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12,1412,14-PGJ2 Induces IL-8 Production in Human T Cells by a Mitogen-Activated Protein Kinase Pathway1
Cancer Center, University of Rochester, Rochester, NY 14642
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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12,14-PGJ2 (15-d-PGJ2).
The lipid mediator 15-d-PGJ2 regulates numerous processes,
including adipogenesis, apoptosis, and inflammation. The
15-d-PGJ2 has been shown to both inhibit as well as induce
the production of inflammatory mediators such as TNF-
, IL-1
, and
cyclooxygenase, mostly occurring via a nuclear receptor called
peroxisome proliferator-activated receptor-
(PPAR-). Data
concerning the effects of 15-d-PGJ2 on human T cells and
immune regulation are sparse. IL-8, a cytokine with both chemotactic
and angiogenic effects, is produced by T lymphocytes following
activation. Whether 15-d-PGJ2 can regulate the production
of IL-8 in T cells in unknown. Interestingly, 15-d-PGJ2
treatment of unstimulated T cells induces cell death. In contrast, in
activated human T lymphocytes, 15-d-PGJ2 does not kill
them, but induces the synthesis of IL-8. In this study, we report that
15-d-PGJ2 induced a significant increase in both IL-8 mRNA
and protein from activated human T lymphocytes. The induction of IL-8
by 15-d-PGJ2 did not occur through the nuclear receptor
PPAR-, as synthetic PPAR- agonists did not mimic the
IL-8-inducing effects of 15-d-PGJ2. The mechanism of IL-8
induction was through a mitogen-activated protein kinase and NF-
B
pathway, as inhibitors of both systems abrogated IL-8 protein
induction. Therefore, 15-d-PGJ2 can act as a potent
proinflammatory mediator in activated T cells by inducing the
production of IL-8. These findings show the complexity with which
15-d-PGJ2 regulates T cells by possessing both pro- and
anti-inflammatory properties depending on the activation state of
the cell. The implications of this research also include that caution
is warranted in assigning a solely anti-inflammatory role for
15-d-PGJ2. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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12,14-PGJ2 (herein referred to as
15-d-PGJ2)3 is a
potent lipid mediator derived from PGD2 in vivo by
dehydration. PGD2, and thus
15-d-PGJ2, is abundantly produced by mast cells,
platelets, and alveolar macrophages, and has been proposed as a key
immunoregulatory lipid mediator (1). Traditionally,
15-d-PGJ2 was thought to exert its effects on
cells exclusively through the peroxisome proliferator-activated
receptor- (PPAR-) (2, 3). PPARs are a family of ligand-activated nuclear transcription factors, and after ligand binding PPARs form a heterodimer with the retinoic X receptor, and the complex then binds to PPAR-responsive elements in the promoter regions of target genes (>2, 4, 5, 6). To date, PPAR- has been found in adipose tissue, where
it plays a key role in the regulation of adipogenesis (7, 8). Interestingly, a variety of immune cells, including T and B
cells as well as macrophages and dendritic cells, were found to express
PPAR- and are regulated via PPAR--dependent mechanisms
(9, 10, 11, 12). More recently, however, evidence has shown that
there are also effects of 15-d-PGJ2 that are
independent of PPAR- activation. For example, studies with neuronal
cells have shown that 15-d-PGJ2 can promote
neurite outgrowth in PC12 cells (13) and down-regulate
inducible NO synthase in microglial cells (14) through
PPAR--independent mechanisms. Vaidya et al. (15) showed
that 15-d-PGJ2 can inhibit the production of
oxygen-free radicals from neutrophils, also in a non-PPAR--dependent
manner.
The 15-d-PGJ2 seems to possess both
anti-inflammatory and proinflammatory functions. For example,
15-d-PGJ2 has been shown to inhibit inducible NO
synthase, TNF-, and IL-1 production from mouse and human
macrophages (11, 16), suggesting a role in the inhibition
of inflammation. The 15-d-PGJ2 can induce
apoptosis in B lineage cells, supporting a role in clonal deletion and
down-regulation of immunity (10). There is also evidence,
however, that 15-d-PGJ2 can promote inflammation.
