The pathogenesis of septicemia can be triggered by LPS, a potent stimulus for PG synthesis. The enzyme cyclooxygenase (COX) is a rate-limiting step in PG production. COX exists as two isoforms: COX-1, which is constitutively expressed in most cell types, and COX-2, which is inducible by LPS and cytokines in a variety of cells. In this study we determined the role of the proinflammatory cytokines IL-1β and TNF-α released by LPS-stimulated U937 human macrophages in the regulation of COX-2. Macrophages exposed to LPS showed a rapid and sustained expression of COX-2 mRNA and protein for up to 48 h, whereas PGE2 production was notably enhanced only after 12 h. LPS increased COX-2 gene transcription and activation of the transcription factor NF-κB in a transient manner. LPS-treated macrophages produced high levels of TNF-α and moderate amounts of IL-1β protein. However, neutralizing Abs against these cytokines had no effect on COX-2 mRNA and protein expression, nor did they affect the stability of COX-2 mRNA. Interestingly, in the presence of LPS or exogenous IL-1β, COX-2 transcripts were stabilized, and actinomycin D inhibited their degradation. Only when LPS or IL-1β was removed did COX-2 mRNA decay with a t1/2 of ≥5 h. In contrast, dexamethasone promoted a faster decay of the LPS-induced COX-2 transcripts (t1/2 = 2.5 h). These results clearly demonstrate that LPS can regulate COX-2 at both transcriptional and posttranscriptional levels independently from endogenous IL-1β and TNF-α in human macrophages.

Lipopolysaccharide is the triggering factor for multiple organ system failure and death during septic shock. However, the observed systemic effects are caused by the host’s endogenous cytokines, mainly TNF-α and IL-1β, and important secondary mediators such as PGs and platelet-activating factor (1). PGs are lipid mediators involved in vasodilation, pain, and fever (2, 3). A major source of PGs during sepsis are macrophages (1). After stimulation with LPS, these cells secrete PGs (mainly PGE2) and proinflammatory cytokines, which, in turn, act in an autocrine or paracrine manner to regulate the host response (4).

A rate-limiting step in the synthesis of PGs is the enzyme cyclooxygenase (COX).3 To date, two isoforms of this enzyme have been described: COX-1, which is constitutively expressed in most human tissues (5), and COX-2, whose expression is readily induced by inflammatory stimuli such as LPS and cytokines in a variety of cells (6, 7). Interestingly, COX-2 is regulated by cytokines in a rather cell type-specific manner. IL-1 is a potent stimulus for the induction of COX-2 expression in human endothelial cells, human synovial fibroblasts, and rat mesangial cells (8, 9, 10). In contrast, TNF-α stimulates COX-2 mRNA expression in mouse osteoblasts and bovine endothelial cells (11, 12), but has no effect by itself on human monocytes (13). IFN-γ, the main macrophage-activating cytokine (14), can potentiate the effect of LPS on COX-2 expression in mouse macrophages (15), but has no effect on COX activity when simultaneously administered with LPS to human monocytes (16). Furthermore, we have reported that IFN-γ down-regulates COX-2 expression induced by IL-1β in human macrophages (17).

The induction of COX-2 expression by IL-1 occurs at the transcriptional level (8, 17, 18). However, this cytokine also has the ability to stabilize COX-2 transcripts by a still poorly understood mechanism (19, 20). The transcriptional regulation of COX-2 by IL-1β in human cells and by TNF-α in murine osteoblasts involves activation of the transcription factor NF-κB (11, 18, 21). Recent studies have shown that NF-κB is also involved in the induction of COX-2 expression in response to LPS in human and mouse macrophages (22, 23), indicating a transcriptional mechanism for this potent stimulus. LPS-treated macrophages release the proinflammatory cytokines IL-1β and TNF-α. However, the roles these proinflammatory cytokines play in the regulation of COX-2 are not known. We hypothesized that these cytokines may participate in an autocrine manner in the induction or maintenance of COX-2 expression in human macrophages.

To test this hypothesis, we determined the effect of neutralizing polyclonal Abs against IL-1β and TNF-α on COX-2 expression in LPS-stimulated macrophages. LPS induced COX-2 gene transcription in a transient manner and stabilized the mRNA. In the presence of neutralizing Abs against IL-1β and TNF-α there was no effect on COX-2 mRNA and protein expression, and the stability of COX-2 mRNA was unaffected. These results clearly demonstrate that LPS can trigger COX-2 mRNA and protein expression and maintain high production of PGs by macrophages without the influence of the released proinflammatory cytokines, IL-1β and TNF-α.

