numerous inflammatory and
immunological responses, we investigated the capacity of
15d-PGJ2 to modulate chemokine gene expression
and protein production in monocytes/macrophages. Here we show that in
human peripheral blood monocytes/macrophages,
15d-PGJ2 by itself can increase the level of IL-8
mRNA in association with an increased production of IL-8 protein,
whereas it reduces the level of monocyte chemoattractant protein-1
(MCP-1) mRNA and its protein production without any significant effect
on the levels of RANTES mRNA and protein. Furthermore, low
concentrations of 15d-PGJ2 had an additive effect
on LPS-induced IL-8 expression, while 15d-PGJ2
decreased the production of IL-8 in cells treated with PMA. Further
studies demonstrated that 15d-PGJ2 regulated IL-8
gene expression via a ligand-specific and PPAR-dependent
pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nonadherent human monocytes were isolated from PBMC of healthy
donors, after depletion of the adherent population by sequential
incubation at 37°C for 1 h on plastic flasks and nylon wool
columns. The nonadherent PBMC was fractionated by a seven-step Percoll
gradient as previously described (18), and the
enriched monocyte populations (containing 65–85% of
CD14+ cells) were obtained at the very top of the
gradient (top fraction). The cells were collected and washed twice with
ice-cold PBS. After cells were suspended in RPMI 1640 medium
containing 10% (v/v) charcoal/dextran-treated FBS (HyClone, Logan,
UT), 10 mmol/L glutamine, 100 IU/ml penicillin, and 100 µg/ml
streptomycin (Life Technologies, Gaithersburg, MD) and diluted to
5 x 106 cells/ml, 3 ml was transferred to
each well of a six-well tissue-culture plate and cultured in an
atmosphere of 5% CO2 at 37°C for additional
experiments. Experiments were initiated on the same day when cells were
isolated. U937 and THP-1 cells were obtained from American Type Culture
Collection (Manassas, VA). PG derivatives
15d-PGA1, 15d-PGA2, and
15d-PGD2 were purchased from Cayman Chemical (Ann
Arbor, MI). 15d-PGJ2 was obtained from Biomol
(Plymouth Meeting, PA) and Cayman Chemical (Ann Arbor, MI). PPAR
ligand WY-14643 was purchased from Biomol. PMA was obtained from
Calbiochem (La Jolla, CA). LPS was purchased from Sigma (St. Louis,
MO). Absolute ethanol or methyl ester was used as drug vehicles.
Total RNA isolation and multiple-probe RNase protection assay (RPA)
Total RNA was extracted from control (drug-vehicle treated) and treated cells using a single-step phenol/chloroform extraction procedure (TRIzol; Life Technologies). The abundance of RNA was quantitated spectrophotometrically. A total of 5 µg of RNA from each group were used in the assays. Multicytokine templates (BD PharMingen, San Diego, CA) were used to generate 33P-labeled riboprobes. The procedure of RPA including the labeling of probe, hybridization, RNase digestion, and denaturing polyacrylamide gel electrophoresis was performed as previously described (19).
ELISA
Immunoreactive cytokines were assayed in cell culture supernatants bya double-Ab ELISA kit using recombinant cytokines as standards(R&D Systems, Minneapolis, MN) following the instructions from the manufacturer.
Chemotaxis assays
Migration of human neutrophils was assessed using a 48-well microchemotaxis chamber technique. Stimulants were placed in wells of the lower compartment of the chamber (NeuroProbe, Cabin John, MD). The cell suspension was seeded into wells of the upper compartment, which was separated from the lower compartment by a polycarbonate filter (Osmonics, Livermore, CA; 5-µm pore size). After incubation at 37°C for 60 min, the filter was removed, stained, and the number of cells migrating across the filter was counted by light microscopy after coding the samples. The results are presented as the number of migrated cells in one high power field. The significance of the difference in migration in response to stimulants vs medium control was analyzed by Student’s t test.
Transient transfection and IL-8 reporter gene luciferase assay
A 1525-bp fragment containing nucleotides –1481 to +44, a
590-bp fragment containing –546 to +44, and a 177-bp fragment
containing –133 to +44 of the promoter region of the IL-8 gene were
respectively subcloned into the pGL3-basic luciferase expression vector
(Promega, Madison, WI) between KpnI and HindIII
restriction sites. The sequences were confirmed by DNA sequencing.
