INTRODUCTION

Macrophages are known to generate prostaglandins (PGs)¹ in response to various stimuli such as endotoxin (1, 2), phorbol myristate acetate (3), or phagocytic particles (4). In addition to playing important roles in such biologic processes as cell proliferation (5), inflammatory and immune responses (6-8), and the production of extracellular matrix proteins (9), prostaglandins may also act in an autocrine/paracrine fashion to modulate the responses of the macrophages themselves (10-12). We have previously shown that in murine macrophages, prostaglandin E₂ (PGE₂) increased the synthesis of insulin-like growth factor-1 (IGF-1), a growth factor for fibroblasts, by a TNF-independent signaling pathway. The effect of TNF on this PGE₂-induced process was additive (13). Since macrophages are themselves a potent source of PGE₂, autocrine up-regulation of IGF-1 was a distinct possibility. Furthermore, TNF might itself alter the IGF-1 response by, in part, enhancing production of PGE₂. Accordingly, in this study, we addressed the effects of TNF and PGE₂ on PGE₂ production in murine bone marrow-derived macrophages. Since PGE₂ acts by receptor-mediated generation of cyclic AMP and activation of protein kinase A (PKA), the effect of direct addition of dibutyryl cyclic AMP (dbcAMP) was also examined.

The first enzymatic step in eicosanoid synthesis is the release of free arachidonic acid (AA) from membrane phospholipid. Depending on the cell type and stimulus, different forms of phospholipase A₂ (PLA₂), including an 85-kDa cytosolic PLA₂ (cPLA₂) (14, 15), 14-kDa secreted group II PLA₂ (sPLA₂) (16-18), or a calcium-independent cytosolic PLA₂ (19, 20), have each been described to play a role in mediating AA release and subsequent prostaglandin synthesis. Agents that stimulate AA release in macrophages, including zymosan, phorbol 12-myristate 13-acetate, calcium ionophore A23187, and okadaic acid, can activate cPLA₂ by enhancing serine phosphorylation of the enzyme (21). More recently, a role for mitogen-activated protein kinase has been described in mediation of agonist-induced activation of cPLA₂ (22, 23). Cytokine-induced changes in gene expression of sPLA₂ have been reported in rat mesangial cells (24) and human synovial cells (25), but the activity of these enzymes is presumably also regulated by the extent and location of their secretion.

Prostaglandin endoperoxide-synthase or cyclooxygenase (COX) catalyzes the conversion of AA to PGH₂, which is then metabolized by one or more terminal synthases to a variety of biologically active prostanoids (26). Cyclooxygenase is a key enzyme in prostanoid synthesis and possesses both fatty acid cyclooxygenase activity (producing PGG₂ from AA) and PG hydroperoxidase activity (converting PGG₂ to PGH₂). COX2 is a recently described form of cyclooxygenase that is induced in a number of cells by proinflammatory stimuli, which contrasts to the lack of induction seen with the previously characterized constitutive enzyme, COX1 (27). Thus, COX2 is thought to contribute to the generation of prostanoids at sites of inflammation (28), (see Ref. 29 for review).

In the current study, the ability of murine macrophages to synthesize PGE₂ in response to a phagocytic particle (zymosan) was selectively induced by TNF. In contrast, a concomitant decrease in PGD₂ production was observed after TNF priming, suggesting a striking switch from production of PGD₂ to PGE₂ and regulation of PG synthase activity. In addition, we show herein that activators of PKA markedly potentiated the TNF-induced increase in PGE₂ through up-regulation of COX2 gene expression. Our results suggest that (i) PGE₂ might act as an autocrine mediator to stimulate and to maintain the differentiation of uncommitted macrophages into IGF-1 producing cells and (ii) this differentiated cell type may participate in an exocrine fashion to regulate the inflammatory response by its ability to synthesize and release PGE₂.

EXPERIMENTAL PROCEDURES

C3H/HeJ mice were bred in the Biological Resource Center at the National Jewish Center. The C3H/HeJ strain is lipopolysaccharide hyporesponsive and was chosen to minimize the effects of trace lipopolysaccharide contamination (30). Approximately 8-week-old mice were killed by CO₂ narcosis.

