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(Received for publication, June 5, 1997, and in revised form, July 29, 1997)
From the Laboratory of Immunology, Center for Biologics Evaluation
and Research, Food and Drug Administration,
Bethesda, Maryland 20892
ABSTRACT Prostaglandin E2
(PGE2) modulates a variety of physiological processes
including the production of inflammatory cytokines. There are two
cyclooxygenase (Cox) enzymes, Cox-1 and Cox-2, that are responsible for
initiating PGE2 synthesis. These isozymes catalyze
identical biosynthetic reactions but are regulated by different
mechanisms in the cell. This report examines differences in the roles
of Cox-1 and Cox-2 in regulating cytokine synthesis in macrophages. We
employed agents that selectively modulate the activity of each isozyme
and measured their effects on synthesis of interleukin (IL)-6, IL-1,
and tumor necrosis factor- INTRODUCTION Prostaglandins are important mediators of a wide variety of
physiological processes (reviewed in Ref. 1). There are two isozymes,
Cox1-1 and Cox-2, that
initiate prostaglandin synthesis (2-5). Both enzymes utilize
arachidonic acid as the predominant substrate, and both catalyze the
same cyclooxygenase and peroxidase reactions that constitute the first
two steps of eicosanoid metabolism (6). Although indistinguishable in
their biosynthetic catalytic activities, the two isozymes appear to
have different physiological functions. Cox-1 (prostaglandin
synthase-1, EC 1.14.99.1) protein is expressed constitutively in most
cell types and is thought to be responsible for regulating normal
physiological functions such as gastric acid secretion and kidney
function (7, 8). The cellular activity of Cox-1 is regulated primarily
through substrate availability, e.g. from the release of
free arachidonic acid from membrane phospholipids. Cox-2 is an
inducible enzyme expressed in activated macrophages (5, 9-14),
fibroblasts (10, 15-18), and several other cell types (19-23).
In vivo expression of Cox-2 is seen in chronic inflammatory conditions such as arthritis (24, 25), experimental peritonitis (14),
and in human colon cancer tissue (26, 27). Cox-2 expression is induced
in vitro in response to stimuli such as LPS and growth factors (9, 11, 15, 16, 28). Due to small differences in the amino acid
sequences near the cyclooxygenase catalytic site, Cox-1 and Cox-2 can
be inhibited differentially by non-steroidal anti-inflammatory drugs
(NSAIDS (29-31)). For example, aspirin and indomethacin inhibit both
isozymes but second generation NSAIDS such as NS-398 inhibit Cox-2
preferentially (32). These drugs can be used to distinguish between the
activities of Cox-1 and Cox-2 within the cell.
A substantial body of work indicates that PGE2 modulates
production of inflammatory cytokines (reviewed in Ref. 33). Different effects of PGE2 are observed depending on the experimental
system employed. Although there are discrepancies in the literature
(34-38), addition of exogenous PGE2 to macrophages prior
to LPS stimulation generally down-regulates IL-6 and TNF- In previous studies, we found that inflammatory agents that induce IL-6
synthesis in macrophages in vitro also induce expression of
Cox-2 (14). Significantly, both Cox-2 mRNA expression and IL-6
protein were co-elevated in inflammatory exudates in vivo. Inhibition of Cox-2 activity by NS-398 in vitro inhibited
IL-6 synthesis. These results suggested that Cox-2 activity might be responsible for modulating IL-6 production.
The question addressed in this report is whether stimulation of
PGE2 synthesis from Cox-1 has the same effect on macrophage cytokine synthesis as stimulation of PGE2 production from
Cox-2. Because Cox-1 protein is expressed constitutively, prostaglandin synthesis is activated within minutes after addition of an appropriate stimulus (e.g. free arachidonic acid). In contrast,
synthesis of PGE2 from Cox-2 is delayed by several hours
due to the requirement for de novo mRNA and protein
synthesis. The two isozymes are also localized in different membrane
compartments within the cell (47). Thus, it is possible that the
PGE2 synthesized from the two cyclooxygenase enzymes may
control different functions in the cells in which it is formed. We find
that among the three pro-inflammatory cytokines examined here (IL-6,
IL-1 EXPERIMENTAL PROCEDURES BALB/c mice were purchased from Charles River
Laboratories and fed Purina laboratory chow and water ad
libitum. Animals were cared for in accordance with the National
Institutes of Health Animal Care Guidelines. Female mice (8-16 weeks
old) received a single 0.5-ml intraperitoneal injection of pristane
(2,6,10,14-tetramethylpentadecane).