Expression vectors for PPAR and chicken OVA upstream
promoter-transcription factor-II (COUP-TFII) as well as a
pGL3-thymidine kinase (TK) construct containing PPAR response element
(PPRE) were described previously (9, 20). U937
cells were transiently transfected by Fugen 6 following the
manufacturer’s recommendations (Promega). pRL-null construct (Promega)
was used as an internal control for normalization of transfection
efficiency. Cell lysis and luciferase assays were performed using the
dual luciferase assay system from Promega following the instructions of
the manufacturer. All transfection experiments were performed at least
in triplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been previously reported that freshly isolated human
peripheral blood monocytes contained a detectable amount of PPAR
mRNA (7, 8). The prostanoid 15d-PGJ2
is a natural ligand for PPAR that has displayed PPAR agonist
activity at micromolar concentrations (14). To examine the
capacity of 15d-PGJ2 to modulate chemokine
expression in monocytes, we incubated human peripheral blood monocytes
with increasing concentrations of 15d-PGJ2. Total
RNA was extracted from treated cells, and cytokine mRNA expression was
determined by RPA. As shown in Fig. 1
, 15d-PGJ2 significantly induced the mRNA level of
IL-8, down-regulated MCP-1 mRNA, and did not affect RANTES mRNA. By
semiquantitative analyses using phosphorimaging (Fig. 1B),
the optimal concentration of the compound for induction of IL-8 mRNA
was 5–10 µM. Notably, the dose-response curve of IL-8 production was
bell-shaped, i.e., the expression of IL-8 mRNA was reduced when higher
concentrations of the compound (>10 µM) were used. The compound also
down-regulated the mRNA levels of macrophage inflammatory proteins 1
and 1 (data not shown).
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A, treatment
of freshly isolated human monocytes with 15d-PGJ2
increased the production of IL-8 in a dosage-dependent manner with
optimal concentrations at 5–10 µM. The induction of IL-8 could be
detected as early as 4 h after incubation (data not shown), and
further production of IL-8 was observed at 16 h. In contrast,
15d-PGJ2 significantly inhibited the production
of MCP-1 (Fig. 2B), while it had no effect on the production
of RANTES (data not shown).
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Differential effect of 15d-PGJ2 on LPS- and PMA-induced IL-8 production
It has been previously reported that PPAR agonists could
inhibit production of inflammatory cytokines such as TNF-, IL-1,
and IL-6 by PMA- but not LPS-activated human monocytes/macrophages
(7). To evaluate the effect of
15d-PGJ2 on IL-8 production by PMA- or
LPS-activated human monocytes/macrophages, we incubated monocytes with
PMA or LPS in the presence of increasing concentrations of
15d-PGJ2. The cell culture medium was collected
for ELISA while total RNA was prepared for RPA analysis. As shown in
Fig. 3, IL-8 production from the cells
stimulated with PMA alone was augmented by 10-fold. The prostanoid
15d-PGJ2 at low concentrations had no effect on
PMA-induced IL-8 production, whereas at high concentrations it almost
completely inhibited the production of IL-8 induced by PMA.
Similarly, after stimulation with LPS for 16 h, the
production of IL-8 by monocytes was increased by 4-fold. In contrast to
its effect on PMA-induced IL-8 production,
15d-PGJ2 at low concentrations (2.5 µM) had an
additive effect on LPS-induced IL-8 production (up to
10-fold), which was not observed at high concentrations such as 5 and
10 µM.
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, the compound alone induced the
steady-state level of IL-8 mRNA in a dosage-dependent manner. However,
it had an additive effect at a lower concentration on LPS-induced
increase in IL-8 mRNA, while higher concentrations of the compound had
no additive effect on LPS-induced IL-8 mRNA levels (Fig. 4B). The compound displayed inhibitory effects on
PMA-induced IL-8 mRNA level that was well-correlated with the change in
IL-8 protein production (data not shown). Thus, the regulatory effects
of 15d-PGJ2 on IL-8 production induced by either
PMA or LPS are not only at the level of protein production but also at
the steady-state mRNA level.