The 15-d-PGJ2 can induce the proinflammatory
mediators type II secreted phospholipase A (2) and
cyclooxygenase 2 in smooth muscle and epithelial cells, respectively
(17, 18). Therefore, the role of
15-d-PGJ2 in the regulation of inflammation is
complex and remains under intense investigation.
The proinflammatory cytokine IL-8 is a member of the C-X-C family of chemokines, and induces migration in cells such as polymorphonuclear cells, T cells, basophils, and endothelial cells (19, 20). In addition to its properties as a chemotactic agent, IL-8 can also activate neutrophils and monocytes, regulate histamine production from basophils, and induce angiogenesis in endothelial cells (21). In humans, IL-8 is primarily expressed during the inflammatory response to bacterial and viral infections. Therefore, IL-8 production is implicated in a number of human diseases, including cystic fibrosis (22, 23), HIV (24), and pulmonary fibrosis (25).
The production of IL-8 in lymphocytes, namely T cells, is crucial for
the ability of the immune system to fight infection. Although recent
work by Zhang et al. (26) has shown that
15-d-PGJ2 can induce IL-8 production in human
monocytic cells, no other literature describes the effects of
15-d-PGJ2 on IL-8 production. In fact, aside from
recent studies showing that 15-d-PGJ2 can induce
cell death and apoptosis in mouse T cells and inhibit certain aspects
of human T cell activation, there is no information of the roles of
this molecule on T cell cytokine production or function (9, 27, 28). Therefore, we have investigated the effects of
15-d-PGJ2 on IL-8 production in human T cells.
Interestingly, in stimulated human T cells,
15-d-PGJ2 induces a significant increase in the
amount of IL-8 produced. Although many of the effects of
15-d-PGJ2 can occur through the PPAR- nuclear
receptor, we find that the 15-d-PGJ2-induced IL-8
production is PPAR- independent. We also demonstrate that the
mechanism of IL-8 induction in T cells is via a mitogen-activated
protein (MAP) kinase and NF-B pathway. Therefore, in activated human
T cells, 15-d-PGJ2 acts as a proinflammatory
mediator by inducing the production of the chemokine IL-8.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MTT, PGF2, PGE2,
PGI2, and DMSO were purchased from Sigma-Aldrich
(St. Louis, MO). The 15-d-PGJ2, ciglitazone,
SN50, and SN50 M were purchased from BioMol (Plymouth Meeting, MA).
Troglitazone was kindly provided by Parke-Davis (Morris Plains, NJ).
TriReagent was purchased from Molecular Research Center (Cincinnati,
OH). Anti-human CD3 and anti-human CD28 mAbs were purchased from BD
PharMingen (San Diego, CA). PD98059 was purchased from New England
Biolabs (Beverly, MA).
Cells and culture conditions
Jurkat (acute human T cell leukemia) and CCRF-CEM (acute human
lymphoblastic leukemia) were purchased from the American Type Culture
Collection (Manassas, VA). J-Jahn (human T cell lymphoma) cells were
kindly provided by Dr. S. Dewhurst (University of Rochester). All three
cell lines are long-term established tumoral cell lines. Human buffy
coats were purchased from the American Red Cross. Human PBMCs were
isolated using Ficoll according to the manufacturer’s instructions.
Human CD4-positive cells were positively selected with CD4 Dynabeads
and Detachabeads (Dynal, Great Neck, NY), according to the
manufacturer’s instructions. Cell purity was assessed by flow
cytometry with FITC-labeled anti-human CD4 Ab (BD PharMingen).
Cells were >98% CD4 positive. For each experiment, three different T
cell donors were used. T cells were maintained in RPMI 1640 tissue
culture medium (Life Technologies, Grand Island, NY) supplemented with
10% FBS (Life Technologies). T cells were plated into 96-well plates
at a density of 1 x 106 cells/well with 200
µl final volume for 24 h. At the initiation of cultures, cells
were treated with various concentrations of PGs. T cells were
additionally cultured with anti-CD3 (1 µg/ml) and
anti-CD28 (250 ng/ml). In some cases, PD98059 was added at a
concentration of 50 µM. This concentration was determined using
manufacturer’s instructions and titration studies and does not inhibit
cell viability (data not shown). Also, some cells were treated with
SN50 or SN50M at a concentration of 37 µg/ml. This concentration was
found to specifically inhibit NF-B activity in T lymphocytes,
without inhibiting cell viability (data not shown). In cells treated
with PGF2, 100 µM PG was added. This
concentration is reported to induce MAP kinase activity in many systems
(29, 30, 31, 32, 33). IL-8 production was measured by an IL-8 ELISA,
with Ab pairs from BD PharMingen. Cell viability was measured by MTT.