Dulbecco's modified Eagle's medium was obtained from Bio-Whittaker (Walkersville, MD). Fetal bovine serum (Hybri-Max) as well as all chemical compounds were purchased from Sigma except for valeryl salicylate and NS-398, which were purchased from Cayman Chemical (Ann Arbor, MI). Nonidet P-40 was purchased from Boehringer Mannheim, and nitrocellulose membranes were purchased from Bio-Rad. Recombinant murine TNF was generously provided by Genentech, Inc. Zileuton (A64077) was obtained from Abbott Laboratories (North Chicago, IL). Zymosan was purchased from Sigma and opsonized using a human serum pool as described (3). The antibodies used included generous gifts of antibody to PGE₂ and PGD₂ (Dr. J. Maclouf, INSERM, Paris), 6-keto-PGF₁ (Dr. K. Allen, Colorado State University, Fort Collins, CO) and TXB₂ (Dr. Frank Fitzpatrick, University of Colorado Health Science Center, Denver). Antibodies for LTB₄ and LTC₄ were purchased from Advanced Magnetics (Boston, MA). RNAzol B was purchased from Tel-Test, Inc. (Friendswood, TX), and BCA protein assay was purchased from Pierce. Murine COX1 cDNA probe was purchased from Cayman Chemicals (Ann Arbor, MI), and murine COX2 mRNA analysis was performed using a 1.8-kilobase pair cDNA insert kindly provided by Dr Voelkel (UCHSC, Denver). Human glyceraldehyde 3-phosphodehydrogenase (GAPDH) cDNA was provided by J. Shannon (National Jewish Center, Denver). Both monoclonal antibodies were kindly provided by Dr. J. Maclouf (INSERM, Paris).

Murine bone marrow-derived macrophages were obtained using a technique previously described in detail (31). Dulbecco's modified Eagle's medium containing 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin, 0.37% (w/v) NaHCO₃, 10% (v/v) heat-inactivated fetal bovine serum, and 10% (v/v) L929 cell-conditioned medium as a source of colony-stimulating factor-1 was used for the isolation, culture, and stimulation of murine bone marrow-derived macrophages. Bone marrow cells were flushed asceptically from the dissected pelvises, femurs, and tibias of C3H/HeJ mice with a jet of complete medium directed through a 25-gauge needle to form a single cell suspension. The bone marrow cells were dispensed at a concentration of 0.45 × 10⁶ cells/well in 24-well tissue culture plates for the eicosanoid study, 11 × 10⁶ cells/100-mm diameter culture dishes for protein immunoblotting, and at a concentration of 1.8 × 10⁶ cells/well in 6-well tissue culture plates for RNA analysis. The cells were matured at 37 °C under a 10% (v/v) CO₂ atmosphere for 5 days before use.

After incubation of macrophages for the indicated period of time with various stimuli, the cell monolayers were washed twice in Ca²⁺, Mg²⁺-free phosphate-buffered saline, pH 7.4; then, cells were incubated for 1 h with the phagocytic stimulus (50 particles of opsonized zymosan per cell) or with 10 µM exogenous AA in 2% BSA-Dulbecco's modified Eagle's medium. The culture medium was centrifuged to remove nonadherent cells and assayed for AA metabolites using enzyme immunoassay methods previously described (32, 33). LTB₄, LTC₄, TXB₂, and 6-keto-PGF₁ (the stable metabolite of PGI₂) were assayed directly from fresh supernatants while PGD₂ and PGE₂ were measured as their methyl oximes after derivatization with methoxamine as described by Kelly et al. (34). Macrophage monolayers were scraped in 500 µl of 0.5 N sodium hydroxide, and total protein was determined using a BCA protein assay. Eicosanoid levels were all expressed as picograms per microgram of macrophage protein. Total content of free AA in both cell lysates and supernatants was measured using a quantitative mass spectrometric assay (35). Briefly, macrophage monolayers maintained in culture medium (0.45 × 10⁵ cells/ml) were lysed by adding 1 ml of MeOH directly in the well, and lysates (2 ml) were acidified to pH 3 with 100 µl of 1 N HCl. Free AA was extracted twice with one volume of hexane and dried under vacuum. The pentafluorobenzyl ester was synthesized by adding a 10% solution of pentafluorobenzyl bromide in acetonitrile (50 µl) and an equal volume of a 10% solution of diisopropylamine in acetonitrile and then allowed to stand at room temperature for 10 min. After vacuum evaporation of reagents, the derivative was dissolved in 100 µl of hexane for electron capture ionization mass spectrometric analysis (36). Free arachidonic acid levels were expressed as nanograms per well using a calibration curve.