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To investigate whether the induced IL-8 in the supernatants of
15d-PGJ2-treated monocytes is biologically
active, we tested the capacity of the supernatants to induce in vitro
migration of human neutrophils, a function thought to be relevant to
neutrophil recruitment. As shown in Fig. 5, significant neutrophil chemotactic
activity could be measured in the supernatants from monocytes treated
with 1–10 µM of 15d-PGJ2 (Fig. 5B).
The optimal migration was observed in the supernatants from monocytes
treated with 2 and 5 µM of 15d-PGJ2, which
corresponded to 10 ng/ml IL-8 protein as measured by ELISA (Fig. 5A). The activity of IL-8 contained in the monocytes
supernatant was comparable to that of recombinant human IL-8 at
corresponding concentrations (Fig. 5C). Because
15d-PGJ2 by itself is not chemotactic for
neutrophils at any concentrations tested and the neutrophil chemotactic
activity in supernatants of 15d-PGJ2-treated
monocytes was completely neutralized by an anti-IL-8 polycolonal Ab
(data not shown), we conclude that 15d-PGJ2
induced the production of biologically active IL-8 protein.
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U937 and THP-1 are two human myeloid cell lines that are widely
used as in vitro models for studies related to monocytes. To determine
whether these two cell lines are responsive to
15d-PGJ2, we extracted total RNA from cells
treated with different concentration of 15d-PGJ2
for 24 h and performed RPA analyses. As shown in Fig. 6, IL-8 mRNA levels of both cell lines
were elevated in response to the compound. However, a much higher
concentration of 15d-PGJ2 was needed for
induction of IL-8 mRNA in THP-1 cells than in U937 cells. In U937
cells, induction of IL-8 mRNA initiated at 5 µM of
15d-PGJ2 (Fig. 6A), while in THP-1
cells elevation of IL-8 mRNA was observed at 20 µM (Fig. 6B). There was no significant change for RANTES mRNA in
these two cell lines upon treatment of 15d-PGJ2.
The lower concentrations of 15d-PGJ2 needed for
activation of IL-8 gene expression in U937 cells may be due to the
presence of endogenous PPAR.
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The PPAR family contains three isoforms (, -
, and )
that differ in their ligand binding domains (14). It has
been reported that the activation of PPAR by its specific ligand
resulted in the inhibition of IL-1-induced cyclooxygenase-2 expression
in smooth muscle cells (21). Therefore, we examined
whether ligands specific for other PPARs, such as PPAR, could also
influence the level of IL-8 mRNA. THP-1 cells were treated with
increasing concentrations of WY-14643, a PPAR ligand, for 24 h.
Total RNA was extracted, and IL-8 mRNA levels were determined by RPA.
Meanwhile, the supernatant was collected for ELISA to determine IL-8
protein levels. As shown in Fig. 6
, B–D, we did not observe
changes in IL-8 mRNA and protein levels in WY-14643-treated cells,
whereas both IL-8 mRNA and protein levels were elevated in response to
15d-PGJ2. Likewise, WY-14643 also has no effect
on IL-8 protein level in human monocytes. Thus, the induction of IL-8
mRNA and protein in monocytes is specific for
15d-PGJ2, the PPAR ligand.
Other analogs of 15d-PGJ2 have no significant effect on IL-8 protein production
To further determine whether 15d-PGJ2
specifically affects IL-8 production, we tested how other analogs of
the compound affect IL-8 production. All three compounds selected for
assay, 15d-PGA1, 15d-PGA2,
and 15d-PGD2, share common structural features
with 15d-PGJ2. As shown in Fig. 7, induction of IL-8 protein was only
observed when monocytes were treated with
15d-PGJ2. We did not observe any induction effect
of other analogs at the same dose as 15d-PGJ2 on
IL-8 protein production from monocytes.