MTT was added for the final 4 h of culture. After incubation, 200
µl of DMSO was added to each well to dissolve the formazan product.
Absorbance was read at 510 nm using a Titretek Multiskan ELISA plate
reader (Flow Laboratories, McLean, VA).
RNA isolation and RT-PCR
RNA was isolated with TriReagent according to the
manufacturer’s instructions. A total of 2 µg of RNA was added to 1
µg of oligo(dT) (Pharmacia, Piscataway, NJ) in diethyl
pyrocarbonate-treated water and incubated at 60°C for 5 min and 4°C
for 3 min. A total of 1 mM each of the four deoxynucleic acids (dNTPs;
Pharmacia), 8 µl of 5x cDNA synthesis buffer (Life Technologies),
and 400 U of Maloney murine leukemia virus reverse transcriptase (Life
Technologies) were added, and the reactions were incubated at 37°C
for 1 h, 95°C for 5 min, and stored at 4°C. For each cDNA
synthesis reaction, a reaction was performed without reverse
transcriptase and used as a negative control in the PCR. An aliquot of
cDNA synthesis reaction was added to 5 µl of 10x PCR buffer
(Boehringer Mannheim Biochemica, Indianapolis, IN), 1 mM dNTPs,
oligonucleotide primers specific for human IL-8
(5'-ATGACTTCCAAGCTGGCCGTGGCT and 3'-TCTCAGCCCTCTTCAAAAACTTCTC)
or the housekeeping gene -actin (5'-GTGGGGCGCCCCAGGCACCA and
3'-CTCCTTAATGTCACGCACGATTTC) at a concentration of 1 µM, 2.5 U of
Taq DNA polymerase (Boehringer Mannheim), and water to a
final volume of 50 µl. PCR samples were run for 35 cycles (94°C for
50 s, 60°C for 50 s, 72°C for 90 s) with a final
extension at 72°C for 7 min in a DNA thermal cycler. Samples were
analyzed by gel electrophoresis on 1% agarose gels and stained with
ethidium bromide. The IL-8 PCR product is 289 bp, and the -actin
product is 550 bp. A normal strain of human lung fibroblasts (L828) was
used as a positive control. Densitometry was performed using the Kodak
Digital Sciences 1D program from the Scientific Imaging Systems
Division (New Haven, CT). Relative sum intensity was calculated by
normalizing the sum intensity of IL-8 product to its -actin
control.
MAP kinase Western
T cell lines were cultured in six-well plates at a concentration of 5 x 106 cells/well. Cells were pretreated with 50 µM MAP/extracellular signal-regulated kinase (ERK) kinase inhibitor PD98059 for 2 h. Cells were then treated with anti-CD3 (1 µg/ml) and anti-CD28 (250 ng/ml) with either DMSO (diluent) or 15-d-PGJ2. Cultures were incubated for 1–20 min, and cell lysates were harvested with Nonidet P-40 lysis buffer. Protein was quantitated with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL), according to the manufacturer’s instructions. A total of 10 µg of protein was electrophoresed on 10% denaturing polyacrylamide-stacking gels for 1–2 h. The gels were transferred onto polyvinylidene difluoride membranes overnight at 4°C. Membranes were blocked for 2 h at room temperature with blotto (PBS/0.05% Tween 20/10% milk) and washed with PBS/Tween 20. Primary Ab (New England Biolabs anti-phophorylated ERK1/ERK2 Ab) was added at a 1/2000 dilution in PBS-Tween 20 overnight at 4°C, the membranes were washed with PBS/Tween 20, and goat anti-rabbit IgG-HRP (Caltag Laboratories, Burlingame, CA) was added at 1/2000 in PBS/Tween 20 for 1 h at room temperature. Membranes were washed in PBS/Tween 20 and developed with an ECL kit (Pierce).