Mouse macrophages were isolated, cultured, and stimulated as described above. Prostaglandin synthase activity was performed according to Kelner and Uglik (37). After a 16-h incubation period, cells (2 × 10⁶) were collected by scraping and resuspended in 100 µl of cold buffer (50 mM TRIS, pH 7.5, 0.1 M sodium chloride, 0.1 M EDTA). The cells were lysed by freezing/thawing them twice in a acetone-dry ice bath and centrifuged at 14,000 rpm in a microcentrifuge. Supernatants were warmed to 37 °C in a water bath for 1 min, and reduced glutathione (6 µg in 10 µl) was added for 1 min. Then, 150 ng of PGH₂ was added for 1 min. The reaction was terminated by adding 10 µl of a 25 mM FeCl₂ solution. Spontaneous conversion was evaluated by incubating PGH₂ in buffer alone with no cell. The concentrations of PGE₂ and PGD₂ at 0 and 1 min were determined by enzyme immunoassay.

The expression of COX1, COX2, and GAPDH mRNA transcripts was determined by Northern analysis. Total cellular RNA was extracted from macrophage monolayers with 1 ml of RNAzol B for 1.8 × 10⁶ cells (38). 10 µg of total RNA was electrophoresed under denaturing conditions through a 1.2% agarose-formaldehyde gel and then transferred to NYTRAN hybridization filters as described (39). Total RNA was covalently linked to the membrane by UV cross-linking (0.12 joules) using a Stratalinker 1800 (Stratagene). The blots were hybridized with 10⁶ dpm/ml of ³²P-labeled cDNA probes for 18 h and washed to a final stringency of 0.2 × SSC at 42 °C, and autoradiograms were prepared by exposure to Kodak X-OMAT AR film at 70 °C. To ensure that the differences in transcript expression were specific to COX1 and COX2 mRNA and not due to differential loading of RNA, the blots were stripped in 2% glycerol for 3 min at 100 °C and reprobed with GAPDH. The density of the autoradiographic signals was quantified by scanning densitometry combined with integration using the "Image 1.35" software run on a Macintosh microcomputer. The results were expressed as a ratio of COX to GAPDH expression as indicated.

Macrophage monolayers were scraped into ice-cold lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM dithiothreitol, and 1 mM aprotinin. 50 µg of total protein determined using a BCA protein assay were separated in SDS/10% polyacrylamide gels and transferred onto nitrocellulose membranes. The blots were washed in Tris-buffered saline (20 mM Tris, pH 7.6, 137 mM NaCl) with 0.05% (v/v) Tween 20 (TBST), blocked overnight with 5% (w/v) fat-free dry milk in TBST. The same blot was probed with a monoclonal antibody for COX1, stripped, and then reprobed with a monoclonal antibody for COX2.

Results are presented as the mean ± S.E. for at least three separate experiments. Comparisons between groups were made using the student's paired t test.