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To examine the effect of 15d-PGJ2 on IL-8
gene promoter activity, we performed transient transfection analysis
with IL-8 promoter constructs. Because it is very difficult to
transfect primary cells, we used U937 cell line as the target cells for
transfection. Previous studies demonstrated that the regions of
nucleotides –133 bp relative to the initiation site of IL-8 gene
transcription are essential for regulation of IL-8 gene promoter
activity (31). This region contains an AP-1 site, an
NF-
B site, a STAT site, and an NF-IL-6 site. We performed transient
cotransfection analysis using luciferase constructs containing
nucleotides in the –133-bp, –546-bp, and –1481-bp region of the IL-8
gene promoter, respectively, with a PPAR expression vector. As shown
in Fig. 8, the promoter activity of
–133-bp and –546-bp nucleotide constructs did not respond to
stimulation with 15d-PGJ2 regardless of PPAR
expression, while the –1481-bp construct cotransfected with PPAR
expression vector responded to the treatment with
15d-PGJ2 in a dose-dependent manner. As a
positive control, the PPRE-containing construct transfected with the
PPAR expression vector displayed significant reporter activity in
response to treatment with 15d-PGJ2. The
observation suggests that the region between –1481 bp and –546 bp of
the IL-8 promoter contain element(s) that respond to
15d-PGJ2 treatment. Furthermore, the AP-1,
NF-B, STAT, and NF-IL-6 sites in the region of previous identified
IL-8 promoter (nucleotides –133 bp upstream from initial site of IL-8
gene) may not be sufficient for 15d-PGJ2 to
enhance IL-8 gene expression.
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in IL-8 promoter activity induced by
15d-PGJ2
Because 15d-PGJ2 is a naturally occurring
ligand for PPAR, we tested whether blockade of PPAR will affect
the promoter activity of the IL-8 gene upon stimulation with the
compound. COUP-TFII is another member of the nuclear hormone receptor
superfamily that can form a heterodimer with retinoid X receptor
(RXR) (20). RXRs are essential partners for PPAR
and form heterodimers that interact with a cis-element on
the promoter of the target gene. Thus, overexpression of COUP-TFII will
deplete endogenous RXRs, leading to the dysfunction of PPAR. As
shown in Fig. 9, the reporter construct
containing the –1481-bp region of the IL-8 promoter cotransfected with
the PPAR expression vector displayed higher promoter activity under
stimulation with 15d-PGJ2. However,
overexpression of COUP-TFII by cotransfection resulted in diminished
15d-PGJ2-induced-IL-8 promoter activity. As a
negative control, we did not observe any significant change of the
promoter activity of the –546-bp IL-8 gene construct either in the
presence of the PPAR expression vector or upon overexpression of
COUP-TFII. Thus, our data further demonstrate that PPAR plays an
important role in the 15d-PGJ2-induced IL-8
promoter activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, IL-6, and gelatinase
B/matrix metalloproteinase-9 (7, 8, 12). However, other
studies showed that activation of PPAR did not inhibit the
production of proinflammatory cytokines induced by LPS stimulation
(13). Therefore, whether 15d-PGJ2
and its synthetic analogs can be safely used as antiinflammatory agents
remains elusive. Our current study attempts to clarify the role of
15d-PGJ2 on gene expression of proinflammatory
cytokines in cells from human monocytes/macrophage lineage.
Our data demonstrated that 15d-PGJ2 could
selectively regulate expression of different chemokines genes:
induction of IL-8 gene expression, but inhibition of MCP-1. Our
experiments were conducted using freshly prepared nonadherent human
monocytes. In addition, we obtained identical results using freshly
prepared adherent human monocytes (data not shown). A previous study in
a colon epithelial cell line (Caco cells) demonstrated that
15d-PGJ2 reduced IL-1-induced IL-8 gene
expression possibly through inhibition of NF-B activation by a
PPAR-dependent pathway (10). Notably,
15d-PGJ2 at low concentrations had no effect on
IL-8 gene expression in a human monocytic cell line (THP-1) as reported
by a previous study (12) and our current study (Fig. 5),
while it induces IL-8 gene expression at higher concentrations (Fig. 5). Furthermore, we demonstrated in the current study that
15d-PGJ2 at pharmacological concentrations could
induce IL-8 gene expression in cells from the monocytes/macrophage
lineage, including freshly prepared human monocytes/macrophages and a
human myeloid cell line (U937). Monocytes/macrophages can be induced to
produce proinflammatory cytokines by numerous agents including LPS and
PMA. As reported in a recent study, 15d-PGJ2 was
able to block PMA-induced TNF- synthesis whereas LPS-induced TNF-
production was unaffected by the agent (8). We also found
that PMA-induced IL-8 synthesis was subjected to the inhibitory effect
of 15d-PGJ2. LPS-induced IL-8 gene expression was
not affected by high concentrations of 15d-PGJ2,
which was in agreement with the results from LPS-induced TNF-
production. However, we unexpectedly found that lower concentrations of
15d-PGJ2 additively increased LPS-induced IL-8
gene expression. It has been reported that
15d-PGJ2 also reduced gene expression of MCP-1 in
Caco cells (10). We similarly observed that this compound
reduced MCP-1 gene expression in human monocytes/macrophages. In
contrast, 15d-PGJ2 had no effect on MCP-1 gene
expression in human saphenous vein endothelial cells (12).