LPS (from Escherichia coli, serotype 0111, 4% phenol extract), phorbol 12-myristate 13-acetate (PMA), dexamethasone (DEX), 3-(4,5-dimethyl-thiazol-2-yl-)2,5-di-phenyltetrazolium bromide (MTT), and 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) diammonium salt (ABTS) were purchased from Sigma (St. Louis, MO). Actinomycin D (AD) was obtained from Life Technologies (Burlington, Canada). Recombinant human IL-1β and TNF-α, goat anti-human IL-1β, goat anti-human TNF-α, rabbit anti-human IL-1β, and normal goat IgG Abs were purchased from R&D Systems (Minneapolis, MN). Rabbit anti-human TNF-α Ab was purchased from Endogen (Woburn, MA). RPMI 1640 with l-glutamine was obtained from Life Technologies and was completed with 100 U/ml of penicillin, 100 μg/ml streptomycin sulfate, 20 mM HEPES (Sigma), and 10% heat-inactivated FCS (HyClone, Logan, UT).

The human macrophage cell line U937 was grown in complete RPMI 1640. Cells were kept at 37°C in 5% CO2-95% air and harvested at the log phase of growth. To differentiate the cells into adherent macrophages, they were adjusted to 1 × 106 cells/ml and incubated in 24-well plates (1 × 106 cells/well) for 3 days in the presence of 10 nM PMA. Macrophages were made quiescent in fresh complete RPMI without PMA for 24 h before stimulation with LPS or cytokines. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2-yl-)2,5-di-phenyltetrazolium bromide (MTT) method (24) and was 90% after 12 h of incubation with AD (10 μg/ml).

The quantification of COX-2 and COX-1 mRNA was performed by Northern blot analysis as previously described (17), using actin as a housekeeping gene to normalize the values obtained for COX-2. The expression of COX-2 and COX-1 proteins was assessed by immunoblot as previously reported (17), using polyclonal Abs prepared against the complete ovine COX-1 and COX-2 proteins. The Abs were gifts from Dr. G. O’Neill (Merck Frosst, Kirkland, Canada).

The protocol was previously described (17). Briefly, the cells (5 × 107) were lysed, and the nuclear pellets were obtained by centrifugation. The nuclei were resuspended in 100 μl of storage buffer and frozen in liquid nitrogen until used. The nascent chains of newly synthesized mRNA were elongated in vitro and labeled with [α-32P]UTP. The radiolabeled RNA was then extracted with Trizol (Life Technologies) and hybridized onto nitrocellulose membranes containing the following linearized plasmids: pcDNACOX-1, pcDNACOX-2, pBA-1, pcDNA1Amp, and pBR322. The latter two plasmids were the controls without the COX and actin fragments, respectively. The membranes were prehybridized for 3 h and then hybridized for 48 h; both steps were conducted at 65°C. The membranes were exposed for autoradiography for 15 days. The films were scanned, and densitometric analysis was performed as previously reported (17).

The protocol for nuclear protein extraction was as described previously (25) with the modifications reported by Zhang et al. (26). For the DNA-protein binding reaction, nuclear extracts (10 μg) were incubated for 30 min at room temperature with 1–2 ng (200,000–600,000 cpm) of 32P-labeled double-stranded oligonucleotides in a binding buffer (20 μl final volume) containing 10 mM Tris-HCl (pH 7.4), 40 mM NaCl, 1 mM EDTA, 1 mM 2-ME, 0.1% Nonidet P-40, 4% glycerol, 1 μg/μl BSA, and 0.2 μg/μl poly(dI/dC) (Pharmacia Biotech, Piscataway, NJ). For cold competition binding assays, the nuclear extracts were first incubated with a 10- or 50-fold excess of unlabeled double-stranded oligonucleotides for 20 min, followed by the addition of the radiolabeled oligonucleotides, and incubated for 30 min more at room temperature. Reactions were stopped with 5 μl of loading buffer (25 mM Tris-HCl (pH 7.5), 50% glycerol, and bromophenol blue). Samples were subjected to electrophoresis in 5% polyacrylamide gels in 0.25× Tris-borate/EDTA (TBE) buffer at 150 V for 2 h at 4°C. Gels were pre-electrophoresed for 1 h at 140 V. The synthetic single-stranded oligonucleotides (Life Technologies) were annealed by boiling the complementary oligonucleotides for 10 min in a buffer containing 10 mM Tris-HCl (pH 7.5) and 50 mM NaCl, and were cooled to room temperature overnight. The sequences of the oligonucleotides used were as previously described (18), and the consensus sequences for NF-κB are underlined: distal (upstream) NF-κB site forward, 5′-GGA GAG GGG ATT CCC TGC GCC-3′; distal NF-κB site reverse, 5′-CAG GGA ATC CCC TCT CCC GCC G-3′; proximal NF-κB site forward, 5′-AGT GGG GAC TAC CCC CTC TGC TCC-3′; and proximal (downstream) NF-κB site reverse, 5′-CAG AGG GGG TAG TCC CCA CTC TCC T-3′. Once annealed, the double-stranded oligonucleotides were labeled by filling the overhangs with [32P]GTP (>3000 Ci/mmol; ICN, Costa Mesa, CA) and 2 U of Klenow polymerase (Life Technologies) in a low salt buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, and 50 μg/ml BSA) plus a mixture of dATP, dCTP, and dTTP.