NF-B and AP1 EMSAs
Nuclear protein extracts were prepared as previously described
(34). Briefly, cells were washed in cold PBS and then
incubated on ice for 10 min in 400 µl of an ice-cold hypotonic buffer
(10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 10 mM
KCL, 0.5 mM DTT, 0.5% Nonidet P-40, and 0.2 mM PMSF). Lysates were
vortexed for 10 s and then centrifuged for 30 s. Supernatants
were discarded and pellets resuspended in 50 µl of cold hypertonic
buffer (20 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2,
25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF).
Samples were incubated on ice for 20 min, then centrifuged for 2 min at
4°C. Nuclear protein-containing supernatants were removed and
quantified by bicinchoninic acid protein assay (Pierce). A consensus
sequence for the NF-B DNA binding site
(5'-AGTTGAGGGGACTTTCCCAGGC-3') (Promega, Madison, WI) or AP1
binding site (5'-CGCTTGATGAGTCAGCCGGAA) was labeled with
[-32P]ATP using T4 polynucleotide kinase
(Life Technologies). Labeled DNA was purified over a G-25 column
(Bio-Rad, Hercules, CA) to remove unbound nucleotides. Nuclear protein
extracts, at a concentration of 500 ng, were incubated at room
temperature for 20 min with 
50,000 cpm (0.06 pmol) of the labeled
oligonucleotide suspended in binding buffer (10 mM Tris-HCl (pH 7.5),
50 mM NaCl, 4% glycerol, 1 mM MgCl2, 0.5 mM
EDTA, 0.5 mM DTT, and 0.05 mg/ml poly(dI-dC)). Samples were resolved on
a 4% nondenaturing polyacrylamide gel at 100 V and exposed to
film.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 15-d-PGJ2 has been suggested to play
roles in both the initiation of inflammation and the inhibition of
inflammation, depending on the system being studied. To determine what
effect 15-d-PGJ2 has on the production of the
proinflammatory chemokine IL-8 in human T cells, the human T cell
leukemia CCRF-CEM was treated with 15-d-PGJ2.
Fig. 1
shows that untreated CCRF-CEM
cells do not express mRNA for IL-8. When the T cells are stimulated
with anti-CD3 and anti-CD28 to mimic activation signals
received in vivo, there is now detectable IL-8 mRNA produced. This
induction of IL-8 in T cells through TCR stimulation has been shown by
several other groups, and was expected (24, 35). The
addition of 15-d-PGJ2 at either 10 or 25 µM,
however, increased the detectable IL-8 mRNA 2- to 5-fold over
stimulated cells alone. The apparent increase in IL-8 mRNA was observed
as early as 5 h, and continued through 48 h of culture.
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To determine whether 15-d-PGJ2 could enhance
the synthesis of IL-8 protein, malignant T cell lines were cultured
with anti-CD3 and anti-CD28 and varying concentrations of the
lipid mediator. As shown in Fig. 2A, CCRF-CEM T cells produced
a statistically significant increase in the amount of IL-8 when treated
with 15-d-PGJ2. A difference was seen at
concentrations as low as 6 µM. To verify that these results were not
anomalous to CCRF-CEM cells, the human T cell lines J-Jahn and Jurkat
were also evaluated (Fig. 2, B and C). In all
three cell lines, treatment with 15-d-PGJ2
drastically enhanced IL-8 production from stimulated T cells. To
confirm that the production of IL-8 from activated T cells was not
anomalous to T cell lymphomas, freshly purified peripheral blood T
cells from normal human donors were also used. Fig. 3 clearly shows that
15-d-PGJ2 can induce IL-8 production from normal
human T cells that are activated with anti-CD3 and anti-CD28,
much like occurs with transformed T cell lines. Therefore, both normal
T cells and multiple malignant T cell lines are susceptible to
15-d-PGJ2 induction of IL-8. For the remainder of
our studies, we focused on CCRF-CEM T cells, as they are
15-d-PGJ2 sensitive, induce for IL-8, are a pure
line of T cells, and are amenable to molecular analysis.