RESULTS

Effect of TNF, PGE₂, and dbcAMP on PGE₂ Synthesis and Release

Murine bone marrow-derived macrophages were cultured for 5 days in the presence of colony-stimulating factor-1, followed by priming with TNF, PGE₂, or dbcAMP alone or in combination for a period of 16 h. After the culture medium was removed and cell monolayers washed, macrophages were stimulated for 1 h with 50 particles per cell of opsonized zymosan in fresh culture medium to promote production and release of arachidonic acid metabolites. Previous analysis had shown this amount and time of exposure to zymosan to be optimal (data not shown). As shown in Fig. 1, 10 ng/ml TNF led to a 5-fold increase in PGE₂ production, whereas no change was observed in response to either 0.1 mM dbcAMP or 1 µM PGE₂ treatment alone. However, when cells were incubated with the combination TNF+dbcAMP or TNF+PGE₂, synergistic responses were observed corresponding to a 4-fold increase and a 2-fold increase compared with TNF alone for dbcAMP and PGE₂, respectively.

Production of PGE₂ in murine macrophages in response to TNF, PGE₂, and dbcAMP.

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To determine the point at which the TNF and PGE₂ were exerting their effect, the release of free AA was analyzed to explore the possibility that these effectors may affect the balance of deacylation and reacylation. To block metabolism of free AA released by COX and 5-lipoxygenase (5-LO), cells were incubated in the presence of 5 µM indomethacin to block the COX activity (40) and 5 µM zileuton to inhibit 5-lipoxygenase (41) for the 1-h stimulation period with zymosan. In Fig. 2, intracellular plus extracellular AA content is shown, as determined by mass spectrometry and expressed as nanogram/0.45 × 10⁶ cells. Independently of the presence of the inhibitors, no significant variations in AA release were observed in response to any of the stimuli. Differences observed in AA release between the presence and absence of the COX and 5-lipoxygenase inhibitors are likely due to utilization of AA as substrate. Similar results were obtained when AA release was analyzed after the cells had been labeled with [³H]AA (data not shown). These results suggest that the effect of TNF on PGE₂ levels with or without dbcAMP is not due to enhanced phospholipase activity.

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Effect of TNF, PGE₂, and dbcAMP on Synthesis and Release of Other Eicosanoids

To determine whether the observed induced increase in PGE₂ production was exclusive to PGE₂, the effect on other AA metabolites known to be produced by macrophages was examined. The production of two cyclooxygenase metabolites, TXB₂ and 6-keto-PGF₁ (the stable PGI₂ metabolite), as well as two 5-lipoxygenase products (LTC₄ and LTB₄), were quantified in the supernatant of cells primed for 18 h with the various stimuli and then stimulated with zymosan. Table I shows that untreated cells released high levels of TXB₂ (12.95 pg/µg), whereas release of the other eicosanoids was low. In contrast to PGE₂ (Fig. 1), TNF alone or in combination with dbcAMP did not significantly increase the production of any of these four AA metabolites studied. Interestingly, a slight but significant increase in TXB₂ and LTB₄ release was observed in dbcAMP-treated cells.

Table I. TXB2, 6-keto-PGF1, LTC4, and LTB4 production in murine macrophages Release of arachidonic acid metabolites was quantified in the supernatant as described in Fig. 1 and under "Experimental Procedures." Results are expressed as picograms per microgram protein, and values represent the mean ± S.E. of 5 (TXB2), 4 (LTB4), and 3 (LTC4) different cultures or represent the mean ± variation of two independent experiments (6-keto-PGF1). *, p < 0.05 compared with values of untreated. TXB2 6-keto-PGF1 LTC4 LTB4 Untreated 12.95  ± 1.86 0.46  ± 0.04 1.29  ± 0.54 0.33  ± 0.03 10 ng/ml TNF 7.91  ± 1.20 0.88  ± 0.51 0.717  ± 0.39 0.29  ± 0.11 0.1 mM dbcAMP 16.93  ± 3.09* 1.10  ± 0.80 1.74  ± 0.98 0.56  ± 0.05* TNF + dbcAMP 10.57  ± 0.83 1.95  ± 1.24 0.78  ± 0.46 0.71  ± 0.25