The amounts of IL-1β and TNF-α released in the supernatants of stimulated and nonstimulated macrophages were measured by ELISA as previously described (27) with minor modifications. We used 3% BSA in PBS to block the nonspecific binding sites and 1% BSA in PBS to dilute the Abs instead of FCS in PBS. The standard curves were prepared with twofold dilutions of rIL-1β (4–500 pg/ml) or TNF-α (8–1000 pg/ml) in 3% BSA-PBS. The supernatants were diluted 1:3 in 3% BSA-PBS for the TNF-α determination, whereas undiluted supernatants were used to quantify IL-1β.

PGE2 production was measured by enzyme immunoassay (Cayman Chemicals, Ann Arbor, MI) after different times of incubation with LPS.

We and others (7, 17) have shown that IL-1β can stimulate a sustained accumulation of COX-2 mRNA for up to 12 h. To determine whether LPS can induce a similar expression of COX-2 mRNA, we investigated the kinetics of COX-2 mRNA and protein expression in human macrophages stimulated with LPS for different times. LPS induced a time-dependent increase in COX-2 mRNA levels (Fig. 1,A). COX-2 transcripts were detected as early as 2 h, peaked at 6–12 h, and started to decline slightly after 24 h. Detectable levels of COX-2 protein appeared after 4–6 h and remained stable up to 24 h (Fig. 1,B). Regardless of conditions, COX-1 protein remained stable throughout the incubation period (Fig. 1,B) and correlated with the levels of COX-1 mRNA (Fig. 1 A). The amounts of PGE2 released in the supernatants of stimulated cells were 395 and 760 pg/ml after 12 and 24 h, respectively, whereas nonstimulated cells released 280 pg/ml after 6 h. These results indicate that LPS is a potent stimulus for the induction and sustained expression of COX-2 mRNA and protein and for PGE2 production by human macrophages.

FIGURE 1.

Expression of COX-2 mRNA and protein in macrophages stimulated with LPS. A, PMA-differentiated U937 macrophages were treated with 100 ng/ml of LPS for 2–24 h. Total RNA was isolated to perform Northern blot analysis using specific cDNA probes for COX-2, COX-1, and β-actin. The densitometric units in the histogram reflect the quantitative levels of COX-2 normalized to the constant levels of actin. Lane designations are identical for the blots and histogram. B, Whole lysates of U937 cells (1 × 105) treated as described in A were subjected to immunoblot analysis using polyclonal Abs against purified ovine COX-2 and COX-1 proteins (std.; purified ovine proteins). Figures are representative of three independent experiments with similar results.

FIGURE 1.

Expression of COX-2 mRNA and protein in macrophages stimulated with LPS. A, PMA-differentiated U937 macrophages were treated with 100 ng/ml of LPS for 2–24 h. Total RNA was isolated to perform Northern blot analysis using specific cDNA probes for COX-2, COX-1, and β-actin. The densitometric units in the histogram reflect the quantitative levels of COX-2 normalized to the constant levels of actin. Lane designations are identical for the blots and histogram. B, Whole lysates of U937 cells (1 × 105) treated as described in A were subjected to immunoblot analysis using polyclonal Abs against purified ovine COX-2 and COX-1 proteins (std.; purified ovine proteins). Figures are representative of three independent experiments with similar results.

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Nuclear run-on analysis was performed to determine whether the sustained expression of COX-2 mRNA induced by LPS was the result of a steady increase in gene transcription. Macrophages were exposed to LPS for 1 or 12 h, and newly synthesized mRNA chains were radiolabeled and hybridized to specific probes as described in Materials and Methods. As shown in Fig. 2,A, LPS caused a 5.4-fold increase in the transcription rate of the COX-2 gene after 1 h, which returned to basal levels after 12 h of stimulation. In contrast, the transcription rate of COX-1 remained unaltered following LPS treatment. To determine whether the transcriptional induction correlated with the activation of the transcription factor NF-κB, EMSA was performed using the distal (upstream) NF-κB sequence in the COX-2 promoter as a probe (18). A DNA-protein complex was formed by the nuclear protein extracts of cells treated with LPS for 1 h, but disappeared by 12 h of incubation. The complex was specific, as its formation was inhibited by preincubation of the nuclear extracts with a 10- or 50-fold excess of unlabeled NF-κB probe (Fig. 2 B). Under these conditions we did not observe any DNA-protein complex when the proximal NF-κB sequence was used as a probe (data not shown). These data indicate that the induction of COX-2 gene transcription by LPS is rapid but transient and correlates with the activation of the transcription factor NF-κB.

FIGURE 2.