Peritoneal lavage
samples were collected 2-4 months after pristane treatment (48). The
peritoneal macrophage population was isolated as described previously
(48). Briefly, neutrophils and macrophages were separated by density
gradient centrifugation (49). Macrophages were purified further by
plating at a concentration of 2.5 × 106 cells/ml
(1.25 × 106 cells/well) in 24-well plastic tissue
culture plates in DMEM containing antibiotics and allowing cells to
adhere at 37 °C for 2-4 h. Nonadherent cells were removed by
washing with DMEM. Cell numbers and viability were quantified by
hemocytometry with trypan blue. Cell differentials were analyzed from
Diff-Quick stained cytocentrifuge slides.
Macrophages were stimulated
with a bovine serum albumin (pAlb) preparation from Boehringer Mannheim
(catalog number 100 069; lot 108303) that contains roughly 4% albumin
polymers. This material was characterized previously and shown to
stimulate macrophage IL-6 production both in vitro and
in vivo (50). PGE2 synthesis was also stimulated
with exogenous arachidonic acid (Sigma), prepared freshly as a 400 µM stock in 50% ethanol.
Macrophages were cultured in DMEM at 37 °C in a humidified
atmosphere containing 5% CO2 and stimulated by addition of
either pAlb (50-200 µg/ml) or arachidonate (10 µM).
Where indicated, indomethacin (1 µM) or NS-398 (1 µM) was added to the cell cultures 30 min before adding
the stimulus. After various times of incubation, the culture medium was
removed from each well and centrifuged. In experiments that contained
no added protein, ultrapure bovine serum albumin (Boehringer Mannheim,
catalog 238 031) was added to each macrophage supernatant at the time
of collection to decrease the adsorption of cytokines to the walls of
the collection tube. Supernatants were stored frozen at Total RNA
was purified from in vitro plated macrophages after various
times of incubation using TRIzol® (Life Technologies, Inc.) following
the protocol recommended by the manufacturer. RNA samples (4 or 15 µg
each) were run on 1% agarose, 0.7% formaldehyde gels containing
ethidium bromide and transferred to nitrocellulose. 32P-Labeled IL-6, Cox-2, and actin cDNA probes were
prepared using a random priming system (Life Technologies, Inc.) and
[ Cells were washed in phosphate-buffered saline, lysed
in sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10%
glycerol, 5% IL-6-dependent B9 hybridoma
cells (51) were cultured in a serum-free medium (52) supplemented with
5% heat-inactivated fetal calf serum and IL-6. Prior to the assay, the
B9 cells were washed to remove IL-6 and were then cultured in
flat-bottom 96-well plates at 3000 cells/well. Samples to be assayed
for IL-6 activity were added at serial 2-fold dilutions. Standard
curves with recombinant murine IL-6 were run to control for interassay
variation. After 3 days of culture at 37 °C, the number of viable
cells was assayed using the colorimetric reagent 3-(4,5-dimethyl
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 0.55 mg/ml (48, 53).
The data were calculated using a four-parameter fit analysis. A unit of
activity is defined as the dilution that gives half-maximal B9 cell
growth in 3 days and corresponds to approximately 2 pg/ml homogeneous
murine recombinant IL-6. Specificity of the assay was confirmed by
blocking all activity with a polyclonal anti-IL-6 antibody. Results of
the B9 bioassay were very similar to those obtained using an
enzyme-linked immunosorbent assay specific for mouse IL-6 (Endogen,
Cambridge, MA).