Thus, our data demonstrate that 15d-PGJ2 itself
has a differential effect on chemokine gene expression in human cells
and illustrated the complexity of regulation of chemokine gene
expression by 15d-PGJ2.
Induction of IL-8 gene expression in human monocytes appears to be
specific for 15d-PGJ2, the ligand for PPAR.
The analogs with a similar structure such as
15d-PGA1, 15d-PGA2, and
15d-PGD2 did not have any inducing effect on IL-8
gene expression in human monocytes. Although these compounds have same
antimitotic and antitumor activities as 15d-PGJ2,
whether they can function through a PPAR-dependent pathway still
remains unknown (35). A recent study demonstrated that
activation of PPAR with the synthetic ligand WY-14643 stimulates the
synthesis of IL-8 and MCP-1 by human aortic endothelial cells
(22). In our current study of monocytes/macrophages
models, we did not observe any significant induction effect of WY-14643
on IL-8 gene expression. Thus, it is possible that the effect of
15d-PGJ2 on gene expression of IL-8 in
monocytes/macrophages at least functions in part via a
15d-PGJ2-specific PPAR-dependent pathway at
the transcriptional level.
The precise molecular mechanisms by which
15d-PGJ2 differentially regulates IL-8 and MCP-1
in human monocytes/macrophages remain to be determined. The balance of
multiple intracellular factors functioning in various cell types may
determine how a specific cell functions in response to a variety of
stimuli. PPAR belongs to the nuclear hormone receptor superfamily
that is composed of ligand-dependent transcription factors
(14). 15d-PGJ2 is the naturally
occurring ligand for PPAR to function on the promoter of target
genes (14, 16). Transient transfection experiments using
pGL3-derived luciferase reporter constructs indicate that the
PPAR-dependent pathway is one of the key players needed to exert the
effect of 15d-PGJ2 on the induction of IL-8 gene
expression. Reporter constructs containing –1481 to +44 bp upstream
from the transcription start site (+1) of the IL-8 gene showed a
significant 5- to 8-fold increase in luciferase activity in the
presence of PPAR and 15d-PGJ2. However, a
reporter construct containing –546 bp of the IL-8 gene promoter was
not activated in the presence of PPAR and
15d-PGJ2. Thus, the essential element(s) required
for 15d-PGJ2 activity on the promoter of the IL-8
gene are located between nucleotides –1481 bp and –546 bp. Further
studies will be needed to identify the cis-elements and
trans-factors responsible for the effect of
15d-PGJ2. Cotransfection with COUP-TFII further
demonstrated the involvement of PPAR in the
15d-PGJ2-mediated induction of IL-8 promoter
activity. COUP-TFII is an orphan member of the nuclear hormone receptor
superfamily. Previous study demonstrated that COUP-TFII did not
activate transcription of PPRE-linked reporter genes in mammalian cells
but rather strongly inhibited induction mediated by PPAR
(20). In the current study,
15d-PGJ2-induced IL-8 promoter activity was
blocked by overexpression of COUP-TFII. Possibly, COUP-TFII competes
with PPAR to form a heterodimer with RXR, an essential partner for
PPAR function. It is also possible that the heterodimer of
COUP-TFII/RXR may compete with binding of PPAR/RXR to a
cis-element in the promoter. While our data demonstrates
that PPAR plays a very important role in the
15d-PGJ2-mediated regulation of IL-8 gene
expression, we cannot rule out the possibility that a
PPAR-independent pathway at transcriptional/posttranscriptional
levels may also play a role in the
15d-PGJ2-mediated regulation of IL-8 gene
expression (23, 24, 25, 26, 27, 28, 29, 30).