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-independent mechanism is responsible for IL-8 induction
in T cells
The 15-d-PGJ2 is believed to exert its
effects on cells by activating the PPAR- transcription factor
(7, 36). Recently, a number of reports demonstrate that
PPAR--independent effects of 15-d-PGJ2 can
also occur (13, 14). To evaluate whether the mechanism of
action of 15-d-PGJ2 was through PPAR-, two
PPAR- agonists (ciglitazone and troglitazone) were tested on
CCRF-CEM T cells. Fig. 4A
shows that neither of the potent PPAR- agonists was able to mimic
the effects of 15-d-PGJ2 by increasing IL-8
protein production. This suggests that 15-d-PGJ2
is enhancing IL-8 in activated T cells by a PPAR--independent
mechanism. It was possible that the PPAR- agonists ciglitazone and
troglitazone are not functional in T lymphocytes at the concentrations
used in this assay. To verify that the PPAR- agonists are
functional, MTT cell viability assays were performed. Fig. 5 shows that ciglitazone and troglitazone
drastically inhibit the viability of CCRF-CEM cells in a similar
concentration range as used for IL-8 induction experiments. Therefore,
although the activation through PPAR- can induce killing in CCRF-CEM
cells, the induction of IL-8 is most likely occurring through a
PPAR--independent pathway. Since 15-d-PGJ2 is
not acting through PPAR-, it is possible that it could act through
one of the other known PG cell surface receptors. If this is the case,
other PGs may also induce IL-8 production. To determine whether this is
occurring, PGE2, PGI2, and
PGF2 were tested for their ability to increase
IL-8 in activated human T cells. It is clear from Fig. 4B
that none of the other PGs tested induced IL-8 production. Although
several concentrations were evaluated (1–20 µM), none showed an
increase in IL-8. Therefore, the action of
15-d-PGJ2 on T lymphocytes is specific, and most
likely does not occur via the prostacyclin, PGE2, or
PGF2 receptors.
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agonists do not inhibit the
viability of activated human T cells
Recent reports show that PPAR- agonists can inhibit the
viability of unstimulated lymphocytes, including both mouse and human B
and T cells (9, 10). Fig. 5
shows that when the human T
cell leukemia CCRF-CEM was stimulated with anti-CD3 and
anti-CD28 and 15-d-PGJ2, or the PPAR-
agonists ciglitazone or troglitazone, however, there was no inhibition
of viability as judged by a MTT assay (cells treated with
anti-CD3/anti-CD28 alone also showed no change in viability).
Treatment of freshly isolated normal human T cells from PBMCs also
showed no inhibition of viability when treated with
15-d-PGJ2 after anti-CD3 and anti-CD28
stimulation (data not shown). Therefore, stimulation of human T
lymphocytes protects the cells against
15-d-PGJ2-mediated cell death, while
simultaneously promoting IL-8 production.
A MAP kinase pathway activator enhances 15-d-PGJ2-induced IL-8 production
The mechanism of IL-8 induction is reported to be mediated through
a MAP kinase phosphorylation pathway. Stimulation of T lymphocytes with
anti-CD3 and anti-CD28 activates MAP kinase phosphorylation,
and it is possible that 15-d-PGJ2 is
synergistically enhancing that effect. To determine whether
15-d-PGJ2 would act in the same way in
conjunction with another MAP kinase stimulator, cells were treated with
PGF2 rather than anti-CD3 and
anti-CD28. PGF2 is a PG known to stimulate
the phosphorylation of MAP kinases (37). As Fig. 6 shows, 15-d-PGJ2
not only enhances the production of IL-8 protein from anti-CD3- and
anti-CD28-stimulated CCRF-CEM cells, but also from
PGF2-stimulated cells. It is therefore likely
that the induction of IL-8 production in human T cells is occurring
through a MAP kinase-mediated pathway.
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To verify that 15-d-PGJ2 is enhancing IL-8
production from stimulated human T cells via a MAP kinase pathway, the
MAP/ERK kinase inhibitor PD98059 was added to cultures before
15-d-PGJ2 treatment. Fig. 7A shows that the inhibitor
significantly reduces (by 79–88%) the production of IL-8 from
15-d-PGJ2-treated T cells at the 6 and 12.5 µM
concentrations. As further evidence that the MAP kinase pathway is
involved, CCRF-CEM cells treated with 15-d-PGJ2
were subject to Western blotting with an Ab specific for the active, or
phosphorylated, forms of ERK1 and 2. Treatment of cells with
15-d-PGJ2 increased the amount of the active
kinases, and the inhibitor PD98059 blocked that activation (Fig. 7B). Therefore, 15-d-PGJ2 enhances the
production of IL-8 from human T cells by activating a MAP kinase
pathway.