IL-6, IL-1, and TNF- PGE2 was assayed
using a monoclonal antibody/enzyme immunoassay kit from Cayman
Chemical. The linear range of the assay was from 10 to 1000 pg/ml.
Escherichia coli LPS 055:B5 was from Difco.
Indomethacin, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium
bromide, PGE2, and arachidonate were from Sigma. NS-398 was
purchased from Biomol (Plymouth Meeting, PA). Pristane was from
Aldrich. A cDNA probe for murine IL-6 (0.65 kb) was a gift from Dov
Pluznik (FDA/CBER). A cDNA probe for murine Cox-2 (1.7 kb (11)) was
a gift from Joshua Farber (NIAID, National Institutes of Health). A
cDNA probe for murine RESULTS Pristane-elicited
macrophages spontaneously secrete low levels of IL-6 and
PGE2 (48). Stimulation with either LPS or polymerized albumin (pAlb) promotes an increase in the expression of the Cox-2 and
IL-6 genes (Fig. 1). We employ pAlb for
our in vitro studies because it is pro-inflammatory in
vivo and yet is less toxic than LPS to macrophages (50). The time
course of expression of IL-6 and Cox-2 mRNAs after induction by
pAlb is shown in Fig. 2. Elevated Cox-2
mRNA expression is first seen 30 min after addition of pAlb to the
cells. By comparison, the elevation in IL-6 expression is not detected
until 45 min after stimulation.
PGE2 is the predominant eicosanoid
produced by pAlb- and LPS-stimulated
macrophages2 and hence is the
focus of these studies. We have hypothesized that induction of
macrophage IL-6 mRNA expression is controlled, in part, by
PGE2 derived from Cox-2 (14). This conclusion was drawn
from the finding that induction of IL-6 mRNA expression is
inhibited 50-75% in the presence of NS-398, a non-steroidal anti-inflammatory drug that selectively inhibits Cox-2 activity (14,
54). Since PGE2 can modulate production of other
inflammatory cytokines, we sought to determine how inhibition of Cox-2
activity would affect macrophage production of IL-1 and TNF-
To test further whether Cox-1 might be involved in
pAlb-stimulated synthesis of PGE2 and IL-6, we looked for
Cox-1 protein induction by pAlb. Cox-2 protein is induced approximately
300-fold in 5 h by stimulation with pAlb (Fig.
4A). The levels of Cox-1 protein are low but remain constant over the same time period (Fig.
4B). Since Cox-1 activity is generally regulated through modulation of substrate availability and not through gene induction (55), the absence of a change in Cox-1 protein levels does not preclude
a change in Cox-1 activity. To test whether pAlb stimulates any Cox-1
activity, macrophages were pretreated with aspirin, washed, and then
stimulated for 6 h with pAlb. Aspirin irreversibly inhibits
cyclooxygenases by acetylating a serine residue near the cyclooxygenase
active site (6). Since Cox-2 is not present at the time of the aspirin
pretreatment, the drug only inactivates pre-existing Cox-1. As shown in
Fig. 5, pAlb-induced PGE2
synthesis was lowered by roughly 16% following pretreatment with
aspirin, and IL-6 synthesis was decreased by approximately 20%. This
small effect may have derived from incomplete elimination of aspirin from the cells by the washing procedure. In contrast, when aspirin was
present throughout the incubation period with pAlb, PGE2
production was 98% inhibited (consistent with the ability of aspirin
to inhibit the activities of both Cox-1 and Cox-2), and IL-6 synthesis
was reduced by 56%. Taken together, the data presented thus far
suggest that pAlb stimulates IL-6 synthesis by inducing Cox-2 activity and that Cox-1 is not involved in this particular process.
Because pAlb stimulates PGE2 synthesis by activating Cox-2
selectively, the above results do not reveal whether Cox-1 activity would induce IL-6 synthesis if the enzyme became activated. To examine
this possibility, we tested the effect of arachidonic acid on
PGE2 and IL-6 synthesis. Arachidonate is the substrate for
cyclooxygenases, and it stimulates PGE2 synthesis by Cox-1 in macrophages (56). Treatment of cells with 10 µM
arachidonic acid did not induce any measurable IL-6 production even
though substantial PGE2 was generated (Fig.