The transcriptional activation of COX-2 gene by LPS is transient. A, Nuclear run-on analysis was performed on macrophages exposed to medium (1 h) or to 100 ng/ml of LPS (1h and 12 h). Nuclei were isolated, and nascent RNA strands were elongated and radiolabeled in vitro. The labeled RNA was hybridized to specific cDNA probes (COX-2, COX-1, and β-actin) fixed on nitrocellulose membranes. Full-length plasmids without the probes were also blotted on the membranes to evaluate background hybridization. The numbers under each lane represent the ratio between the densitometric values of COX-2 and actin (COX-2/actin) and are the measure of the relative level of transcription. The figure represents results from two independent experiments. B, EMSA for the distal NF-κB probe was performed with nuclear extracts of cells treated with medium only (lane 2) or stimulated with 100 ng/ml of LPS for 1 h (lane3) or 12 h (lane 4). Specificity of binding was determined from cell extracts treated with LPS for 1 h by competition using a 10-fold (10×) or 50-fold (50×) excess of unlabeled distal probe (lanes 5 and 6). Lane 1 contained no nuclear extracts. Results are representative of three independent experiments.

FIGURE 2.

The transcriptional activation of COX-2 gene by LPS is transient. A, Nuclear run-on analysis was performed on macrophages exposed to medium (1 h) or to 100 ng/ml of LPS (1h and 12 h). Nuclei were isolated, and nascent RNA strands were elongated and radiolabeled in vitro. The labeled RNA was hybridized to specific cDNA probes (COX-2, COX-1, and β-actin) fixed on nitrocellulose membranes. Full-length plasmids without the probes were also blotted on the membranes to evaluate background hybridization. The numbers under each lane represent the ratio between the densitometric values of COX-2 and actin (COX-2/actin) and are the measure of the relative level of transcription. The figure represents results from two independent experiments. B, EMSA for the distal NF-κB probe was performed with nuclear extracts of cells treated with medium only (lane 2) or stimulated with 100 ng/ml of LPS for 1 h (lane3) or 12 h (lane 4). Specificity of binding was determined from cell extracts treated with LPS for 1 h by competition using a 10-fold (10×) or 50-fold (50×) excess of unlabeled distal probe (lanes 5 and 6). Lane 1 contained no nuclear extracts. Results are representative of three independent experiments.

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The kinetics of IL-1β and TNF-α protein released were determined by ELISA on the supernatants of macrophages stimulated with LPS for different times. TNF-α was secreted early (400 pg/ml after 2 h; data not shown), reached maximum levels by 6 h, and remained high even after 48 h (Table I). In contrast, IL-1β increased significantly only after 12 h and continued to accumulate for up to 48 h. These results indicated that in response to LPS, U937 human macrophages release proinflammatory cytokines (IL-1β and TNF-α) in a similar fashion as blood-derived monocytes (27). Based on the kinetics of the release of these cytokines, it is unlikely that they participate in the early transcriptional activation of COX-2. However, they may play a role in the maintenance of high COX-2 levels following LPS stimulation.

Table I.

IL-1β and TNF-α production by macrophages stimulated with LPS

Time (h)IL-1β (pg/ml)aTNF-α (pg/ml)a
<4 <8 
<4 1737.6 ± 528.9* 
12 50.6 ± 10.4* 1618.5 ± 186.3* 
24 116.5 ± 23.5* 1380.4 ± 257.8* 
48 180.5 ± 49.7* 1824.5 ± 142.1* 
Time (h)IL-1β (pg/ml)aTNF-α (pg/ml)a
<4 <8 
<4 1737.6 ± 528.9* 
12 50.6 ± 10.4* 1618.5 ± 186.3* 
24 116.5 ± 23.5* 1380.4 ± 257.8* 
48 180.5 ± 49.7* 1824.5 ± 142.1* 
a

Values are expressed as the mean ± SE from triplicates of three independent experiments. Macrophages were stimulated with 100 ng/ml of LPS for different times, and the supernatants were analyzed by ELISA.

b

, p < 0.01 when compared to control values by the Student t test.

Exogenous IL-1β stimulates a dose- and time-dependent increase in COX-2 mRNA and protein expression in human macrophages (17). To determine whether IL-1β released by LPS-stimulated macrophages could have an autocrine effect on the expression of COX-2 mRNA, the following experiment was performed. Macrophages were stimulated for different times with LPS (6, 12, and 24 h) or with IL-1β (only for 12 h) in the presence or the absence of neutralizing anti-IL-1β Abs. As shown in Fig. 3,A, neutralizing Abs to IL-1β did not affect COX-2 mRNA expression induced by LPS regardless of the time of stimulation. In contrast, the neutralizing Abs inhibited the expression of COX-2 induced by IL-1β after 12 h. As expected, the control Ab (normal goat IgG) had no effect on either LPS- or IL-1β-induced COX-2 mRNA expression. Similar results were observed when the levels of COX-2 protein were evaluated by immunoblot (Fig. 3 B). These data clearly suggest that endogenous IL-1β does not contribute to the maintenance of the steady state levels of COX-2 mRNA induced by LPS.