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B and AP-1 activation in
stimulated T cells
To determine which transcription factors are involved in
15-d-PGJ2-induced IL-8 production in human T
cells, EMSAs for NF-B and AP-1 were performed. Binding sites for
both transcription factors are present in the promoter region of the
IL-8 gene, and both have been implicated in IL-8 production in T
cells, as well as other cell types. Fig. 8 shows that both transcription factors
are induced by 15-d-PGJ2 treatment of stimulated
T cells when compared with vehicle control (DMSO). The induction of
AP-1, however, is much less than that of NF-B. We therefore conclude
that NF-B is the predominant transcription factor induced by
15-d-PGJ2.
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B results in the blockage of IL-8 production
To verify that NF-B activation is essential in the production
of 15-d-PGJ2-induced IL-8 from activated human T
cells, the NF-B-blocking peptide SN50 was used. This peptide
inhibits nuclear transport, and thus function of NF-B (38, 39). CCRF-CEM T cells were treated with anti-CD3 and
anti-CD28, 15-d-PGJ2, and the
NF-B-blocking peptide SN50, as described in Materials and
Methods. Fig. 9 clearly shows that
the addition of the blocking peptide results in the abrogation of IL-8
production from human T cells. As a control, the mutant SN50 peptide
(SN50M) was also used. Although this peptide binds to NF-B, it does
not inhibit nuclear translocation. From Fig. 8 it is evident that the
mutant peptide had no effect on IL-8 production. Therefore, the
induction of IL-8 by 15-d-PGJ2 in human T cells
is NF-B dependent.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). However, it is quite clear
that when activated, the cells display no inhibition of viability (Fig. 5). Although it is unclear what is responsible for the differential
effects on cell death, it is likely that the activation of MAP kinases
and NF-B by TCR stimulation may act as a survival signal to the T
cells. At the same time, unactivated T lymphocytes make undetectable
amounts of IL-8, but activated cells treated with
15-d-PGJ2 show massive IL-8 induction. Therefore,
15-d-PGJ2 may act by preventing the initial
induction of inflammation (inducing cell death from unactivated T
cells), but helping to maintain inflammation that already exists
(inducing IL-8 production from activated T cells). It is very likely,
in fact, that 15-d-PGJ2 would play some role in
the stimulation of inflammation. During allergic and asthmatic
responses, for example, PGD2 is produced in high
quantity from mast cells after degranulation (1, 43).
PGD2 is broken down into
15-d-PGJ2 in vivo, which can then act on nearby
cells recruited by the other proinflammatory factors released. Our data
show that the action of 15-d-PGJ2 on recruited,
newly activated T cells (which are quite prevalent during an asthmatic
response) serves to induce the production of IL-8. This agrees with
published findings that IL-8 is, in fact, found in much higher
concentrations in the epidermis of psoriasis patients as well as in the
bronchoalveolar lavage fluid of asthmatics (44, 45). This
increased IL-8 then induces the chemotaxis and subsequent activation of
cells such as neutrophils. Neutrophils play a major role in allergic
inflammation (46). In fact, the level of
neutrophil-mediated inflammation in bronchoalveolar lavage fluid often
reflects the severity of asthma in the patient (44).
Therefore, 15-d-PGJ2 could be playing an integral
role in the development of inflammation associated with allergies and
asthma.
This is the first report demonstrating that
15-d-PGJ2 induces chemokine production in human T
lymphocytes. The production of IL-8 mRNA and protein in activated human
T cells is consistent with a PPAR--independent pathway, as the
PPAR- agonists ciglitazone and troglitazone do not induce IL-8
protein (Fig. 4). These data support the emerging concept that
15-d-PGJ2 does not exclusively act through the
PPAR- receptor. Although 15-d-PGJ2 can bind to
and signal through the PGD2 receptor (DP-R)
(47), it has been hypothesized that there is a separate cell surface receptor for 15-d-PGJ>2
yet to be identified. It is also possible, however, that
15-d-PGJ2 is entering cells through a
nonreceptor-mediated pathway, and simply acting on another
transcription factor complex once inside the cell. The non-PPAR-
mechanism of action of 15-d-PGJ2 is currently
under intense study.