TNF Inversely Regulates PGD₂ and PGE₂ Production

In contrast to the effect of the cytokine on thromboxane, PGI₂, or leukotrienes, TNF had a dramatic inhibitory effect on the production of PGD₂. Fig. 3A depicts the dose-dependent variations in PGE₂ and PGD₂ synthesis and release in response to increasing concentration of TNF. Whereas unprimed macrophages, when stimulated with zymosan, synthesize and release high amounts of PGD₂ (22.89 pg ± 4.99 pg of PGD₂/µg of total protein; n = 6), the release of PGE₂ by these same cells remained at a low level (1.75 pg ± 0.34 pg/µg). However, when cells were incubated for a 16-h period with increasing concentration of TNF (0.01 to 100 ng/ml), a simultaneous decrease in PGD₂ release and increase in PGE₂ production was observed that dropped to 7.5 ± 0.88 pg/µg and increased to 10.33 ± 0.3 pg/µg, respectively (at 10 ng/ml TNF). Interestingly, the overall amount of released prostaglandins (PGE₂+PGD₂) remained unchanged (15.2 pg/µg for no TNF versus 15.5 with 10 ng/ml TNF stimulation; Fig. 3A). The same reciprocal pattern of prostaglandin production was seen when cells were incubated with 10 ng/ml TNF for 16 h and then stimulated for 1 h with 10 µM exogenous AA instead of zymosan (Fig. 3B), suggesting that the role of zymosan is to stimulate PLA₂, thus generating the AA, and that the effects of TNF are downstream of this step. All together, the results shown in Fig. 3 suggest that TNF might act by switching the synthesis of prostaglandins from the PGD₂ to the PGE₂ pathway.

Regulation of PGE₂ and PGD₂ production by TNF.

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To further investigate the molecular basis involved in the regulation of PGE₂ and PGD₂ synthesis in murine macrophages, cells were treated with 10 ng/ml TNF alone or in combination with 0.1 mM dbcAMP in the presence or absence of 5 µg/ml cycloheximide or 0.5 µg/ml actinomycin D. As illustrated in Fig. 4, the observed increase in PGE₂ production after stimulation of cells with TNF alone or in combination with dbcAMP was completely abolished by either cycloheximide or actinomycin D, suggesting an absolute requirement for both RNA and protein synthesis. In contrast, neither cycloheximide nor actinomycin D prevented the inhibition of PGD₂ production by TNF. Interestingly, only cycloheximide but not actinomycin D inhibited basal PGD₂ levels. These results suggest that, unlike the TNF+dbcAMP-mediated induction of PGE₂ synthesis, basal production of PGD₂ does not require new RNA synthesis. The observed decrease in PGD₂ synthesis following cycloheximide treatment might reflect the turnover of constitutive enzyme(s) involved in the basal production of this prostaglandin.

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TNF and dbcAMP or PGE₂ Synergistically Up-regulate COX2 Gene Expression and Protein Synthesis

Since protein and RNA synthesis were involved in the enhanced production of PGE₂ by the combination of TNF+dbcAMP and because basal production of PGD₂ was dependent of protein synthesis, we next examined steady state mRNA levels of the constitutive (COX1) and the inducible form (COX2) of cyclooxygenase. Messenger RNA levels were analyzed by Northern blot after a 12-h stimulation period, and hybridization with a GAPDH cDNA probe was performed to ensure for equal loading of total RNA. As shown in Fig. 5, COX1 mRNA was constitutively expressed in control macrophages while COX2 mRNA level remained undetectable. Neither 1 µM PGE₂ nor 0.1 mM dbcAMP affected the expression of either COX1 or COX2, whereas 10 ng/ml TNF inhibited the basal expression of COX1 mRNA and seemed to slightly increase COX2 mRNA levels. When cells were incubated with the combination TNF+PGE₂ or dbcAMP, elevation in COX2 mRNA levels was observed. The TNF-induced inhibition of COX1 mRNA expression was not significantly altered by the presence of PGE₂ or dbcAMP. As seen in Fig. 6, Western blot analysis of macrophage whole cell lysates using specific monoclonal antibodies for COX1 and COX2 showed that the COX2 protein levels paralleled those of the transcript. The low content of COX2 protein in untreated cells was not affected by any of the stimuli used alone but was enhanced when cells were incubated with the combination of TNF+dbcAMP. In contrast, the basal expression of COX1 protein remained unchanged in response to any of the treatments. Although the TNF-induced down-regulation of COX1 mRNA levels was observed at 12 h, no significant change in the corresponding protein was seen after 18 h. This latter observation suggests that the constitutive form of COX may be relatively stable so that protein levels were not yet affected at 18 h despite the absence of transcript at 12 h.