6).
In control experiments, we verified that addition of exogenous
arachidonic acid to these cells stimulates PGE2 synthesis
from Cox-1 and not Cox-2. First, we examined the time course of
PGE2 synthesis by this agent. As shown in Fig.
7, arachidonic acid stimulated rapid
secretion of PGE2 (within 30 min) compared with pAlb (2-3
h). This time course of arachidonate-induced prostaglandin synthesis is
similar to that documented previously by others (57). The production of
PGE2 from arachidonate-stimulated macrophages occurred in
the absence of detectable Cox-2 protein, determined by Western blot
immunoassay of cell extracts collected at each of the time points shown
in Fig. 7 (data not shown). Moreover, pretreatment of the cells with
aspirin for 30 min prior to addition of arachidonate completely
inhibited PGE2 production (Fig.
8). Since there was no Cox-2 protein
present in the cells during the preincubation period, the inhibition
had to derive from an effect on Cox-1. This result can be contrasted to
the effect of aspirin pretreatment on pAlb-stimulated PGE2
production (see Fig. 5). Thus, we conclude that arachidonic acid
stimulates PGE2 synthesis from Cox-1 and that this activity
is not sufficient to induce IL-6.
How can stimulation of PGE2 production from
Cox-2 result in increased IL-6 production while PGE2
derived from Cox-1 has no effect? One explanation could be that agents
such as pAlb that induce Cox-2 gene expression also activate ancillary
signaling pathways that are required for elaboration of the
PGE2 effect. Absent activation of these pathways, the
PGE2 generated by the cells is ineffective at stimulating
IL-6 synthesis. To test this hypothesis, macrophages were stimulated
with pAlb in the presence of NS-398 which was added to inhibit
endogenous PGE2 synthesis from Cox-2. Exogenous
PGE2 was added either 30 min prior to or 4 h after
treatment of the cells with pAlb and NS-398. As shown in Fig.
9, exogenous PGE2 can
stimulate IL-6 synthesis and can overcome the inhibition caused by
NS-398 but only when added to the cells at the delayed time point, at
which time Cox-2 and Cox-2-derived PGE2 levels are normally
elevated (see Figs. 4 and 6). When PGE2 is added to
macrophages prior to stimulation with pAlb, IL-6 levels remain at or
below the NS-398-inhibited level. This is not a generalized effect
because IL-1 synthesis is insensitive to the presence of PGE2, regardless of the time it is added.
DISCUSSION The findings in these studies can be summarized as follows.
Inflammatory agents that stimulate IL-6 expression by macrophages do so
in part by inducing Cox-2 gene expression and Cox-2-derived PGE2 synthesis. Of the three pro-inflammatory cytokines
examined here (IL-6, IL-1, and TNF- The difference in outcome derived from activating Cox-1 or Cox-2 may
lie in part in the time course of production of PGE2 by
each isozyme. That is, PGE2 synthesis from Cox-1 begins
within minutes after addition of an appropriate stimulus and is
complete within 1 h. In contrast, PGE2 synthesis
derived from induction of Cox-2 commences after a delay of more than an
hour following stimulation and continues to accumulate for hours
thereafter. The delayed production of PGE2 by Cox-2 may be
necessary to generate the IL-6 response. Experiments using exogenous
PGE2 demonstrate this point. If the PGE2 is
added early, at roughly the same time as pAlb, then IL-6 synthesis is
slightly decreased. However, if the addition of PGE2 is
delayed to 4 h after pAlb stimulation, then IL-6 synthesis is
greatly augmented. The delayed time frame coincides with the time when
pAlb normally induces maximal Cox-2 synthesis and activity. Thus,
addition of PGE2 at this time may mimic Cox-2 activity. It
should be noted that the concentration of exogenous PGE2
added to the macrophages is roughly equivalent to the amount generated
during an overnight incubation of the cells with pAlb or LPS.