Previous studies indicate that the stimulus-specific and cell
type-specific expression of IL-8 can be mediated by the differential
activation and binding of inducible transcription factors to the IL-8
promoter (31). Subsequent studies based on promoter
mutations demonstrated that the NF-B binding site is the predominant
cis-acting element involved in IL-8 gene expression and is
located in the region of nucleotides –133 bp upstream to the
initiation site of IL-8 gene transcription. This site was critical for
stimulus-mediated IL-8 gene expression via cooperation with other
essential transcription factors for optimal activation in several cell
types (31). This IL-8 promoter region also contains other
cis-elements important for induction of IL-8 gene
expression: AP-1 and NF-IL-6 binding sites. In the current study, a
reporter construct containing nucleotides –133 to +44 bp upstream from
the transcription start site (+1) of the IL-8 gene did not show any
significant changes in luciferase activity under treatment of
15d-PGJ2 regardless of the presence of PPAR.
Thus, our data indicate that PPAR and its natural ligand at
pharmacological concentrations may not affect the basal promoter
activity of the IL-8 proximal promoter in which NF-B plays a
dominant role in regulating gene expression. Furthermore, these results
also ruled out the possible contamination of LPS in the
15d-PGJ2 preparation because NF-B binding in this
region was an indispensable cis-element for conferring the
responsiveness to LPS (32).
Cytokines produced by monocytes/macrophages play pivotal roles in the
development of inflammatory processes. The CXC chemokine IL-8 is the
most potent chemotactic cytokine to attract and activate neutropils
(2, 3, 29), while CC chemokine MCP-1 is one of the most
potent chemotactic cytokines for monocytes (2). Excess
amounts of locally produced chemokines have been shown to have harmful
effects resulting in a number of inflammatory diseases. Acute severe
asthma is characterized by the prominent presence of neutrophils in the
inflammatory airway as a consequence of overproduction of IL-8
(33). IL-8 generated from monocytes/macrophages may also
contribute to the inflammatory process in the early synovitis of
rheumatoid arthritis (34). Because
15d-PGJ2 itself can induce gene expression and
protein production of biologically active IL-8, one should be cautious
about the use of the compound and its synthetic analogs for
antiinflammation therapy. In conclusion, our data provide evidence that
15d-PGJ2, the natural ligand for PPAR,
differentially regulates chemokine genes in monocytes/macrophages,
i.e., up-regulation of IL-8 gene expression and down-regulation of
MCP-1 gene expression in monocytes/macrophages. Moreover, it enhances
IL-8 gene expression in a highly specific manner in which a
PPAR-dependent pathway plays an important role. These results reveal
a previous unappreciated function and mechanism of
15d-PGJ2-mediated regulation of cytokine gene
expression in monocytes/macrophages.
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Acknowledgments |
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Footnotes |
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2 Animal care was provided in accordance with the procedures outlined in the "A Guide for the Care and Use of Laboratory Animals" (National Institutes of Health Publication No. 86-23, 1985).
3 Address correspondence and reprint requests to Dr. Howard A. Young, National Cancer Institute-Frederick Cancer Research Development Center, Building 560, Room 31-93, Frederick, MD 21702-1201. E-mail address: youngh{at}mail.ncifcrf.gov
4 Abbreviations used in this paper: 15d-PGJ2, 15-deoxy-12,14 PGJ2; MCP-1, monocyte chemoattractant protein-1; PPAR, peroxisome proliferator-activated receptor; RPA, RNase protection assay; PPRE, PPAR response element; COUP-TFII, chicken OVA upstream promoter-transcription factor-II; RXR, retinoid X receptor.
Received for publication December 13, 2000. Accepted for publication April 2, 2001.
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, IL-8; 
, drug vehicle; 
, MCP-1; •,
RANTES. Quantitative analyses were conducted using phosphorimaging
system (Molecular Dynamics, Sunnyvale, CA). Nonlinear regression and
linear regression analyses were performed for the best fit.