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To further characterize which COX isozyme was involved in the up-regulated synthesis of PGE₂, inhibitors of COX1 and/or COX2 activities were used. As shown in Fig. 7A, the COX inhibitor indomethacin totally prevented the TNF+dbcAMP-induced increase in PGE₂ synthesis. The COX2 inhibitor NS-398 (40) duplicated the indomethacin results. However, a concentration 10 times higher (10 µM versus 1 µM) was required. In contrast, neither sulindac sulfide (42) nor valeryl salicylic acid (43), which are 10 times more potent inhibitors of COX1 activity, decreased PGE₂ levels even when used at 10 µM. Similar patterns of inhibition were obtained when cells were primed with TNF alone (data not shown). To determine whether the basal levels of PGD₂ were produced as a consequence of COX1 or COX2 activity, PGD₂ release was analyzed in untreated macrophages incubated with increasing concentration of the inhibitors used above. Results shown in Fig. 7B suggest that COX2 and not COX1 is involved in PGD₂ synthesis despite the low basal amounts of both COX2 mRNA and protein.

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Glucocorticoids have been reported to prevent the induction of COX2 (44, 45). To further examine the possibility that only COX2 was involved in production of PGD₂ (before priming) as well as the induced synthesis of PGE₂, the effect of steroids on COX1 and COX2 mRNA levels as well as PGE₂ and PGD₂ production was examined. At 12 h, total RNA was extracted, and the same membrane was consecutively hybridized with COX2, COX1, and GAPDH cDNA probes (Fig. 8). After scanning of the autoradiographs, COX1 and COX2 values were normalized to the density of the GAPDH band. Dexamethasone prevented the TNF+dbcAMP-induced increase in COX2/GAPDH mRNA level by 70%, whereas slight increase in steady state levels of COX1/GAPDH mRNA was observed in control. To analyze the effect of glucocorticoids on PGE₂ and PGD₂ production, cells were primed with stimuli for 16 h as before, but because glucocorticoids inhibit PLA₂ activity (46), cells were incubated in the presence of 10 µM exogenous arachidonic acid rather than zymosan to bypass the requirement for PLA₂. As depicted in Fig. 9, the presence of 1 µM dexamethasone inhibited both constitutive production of PGD₂ (Fig. 9B) and induced increase in PGE₂ synthesis (Fig. 9A), suggesting that expression of COX2 is necessary for both the synthesis and release of these two prostaglandins.

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TNF Increases PGE₂ Synthase Activity

To determine whether PGE₂ and/or PGD₂ synthase activities were modified following TNF treatment, conversion of exogenously supplied PGH₂ to both PGE₂ and PGD₂ was examined. As shown in Fig. 10, a 16-h preincubation period with 10 ng/ml TNF induces a 5.2-fold increase in PGE₂ synthase activity after incubation of cell lysate for 1 min with 150 ng of exogenous PGH₂. This increase in PGE₂ synthesis is similar to the one observed when TNF-primed cells were incubated with zymozan (Fig. 1). When PGD₂ synthase activity was examined, no difference between unstimulated and TNF-treated macrophages was observed, suggesting that TNF increases PGE₂ synthase activity without affecting PGD₂ synthase activity. We next examined the effect of RNA and protein synthesis inhibitors on the activity of the PGE₂ synthase (Fig. 11). TNF-induced increase in PGE₂ synthase activity was abolished using either the protein synthesis inhibitor cycloheximide (CHX) or the RNA synthesis inhibitors actinomycin D (AD) or dichlororibofuranosylbenzimidazole. These results suggest that up-regulation of PGE₂ synthase activity by TNF is dependent of both protein and RNA synthesis.

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TNF-induced increase in PGE₂ synthase activity is dependent of protein and RNA synthesis.