The results also raise the possibility that induction of IL-6 synthesis
by inflammatory agents such as pAlb requires co-induction of ancillary
signals that control the response to PGE2. In previous experiments, we (14) showed that addition of exogenous PGE2 to untreated macrophages stimulates IL-6 synthesis. However, the degree
of induction was relatively low, increasing the levels of IL-6
generated from roughly 20 to 80 pg/ml (14). Here we show that if
PGE2 is added to cells that have been pretreated with pAlb
in the presence of an inhibitor of endogenous prostaglandin synthesis,
then the exogenous PGE2 stimulates the cells to produce an
additional 45,000 pg/ml IL-6 (see Fig. 9), fully restoring the level of
IL-6 synthesis to that which was achieved in the absence of the
cyclooxygenase inhibitor. The augmented level of IL-6 induced by adding
exogenous PGE2 to pAlb-treated cells compared with
untreated cells shows that pAlb primes the cells to respond to the
PGE2. Since agents that stimulate Cox-1 activity result in
the production of PGE2 prior to or in the absence of these ancillary signals, the PGE2 produced from activation of
Cox-1 fails to induce IL-6 synthesis. A similar mechanism for induction of human monocyte responsiveness to cAMP has been proposed by Corcoran
et al. (60). Thus, agents that induce IL-6 synthesis may
have two distinguishing characteristics: induction of delayed PGE2 synthesis due to induction of Cox-2 and not Cox-1, and
activation of secondary signals that control the cellular response to
PGE2. A hypothetical scheme depicting the regulation of
IL-6 synthesis in macrophages by PGE2 is shown in
Scheme 1. An alternative explanation for the different outcomes achieved by stimulation of Cox-1 and Cox-2
is that PGE2 produced by the two isozymes is channeled
differently in the cell. Cox-1 is found primarily in the endoplasmic
reticulum, whereas Cox-2 is also active in the nuclear envelope (47).
This raises the possibility that some of the PGE2 generated
by Cox-2 acts directly on nuclear gene expression without exiting the
cell. This mechanism need not be invoked to explain our results since exogenous PGE2 was able to substitute for Cox-2-derived
PGE2. However, it will be of interest to determine whether
the PGE2 generated from Cox-2 needs to exit the cell to
stimulate IL-6 synthesis.
Scheme 1.
Our data indicate that pAlb stimulates PGE2 production
primarily from Cox-2 even though Cox-1 is present in the cell. This implies that the two isozymes respond to different pools of arachidonic acid, consistent with the experimental results of Reddy and Herschman (56) showing that Cox-1 cannot access arachidonic acid released by LPS.
The same appears to be true for pAlb since aspirin pretreatment had
little effect on the level of PGE2 produced in response to this agent, and NS-398 was equally as effective in inhibiting pAlb-induced PGE2 synthesis as indomethacin (not shown). In
addition, Reddy and Herschman (56) have suggested that exogenous
arachidonic acid is utilized only by Cox-1 in the cell. Our results are
consistent with this conclusion since PGE2 production
induced by arachidonate is rapid and is completely inhibited by
pretreatment of the cells with aspirin. However, our experiments do not
address the question of whether exogenous arachidonic acid could be
utilized by Cox-2 since, under our experimental conditions, Cox-2 is
not present at the time of addition of the arachidonate. Nonetheless,
the results support the conclusion that Cox-1 and Cox-2 are
functionally segregated by differences in the availability of
arachidonic acid to each isozyme (56) and that pAlb stimulates
PGE2 production only from Cox-2 because it generates
arachidonic acid which is not accessible to Cox-1.
IL-1 and TNF- Overall, the results demonstrate that among the three pro-inflammatory
cytokines examined here, IL-6 is unique in being stimulated by
Cox-2-derived PGE2. Thus, chronic overexpression of Cox-2
such as occurs in rheumatoid arthritis (65-67) is expected to be
accompanied by chronic high levels of IL-6 and the symptoms derived
from these abnormally high IL-6 levels (65). Second generation NSAIDS
aimed specifically at inhibiting Cox-2 activity may be useful in
treating chronic inflammatory conditions in which IL-6 is abnormally
elevated.