FIGURE 3.

Inhibition of endogenous IL-1β does not alter COX-2 expression. A, U937 macrophages were stimulated with 100 ng/ml of LPS for 6, 12, or 24 h in the presence or the absence of neutralizing anti-IL-1β Abs (10 μg/ml). The specificity of the Abs was confirmed by inhibition of IL-1β-induced COX-2 mRNA expression after 12 h. Normal goat IgG (10 μg/ml) was used as a control (c) for the effect of the Abs. Northern blot analysis was performed from total RNA as described. B, Immunoblot analysis was performed on samples from A. Similar results were obtained from four independent experiments.

FIGURE 3.

Inhibition of endogenous IL-1β does not alter COX-2 expression. A, U937 macrophages were stimulated with 100 ng/ml of LPS for 6, 12, or 24 h in the presence or the absence of neutralizing anti-IL-1β Abs (10 μg/ml). The specificity of the Abs was confirmed by inhibition of IL-1β-induced COX-2 mRNA expression after 12 h. Normal goat IgG (10 μg/ml) was used as a control (c) for the effect of the Abs. Northern blot analysis was performed from total RNA as described. B, Immunoblot analysis was performed on samples from A. Similar results were obtained from four independent experiments.

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We next determined whether endogenous TNF-α secreted by LPS-stimulated cells could contribute to the expression of COX-2. Macrophages were treated with LPS for 12, 24, and 48 h in the presence or the absence of neutralizing Abs against TNF-α. As shown in Fig. 4, Abs against TNF-α did not alter COX-2 mRNA expression regardless of the time examined. Even when the cells were exposed to LPS and anti-TNF-α Abs for shorter periods (1 and 3 h), the expression of COX-2 mRNA induced by LPS was not affected (data not shown). In contrast to IL-1β, exogenous TNF-α (up to 50 ng/ml) did not stimulate COX-2 mRNA expression in human macrophages. Taken together, these results suggest that the proinflammatory cytokines IL-1β and TNF-α secreted in response to LPS stimulation do not play a costimulatory role in the maintenance of COX-2 mRNA.

FIGURE 4.

Neutralization of endogenous TNF-α has no effect on COX-2 mRNA expression. Macrophages were treated with LPS (100 ng/ml) for 12, 24, or 48 h in the presence or the absence of neutralizing anti-TNF-α Abs (10 μg/ml). As a control for the Abs, cells were incubated with LPS in the presence of 10 μg/ml of normal goat IgG (c). COX-2 mRNA expression was evaluated by Northern blot as described. The figure represents results from three independent experiments.

FIGURE 4.

Neutralization of endogenous TNF-α has no effect on COX-2 mRNA expression. Macrophages were treated with LPS (100 ng/ml) for 12, 24, or 48 h in the presence or the absence of neutralizing anti-TNF-α Abs (10 μg/ml). As a control for the Abs, cells were incubated with LPS in the presence of 10 μg/ml of normal goat IgG (c). COX-2 mRNA expression was evaluated by Northern blot as described. The figure represents results from three independent experiments.

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We and others have shown that IL-1β can increase the rate of transcription of the COX-2 gene (17, 18). Moreover, IL-1β stabilizes the transcripts, inhibiting their rapid turnover by an unclear mechanism (19, 20). The data in Fig. 4 clearly show that COX-2 mRNA levels were elevated after 24 and 48 h of LPS stimulation even though the transcription rate declined to basal levels by 12 h (Fig. 2). Accordingly, we investigated whether LPS can regulate COX-2 expression at the posttranscriptional level. To determine the stability of LPS-induced COX-2 mRNA, macrophages were stimulated with LPS for 12 h followed by the addition of AD, and the fate of the COX-2 mRNA was evaluated at different times. In a second group of cells, LPS was removed from the cell medium after 12-h stimulation, AD was added or not, and the levels of COX-2 mRNA were evaluated thereafter. As shown in Fig. 5,A, only when LPS was removed during the chase period and in the absence of AD did the levels of COX-2 mRNA decrease rapidly and significantly over time. Fig. 5 B shows similar results from experiments in which macrophages were stimulated with IL-1β for 12 h. The half-lives of the COX-2 transcripts following the removal of LPS and IL-1β were 5.04 and 5.30 h, respectively. These results suggest that LPS can stabilize COX-2 mRNA and that active transcription is necessary for its decay.

FIGURE 5.