The production of IL-8 in activated T cells is occurring through a MAP
kinase-mediated pathway (Figs. 6 and 7). The use of
PGF2 in lieu of -CD3 and -CD28
stimulation of T cells demonstrates that T cells need not be activated
through their TCR for 15-d-PGJ2 to induce IL-8
production. A MAP kinase pathway simply needs to be activated by
PGF2 or other means. The inhibition of MAP
kinase activity, and thus IL-8 production, by the compound PD98059
further substantiates the necessity of MAP kinases in the
induction of IL-8 (Fig. 7). In this case, it is possible that MAP
kinases are activating NF-B, which is inducing IL-8 mRNA and
protein. Fig. 8 clearly shows that 15-d-PGJ2
treatment of activated T cells increases NF-B mobilization to the
nucleus, as is evident by the increase in binding to the NF-B
consensus oligos. In addition, inhibiting NF-B nuclear mobilization
with the SN50 peptide potently inhibits IL-8 production (Fig. 9),
further demonstrating the importance of NF-B. Although it has been
shown that 15-d-PGJ2 can inhibit MAP kinases and
NF-B (48, 49, 50), this inhibition would appear to be cell
type and stimulation condition dependent. For example, recent papers by
Reilly (51) and Wilmer (52) show that
15-d-PGJ2 does not inhibit MAP kinases and
NF-B in mesangial cells, but can in fact be stimulatory under
certain conditions. Their results agree with our data that
15-d-PGJ2 induces MAP kinase and NF-B pathways
in activated human T cells. Therefore, we suggest that caution should
be applied when making generalizations of
15-d-PGJ2 being exclusively an
anti-inflammatory lipid.
Cyclopentenone PGs such as PGJ2 and PGA have been postulated to have antiviral functions. For example, the J series PGs can inhibit viral replication and prevent the establishment of persistent viral infections (53). Antiviral effects have been shown against both DNA and RNA viruses, including herpesviruses (54), togaviruses (55), and rhabdoviruses (56). More recent work has focused on the effects of cyclopentenone PGs on the HIV virus. PGJ2 has been shown to inhibit viral replication and subsequent viral mRNA production in T cells. PGJ2 (as well as A series PGs) can protect cells from the cytopathic effects of HIV in either acute or established infections (53). This has led many researchers to propose the use of cyclopentenone PGs in the treatment of HIV infection (53, 57). The data in this study, however, suggest that this may be counterproductive. IL-8 is elevated in the serum of HIV-infected individuals when compared with their normal counterparts (58). The high levels of IL-8 are thought to be key in the pathogenesis of HIV infections. Increased levels of IL-8 could recruit more T lymphocytes to the source of infection, giving the virus more hosts to infect. The newly infected cells then traffic to the lymph nodes, disseminating the virus further (53). As the data presented in this work indicated that 15-d-PGJ2, a cyclopentenone PG, induces a substantial increase in IL-8 production from activated human T cells, the treatment of HIV-infected individuals with this lipid may serve to exacerbate rather than ameliorate the infection.
In conclusion, the findings described in this study are the first to
report that 15-d-PGJ2 can induce activated normal
and malignant human T lymphocytes to produce the chemokine IL-8. This
IL-8 induction is through a MAP kinase and NF-B pathway, and is
PPAR- independent. Our results indicate that not only is
15-d-PGJ2 not always anti-inflammatory, but
it also has a key role in enhancing inflammation via the stimulation of
activated T cells. The 15-d-PGJ2 therefore has
profoundly different effects that are dependent on cell type and
cellular activation state. Caution is therefore urged at solely
assigning anti-inflammatory properties to this complex PG.
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2 Address correspondence and reprint requests to Dr. Richard P. Phipps, Cancer Center, Box 704, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642. E-mail address: Richard_Phipps{at}urmc.rochester.edu
3 Abbreviations used in this paper: 15-d-PGJ2, 15-deoxy-12,14-PGJ2; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; PPAR, peroxisome proliferator-activated receptor.
Received for publication July 31, 2001. Accepted for publication November 1, 2001.
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