FOOTNOTES REFERENCES
Volume 272, Number 41,
Issue of October 10, 1997
pp. 25693-25699
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
DISTINCT ROLES OF CYCLOOXYGENASE-1 AND -2*
by peritoneal macrophages. Among these
three cytokines, only IL-6 synthesis was stimulated by production of
endogenous PGE2. This effect was specifically linked to
activation of Cox-2 and not Cox-1. The specificity derives, partly,
from the timing of the production of PGE2 following
stimulation of each isozyme and from induction of ancillary signals
that control the response to PGE2. The experimental findings demonstrate that the effects of Cox-1 and Cox-2 activity on
macrophage IL-6 synthesis are segregated. This provides a mechanism for
IL-6 to be induced selectively during inflammation.
synthesis
while having no effect on IL-1
(35, 36). However, direct addition of
PGE2 to untreated macrophages induces low levels of both
IL-6 (14) and TNF- (39) synthesis. Since macrophages are a major
source of PGE2 during inflammation (40) and since they also
have receptors for and respond to this eicosanoid (41), the
PGE2 generated by macrophages may regulate cytokine
synthesis in an autocrine fashion (in contrast to paracrine regulation
achieved through adding exogenous PGE2 to cells). Previous
studies have implicated a positive association between endogenous
PGE2 production and IL-6 synthesis in vitro (14,
42, 43), and animal models of chronic inflammation show that
PGE2 is a stimulator of IL-6 production in vivo
(14, 25, 42, 44-46).
, and TNF-), only IL-6 production is stimulated by
PGE2 produced by macrophages. Moreover, the regulation of
IL-6 is uniquely linked to Cox-2 activation; agents that stimulate Cox-1 fail to induce IL-6 synthesis.
20 °C
until assayed for the presence of cytokines or PGE2.
-32P]dCTP (Amersham Corp.). Blots were hybridized
overnight with probe (1 × 106 cpm/ml; 5 × 108 cpm/µg) at 65 °C in Hybrisol II solution (Oncor,
Inc., Gaithersburg, MD) and then washed by standard procedures.
Autoradiography was performed using Kodak XAR film. Films were scanned
(Microtek Scanmaker) and analyzed using the Macintosh densitometry
program NIH Image.
-mercaptoethanol, 1 mM DTPA) and heated at
100 °C for 12 min. Protein (5 × 105 cell
equivalents) was separated by SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes
(Immobilon-P, Millipore, Inc). Membranes were blocked in 3% bovine
serum albumin and then incubated with a polyclonal rabbit anti-murine
Cox-2 antibody (Cayman Chemical Co., Ann Arbor, MI; catalog number
160106) or anti-Cox-1 antibody followed by a horseradish
peroxidase-conjugated goat anti-rabbit IgG (Southern Biotechnologies;
catalog number 4050-05). Immunopositive protein bands were visualized
by chemiluminescence (Renaissance kit, NEN Life Science Products) and
exposure of x-ray film.
were measured using EIA kits from
Endogen (Woburn, MA).
-actin (2 kb) was provided by Konrad Huppi
(NCI, National Institutes of Health). Probes were purified from
plasmids according to standard procedures. Murine recombinant IL-6
purified to homogeneity was a gift of Bob Brown (NCI, National
Institutes of Health).
Fig. 1.
Treatment of macrophages with either
polymerized albumin (pAlb) or bacterial LPS induces expression of both
IL-6 and Cox-2 mRNA. Pristane-elicited peritoneal macrophages
were purified from total peritoneal exudate cells as described under
"Experimental Procedures." The cells were seeded in plastic tissue
culture plates in serum-free DMEM and allowed to adhere for 2 h
followed by washing. The cells were stimulated by addition of either
pAlb (200 µg/ml) or LPS (1 µg/ml). Control cells were cultured
under identical conditions in the absence of any additives. Total RNA
was purified 4 h later and analyzed by Northern blot analysis
using 32P-labeled probes for murine IL-6 and Cox-2.