LPS and IL-1β stabilize COX-2 mRNA. A, Cells were stimulated with LPS (100 ng/ml) for 12 h and then treated or not with 10 μg/ml of AD for 2–8 more h (Chasing time). In another group of cells, LPS was removed after 12 h of stimulation (wsh) and replaced with fresh medium, then AD (10 μg/ml) was added or not during the chase period. The t1/2 (50% stability) after LPS removal was 5.04 h (r2 = 0.9197). B, Macrophages stimulated for 12 h with 10 ng/ml of IL-1β were then treated similarly as in A, except that the length of the chase period with or without AD was 2–12 h. The t1/2 of the transcripts after IL-1β removal was 5.30 h (r2 = 0.9470). Total RNA was isolated at each time point, and COX-2 mRNA levels were analyzed by Northern blot. The half-life of the transcripts was calculated by regression analysis. Results represent the average of three independent experiments.

FIGURE 5.

LPS and IL-1β stabilize COX-2 mRNA. A, Cells were stimulated with LPS (100 ng/ml) for 12 h and then treated or not with 10 μg/ml of AD for 2–8 more h (Chasing time). In another group of cells, LPS was removed after 12 h of stimulation (wsh) and replaced with fresh medium, then AD (10 μg/ml) was added or not during the chase period. The t1/2 (50% stability) after LPS removal was 5.04 h (r2 = 0.9197). B, Macrophages stimulated for 12 h with 10 ng/ml of IL-1β were then treated similarly as in A, except that the length of the chase period with or without AD was 2–12 h. The t1/2 of the transcripts after IL-1β removal was 5.30 h (r2 = 0.9470). Total RNA was isolated at each time point, and COX-2 mRNA levels were analyzed by Northern blot. The half-life of the transcripts was calculated by regression analysis. Results represent the average of three independent experiments.

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To assess whether the proinflammatory cytokines IL-1β and TNF-α released by LPS-stimulated cells can modulate the stability of the COX-2 transcripts, macrophages were exposed to LPS for 12 h followed by the addition of neutralizing Abs against the cytokines. As shown in Fig. 6, the presence of the Abs during the chase period (up to 8 h) did not affect the stability of COX-2 transcripts, clearly indicating that endogenous IL-1β and TNF-α do not participate in the posttranscriptional regulation of COX-2 by LPS.

FIGURE 6.

Endogenous IL-1β and TNF-α do not contribute to LPS-induced stabilization of COX-2 mRNA. Macrophages were treated with 100 ng/ml of LPS for 12 h, and then neutralizing anti-IL-1β (10 μg/ml) plus anti-TNF-α (10 μg/ml) Abs were added or not (Abs); goat IgG (20 μg/ml) was used as a control (cAb). The cells were harvested at the times indicated after the additions to evaluate the levels of COX-2 mRNA by Northern blot. The data represent the average of two independent experiments.

FIGURE 6.

Endogenous IL-1β and TNF-α do not contribute to LPS-induced stabilization of COX-2 mRNA. Macrophages were treated with 100 ng/ml of LPS for 12 h, and then neutralizing anti-IL-1β (10 μg/ml) plus anti-TNF-α (10 μg/ml) Abs were added or not (Abs); goat IgG (20 μg/ml) was used as a control (cAb). The cells were harvested at the times indicated after the additions to evaluate the levels of COX-2 mRNA by Northern blot. The data represent the average of two independent experiments.

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Recent evidence indicates that DEX can inhibit COX-2 expression at the transcriptional level (22), but also increases the turnover rate of the COX-2 transcripts induced by IL-1β (28). To determine whether DEX could promote the decay of COX-2 mRNA induced by LPS, macrophages were stimulated for 12 h followed by the addition of DEX or DEX plus AD, and the levels of COX-2 mRNA were evaluated by Northern blot. Fig. 7 shows that COX-2 transcripts were rapidly degraded in the presence of DEX, whereas their level remained high throughout the chase period in the absence of DEX or in the presence of DEX plus AD. The half-life of LPS-induced COX-2 transcripts was 2.5 h. These results indicate that DEX promotes the rapid decay of the stable COX-2 transcripts induced by LPS and that such an effect depends on active transcription.

FIGURE 7.

DEX promotes the rapid decay of COX-2 mRNA induced by LPS. Macrophages were stimulated with 100 ng/ml of LPS for 12 h and then DEX (1 μM) or DEX (1 μM) plus AD (10 μg/ml) was added or not (CONTROL) for 2–8 h during the chase period. The t1/2 of COX-2 mRNA in the presence of DEX was 2.5 h (r2 = 0.9053). Northern blot analysis was performed to evaluate the levels of COX-2 mRNA after addition of DEX or AD. Only the blots corresponding to COX-2 are shown in the top panels. Results represent the average of three independent experiments.

FIGURE 7.

DEX promotes the rapid decay of COX-2 mRNA induced by LPS. Macrophages were stimulated with 100 ng/ml of LPS for 12 h and then DEX (1 μM) or DEX (1 μM) plus AD (10 μg/ml) was added or not (CONTROL) for 2–8 h during the chase period. The t1/2 of COX-2 mRNA in the presence of DEX was 2.5 h (r2 = 0.9053). Northern blot analysis was performed to evaluate the levels of COX-2 mRNA after addition of DEX or AD. Only the blots corresponding to COX-2 are shown in the top panels. Results represent the average of three independent experiments.

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PGs are important mediators released during septic shock; PGI2 is a vasodilator that can contribute to hypotension, and PGE2 plays a prominent role in fever and pain. LPS is a potent stimulus for the synthesis of high levels of PGs, which occurs through the induction of the enzyme COX-2. In this study we investigated the role of the proinflammatory cytokines TNF-α and IL-1β released by LPS-stimulated human macrophages on the expression of COX-2. We found that neutralizing Abs against TNF-α and IL-1β did not block the induction or the prolonged expression of COX-2 mRNA and protein after LPS stimulation. Furthermore, LPS transiently activated COX-2 gene transcription and stabilized the COX-2 transcripts. Interestingly, COX-2 mRNA decay was inhibited by AD, but was promoted by DEX. Removal of LPS from the medium also promoted the degradation of COX-2 mRNA with a similar turnover rate as IL-1β-induced transcripts.

We have previously shown that LPS can stimulate the expression of COX-2 mRNA in U937 human macrophages (17, 29). In the present report we showed a sustained expression of COX-2 mRNA even after 48 h. Likewise, the COX-2 protein was detected after 6 h, and high levels were still observed after 24 h (Fig. 1 A) and 48 h (data not shown). However, high PGE2 levels were not observed until 12 and 24 h. The lag period between the presence of the protein and the product may reflect the lack of substrate availability or a downstream enzyme. This may be due to a delayed expression of either the cytosolic PLA2 (30, 31) or the putative inducible PGE2 synthase, respectively (30, 32).

The presence of polyclonal Abs against TNF-α and IL-1β had no effect on the expression levels of COX-2 mRNA and protein in LPS-stimulated cells. This is in contrast with other studies that have shown that neutralizing Abs against TNF-α and IL-1β can significantly inhibit COX activity measured by the production of 6-oxo-PGF in bovine endothelial cells (12). In that system TNF-α played a major role in the induction of COX-2 after LPS stimulation. Moreover, exogenous TNF-α enhanced COX activity. In contrast to IL-1β, TNF-α alone did not induce COX-2 expression in our study. Even though large quantities of TNF-α were produced by U937 macrophages following LPS stimulation, there was no apparent participation of this cytokine in COX-2 induction or in the maintenance of the high mRNA levels. Thus, these results indicate that proinflammatory cytokines can exert distinct and specific effects on different cell types. For example, TNF-α stimulates COX-2 expression in human mesothelial cells (33) and synovial fibroblasts (34), while it has no effect on human macrophages (29) and monocytes (13) unless used at very high concentrations (35). This difference in species and cell type may be derived from the diversity in the sequences of the promoter region of the COX-2 gene and the class and number of receptors expressed on the cells. Similarly, the lack of effect of neutralizing Abs against IL-1β on COX-2 mRNA levels also indicated that endogenous IL-1β does not play a role in the regulation of COX-2. Although exogenous IL-1β easily induces COX-2 expression in our system (17), the lag time for IL-1β release may hamper the contribution of this cytokine to the rapid transcriptional activation by LPS. Likewise, the amount of endogenous IL-1β released in response to LPS may not be high enough to participate in the induction of COX-2 mRNA or protein expression. We have found that a concentration of at least 100–500 pg/ml of IL-1β is necessary to induce detectable levels of COX-2 mRNA. Moreover, we did not observe a significant synergistic effect when the macrophages were treated simultaneously with LPS plus IL-1β (our unpublished observations).

At present we cannot rule out the participation of other endogenous cytokines in COX-2 regulation. Recent evidence suggests that endogenous GM-CSF is involved in LPS-stimulated COX-2 protein expression in human monocytes (36). This effect occurs through the delayed activation of STAT5 (90 min). Although endogenous GM-CSF may be playing a role in the maintenance of high levels of COX-2 mRNA or protein, our run-on assays clearly indicated that LPS promptly activates the transcription of the COX-2 gene within 60 min. This was consistent with the activation of NF-κB as shown by EMSA after 1 h of LPS stimulation (Fig. 2 B). The involvement of two NF-κB motifs present in the COX-2 promoter, after LPS or IL-1β stimulation, has been reported in mouse macrophages (23), human pulmonary cells (18), and U937 human macrophages (22). In previous studies, the presence of p50 and p65 NF-κB proteins in the complexes was shown by supershift assays (18, 22). Inoue and Tanabe (22) observed a DNA-protein complex using the proximal NF-κB motif as a probe; interestingly, we did not detect any complex using that same probe. A likely explanation for this is the different binding affinities of the p50/p65 NF-κB heterodimer for the two DNA motifs (18). The involvement of other cis-elements, such as NF for IL-6 expression and cAMP response element sites, in the regulation of the human COX-2 gene by LPS has also been reported (37). The contribution of other endogenous cytokines to LPS-induced COX-2 expression and the transcription factors involved requires further investigation.