Expression of -actin was measured to control for differences in RNA
loading.
[View Larger Version of this Image (40K GIF file)]
Fig. 2.
Time course of induction of Cox-2 and IL-6
mRNAs. Peritoneal macrophages were prepared as described in
the legend to Fig. 1. At time 0, the cells were stimulated by addition
of 200 µg/ml pAlb. Total RNA was isolated at the times indicated. The figure shows a representative Northern blot probed simultaneously for
Cox-2 (1.7-kb probe, 1 × 106 cpm/µg) and IL-6
(0.7-kb probe, 1 × 106 cpm/µg). A longer exposure
of the 32P-labeled blot did not change the timing of
appearance of the bands. Identical results were obtained in two
separate experiments.
[View Larger Version of this Image (65K GIF file)]
Production
by Macrophages
. The
results shown in Fig. 3 indicate that
IL-6 is most significantly affected by NS-398, showing roughly 50%
inhibition by the drug. TNF- synthesis is augmented by 24 ± 12% (mean ± S.D., n = 5), and IL-1 levels are
not significantly affected by the presence of NS-398. These data
suggest that the PGE2 responsible for modulating
pAlb-induced IL-6 secretion derives primarily from Cox-2. In addition,
they show that of the three cytokines examined here, only IL-6 is
induced by endogenous PGE2 production.
Fig. 3.
Effects of NS-398 on pAlb-stimulated
production of IL-6, IL-1, and TNF-. Peritoneal macrophages were
prepared as described in the legend to Fig. 1. The cells were
stimulated by the addition of pAlb (100 µg/ml) in the presence or
absence of NS-398 (1 µM). Following an 18-h incubation,
supernatants were collected and assayed for the presence of IL-6, IL-1,
and TNF-. Bars are mean ± S.D. of triplicate
cultures. Similar results were obtained in three separate
experiments.
[View Larger Version of this Image (13K GIF file)]
Fig. 4.
Induction of Cox-2 protein expression by
pAlb. Pristane-elicited macrophages were prepared and cultured as
described in the legend to Fig. 1. At time 0, the cells were stimulated by the addition of 200 µg/ml pAlb. The time course of Cox-2
(A) and Cox-1 expression (B) with (+) and without
() pAlb stimulation was determined by Western blot immunoassay of
cell lysates prepared at the times indicated in the figure. The level
of Cox-1 protein did not vary at intermediate time points (not
shown).
[View Larger Version of this Image (15K GIF file)]
Fig. 5.
Effect of aspirin pretreatment on
pAlb-induced expression of IL-6 and PGE2.
Pristane-elicited macrophages were treated with aspirin (200 µg/ml)
for 30 min (aspirin-pre), washed thoroughly, and
then stimulated with pAlb (100 µg/ml). Supernatants were collected after a 6-h incubation and assayed for IL-6 and PGE2.
Control cultures were untreated. Aspirin-full indicates that
aspirin was present throughout the entire incubation period.
Bars are mean ± S.D. of triplicate cultures. Similar
results were obtained in two separate experiments.
[View Larger Version of this Image (33K GIF file)]
Fig. 6.
A Cox-1 stimulus is ineffective at inducing
either IL-6 or Cox-2 protein. Pristane-elicited peritoneal
macrophages were stimulated by addition of either pAlb (50 µg/ml) or
arachidonate (10 µM). Following a 5-h incubation, culture
supernatants were collected and assayed for the presence of IL-6
(black bars) and PGE2 (shaded bars)
(A). Cell lysates collected at the same time were analyzed
by Western blotting (B) for the presence of Cox-2 protein.
A shows a densitometric analysis (striped bars)
of the Cox-2 protein blot shown in B. The experiment was
repeated three separate times with similar results. Variation in the
incubation time from 3 to 18 h did not affect the qualitative
outcome of the experiments.