The nuclear run-on assays indicated that LPS exerts a rapid but transient transcriptional activation of the COX-2 gene. The high levels of COX-2 transcripts observed after 48 h of stimulation, however, also suggested a posttranscriptional modulation. COX-2 was discovered as an immediate-early gene that can be induced rapidly and transiently by serum and mitogens (38, 39). Moreover, the 3′-UTR of COX-2 mRNA has 22 copies of the pentamer AUUUA (40), which is considered to be involved in the instability of mRNA for some cytokines and oncogenes (41, 42). Despite these findings, several reports have demonstrated that IL-1 can stabilize COX-2 transcripts (8, 17, 19). Our results indicated that LPS is able to stabilize COX-2 mRNA in a similar manner as IL-1β. We also presented evidence showing that endogenous IL-1β and TNF-α released after LPS stimulation did not contribute to the stabilization of COX-2 transcripts. The addition of AD, which in other systems allows determination of the mRNA turnover rate, inhibited the degradation of COX-2 transcripts induced by LPS or IL-1β. Only when the stimuli were removed did the levels of COX-2 mRNA decline. A potential explanation for this phenomenon is that both LPS and IL-1β induce the expression or activation of labile molecule(s) involved in COX-2 mRNA stabilization. When the stimulus is removed, this unstable molecule(s) is degraded or inactivated, and therefore, COX-2 mRNA is also degraded, probably by a short-lived but constitutively transcribed RNase. However, following removal of the stimulus in the presence of AD, the basal transcription of the RNase is inhibited, and consequently, the degradation of COX-2 transcripts is suppressed. This hypothesis is consistent with evidence indicating the existence of proteins that bind to the 3′-UTR and have the potential ability to stabilize different mRNAs (43, 44, 45). This is supported by the identification of stabilizing cis elements in the 3′-UTR (20) and of cytosolic proteins that bind to COX-2 mRNA after IL-1β stimulation (19). These elements may be acting in concert to prolong the half-life of the transcripts. The inhibition of COX-2 mRNA decay by AD is not specific for our system, because a similar effect has been observed for the urokinase-type plasminogen activator and GM-CSF transcripts, although no mechanism has been proposed for it (46, 47).

In contrast to the stabilizing effect of LPS or IL-1β, DEX may be responsible for the induction or activation of RNases or other proteins that mediate COX-2 mRNA turnover. The destabilizing effect of DEX on IL-1β-induced COX-2 transcripts has been previously shown (28). Interestingly, the half-life of the 4.6-kb COX-2 mRNA isoform in the presence of DEX is ∼3 times shorter in lung fibroblasts (28) than in IL-1β or LPS-stimulated macrophages (Ref. 17 and this report). Although we have no explanation for this, the machinery for mRNA degradation may be differentially expressed depending on the cell type. Recently, it has been reported that the faster degradation of COX-2 transcripts induced by DEX in epithelial cells depends on active transcription and translation, and involves shortening of the COX-2 poly(A) tail (48).

In summary, we have demonstrated that LPS can regulate the COX-2 gene by transcriptional and posttranscriptional mechanisms in human macrophages. First, we showed that the proinflammatory cytokines IL-1β and TNF-α produced by LPS-stimulated cells did not contribute to either the maintenance or the stability of COX-2 mRNA. Second, LPS promoted the transcription of the COX-2 gene and the stability of COX-2 mRNA in a similar manner as IL-1β. Finally, active transcription in the cells or the presence of DEX was required for the fast degradation of COX-2 mRNA. Elucidation of the molecular mechanisms underlying the regulation of COX-2 at both transcriptional and posttranscriptional levels may open new avenues in the design of more specific strategies for the treatment of inflammatory diseases.

We thank Kathy Keller for her excellent technical assistance, and members of Dr. Chadee’s laboratory for helpful discussions and critical review of the manuscript.

1

This work was supported by grants from the Medical Research Council of Canada, the Natural Sciences and Engineering Research Council of Canada, and the Crohn’s and Colitis Foundation of Canada. Research at the Institute of Parasitology is partially funded by the Fonds Pour la Formation de Chercheurs et l’Aide à la Recherche du Québec. M.B.-R. is the recipient of a Ph.D. fellowship from the National Council of Science and Technology of Mexico.

3

Abbreviations used in this paper: COX, cyclooxygenase; PMA, phorbol 12-myristate 13-acetate; AD, actinomycin D; DEX, dexamethasone; 3′-UTR, 3′-untranslated region.

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