[View Larger Version of this Image (29K GIF file)]
Fig. 7.
Kinetics of PGE2 production by
pAlb- and arachidonate-stimulated macrophages. Pristane-elicited
macrophages were treated at time 0 with either pAlb (100 µg/ml,
closed circles) or arachidonate (10 µM,
closed squares). Supernatants were collected at the times indicated, and IL-6 and PGE2 levels were assayed. The data
represent the mean of triplicate cultures. ×, untreated cells.
[View Larger Version of this Image (14K GIF file)]
Fig. 8.
Effect of aspirin pretreatment on
arachidonate-induced expression of PGE2.
Pristane-elicited macrophages were treated with aspirin (200 µg/ml)
for 30 min (aspirin-pre), washed thoroughly, and then
stimulated with arachidonic acid (10 µM). Supernatants were collected after a 90-min incubation and assayed for
PGE2. Control cultures were untreated.
Aspirin-full indicates that aspirin was present throughout
the entire incubation period. Bars are mean ± S.D. of
triplicate cultures.
[View Larger Version of this Image (18K GIF file)]
Fig. 9.
Differential effects of exogenous
PGE2 on macrophage cytokine production when added pre- and
post-stimulation with pAlb. Pristane-elicited macrophages were
cultured as described in the legend to Fig. 1. Cells were treated
either with pAlb only (100 µg/ml), pAlb + NS-398 (1 µM), or pAlb + NS-398 + PGE2 (0.1 µM added 30 min before (PGE-pre) or 4 h
after (PGE-post) pAlb stimulation). Following an 18-h
incubation, culture supernatants were collected and assayed for the
presence of IL-6 or IL-1. The data represent the mean ± S.D. of
triplicate cultures. Similar results were obtained in five separate
experiments.
[View Larger Version of this Image (15K GIF file)]
), only IL-6 is induced by
Cox-2-derived PGE2. Stimulation of PGE2
production by Cox-1 is not sufficient to induce IL-6 synthesis. Our
results suggest that appropriate control of IL-6 synthesis is dependent
upon such segregation of macrophage Cox-1 and Cox-2 activities. In
terms of understanding physiological outcomes, it seems appropriate
that Cox-1 activity does not stimulate IL-6 synthesis since Cox-1 is
expressed constitutively in most cells. If IL-6 could be stimulated by
Cox-1 activity, IL-6 synthesis would be constantly turned on in those
tissues in which Cox-1 is active. Instead, the dependence on Cox-2
synthesis is compatible with IL-6 being induced only when inflammatory
stimuli are present. Collagenase synthesis and matrix metalloprotein
expression represent other examples in which there is a specific
association between Cox-2 activity and activation of
inflammation-associated genes (58, 59).
synthesis are also induced by treatment of macrophages
with inflammatory agents such as LPS and pAlb, but our results indicate
that they are regulated by separate mechanisms that are not dependent
upon co-induction of PGE2 synthesis. Other researchers have
also found that drugs that inhibit cyclooxygenases either have no
effect or cause an increase in TNF- and IL-1 production, suggesting
that autocrine PGE2 synthesis does not affect these cytokines in the same way as it affects IL-6 (38, 42, 61-64, and this
report). These findings may explain recent results with in vivo models showing that NSAIDS that inhibit Cox-2 modulate IL-6 but not TNF- levels (14, 25, 44).
To whom correspondence should be addressed: FDA/CBER, HFM-538,
Bldg. 29A, Rm. 2A-11, Bethesda, MD 20892. Tel.: 301-827-1833; Fax:
301-480-3256.
1
The abbreviations used are: Cox, cyclooxygenase;
IL, interleukin; TNF, tumor necrosis factor; pAlb, polymerized albumin;
LPS, lipopolysaccharide; NSAID, non-steroidal anti-inflammatory drug; DMEM, Dulbecco's modified Eagle's medium; kb, kilobase pair; PGE, prostaglandin E.
2
G. K. Arzadon, L. M. Wahl, and E. Shacter, unpublished results.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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