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DISCUSSION

As shown herein, stimulation of murine macrophages by TNF induced a switch in prostaglandin synthesis from PGD₂ to the PGE₂ without affecting production of other eicosanoids. We also demonstrated that PGE₂ itself as well as the membrane-permeable PKA activator dbcAMP markedly and selectively potentiated the TNF-induced increase in PGE₂ synthesis. This enhancement could not be explained by increasing activity of PLA₂ because no change in AA release was observed. In contrast, we suggest that the enhancing effect of cAMP (PGE₂) is mediated through up-regulation of COX2 gene expression. PGE₂ inhibits macrophage cytocidal activity and can act as an autocrine-negative feedback stimulus to limit this function (11, 12). In the other direction, we have previously shown that in murine macrophages, PGE₂ increased the synthesis of IGF-1 and thus might contribute an autocrine signal to enhance the role of macrophages in wound healing and fibrosis (13). Herein, we show that (i) TNF selectively induced the production of PGE₂ by increasing PGE₂ synthase activity and (ii) PGE₂ itself up-regulated its own synthesis by TNF-primed macrophages. These observations help refine the complex, and mutually exclusive, regulating pathways for synthesis of IGF-1 and for proteins involved in cytocidal macrophage function, respectively (47, 48).

The bone marrow-derived macrophages generated PGD₂ as their predominant prostanoid when stimulated to release arachidonate by uptake of zymosan (or upon addition of free arachidonate). In contrast, when incubated with TNF, the cells switched their production to PGE₂ without any significant changes in the release of other eicosanoids from either the 5-lipoxygenase or the cyclooxygenase pathways. The total level of PGs released (PGE₂ plus PGD₂) was similar in untreated and in TNF-treated cells. Our results suggest that TNF induced a switch in PGD₂ and PGE₂ synthesis from their common precursor PGH₂, i.e. that the cytokine was acting at the level of PGD₂ and/or PGE₂ synthases rather than at the level of active PLA₂ or cyclooxygenase. Such a switch has been reported by others in rat Kupffer cells where in vitro activation of protein kinase C resulted in reduced PGE₂ and enhanced PGD₂ synthase activity (49).

In our study, TNF did not change the amounts of arachidonate released by the uptake of zymosan. Furthermore, the switch was also evident when arachidonate was added directly without any phagocytic stimulus, i.e. in the absence of obvious PLA₂ activation. Our results also indicate that the TNF-induced switch to PGE₂ was not mediated through alteration of COX isotype usage. TNF did not modify either COX1 or COX2 protein levels at 18 h (the time of zymosan addition and prostaglandin measurement) despite a slight increase in COX2 and a decrease in COX1 mRNA steady state levels observed 12 h after TNF treatment. In addition, use of inhibitors of COX1 and COX2 suggested that COX2 but not COX1 was involved in both the production of PGD₂ before TNF treatment as well as the PGE₂ formation afterward.

To further investigate the possibility that TNF might act directly by either increasing PGE₂ synthase activity or by decreasing PGD₂ synthase activity, we incubated cell lysates from control or TNF-treated cultures with PGH₂ as a substrate before the measurement of prostaglandins. Our results provided evidence that TNF priming leads to preferential enzymatic conversion of PGH₂ into PGE₂ while PGD₂ generation remains unchanged (Fig. 10). Thus, the observed switch from PGD₂ to PGE₂ does not seem to be mediated by depletion of glutathione following TNF-induced oxidant stress since both PGE₂ and PGD₂ synthases are glutathione-dependent enzymes (50, 51). We next examined whether the observed TNF-induced increase in PGE₂ synthase activity was dependent on protein or RNA synthesis. Concomitant incubation of macrophages with TNF and either cycloheximide or actinomycin D demonstrated that both inhibitors abolished the TNF-induced effect (Fig. 11). The observation that cycloheximide or actinomycin D prevented the TNF-induced increase in PGE₂ synthesis suggests that TNF acts through up-regulation of PGE₂ synthase itself or of a regulatory protein (Fig. 4). In addition, TXB₂ levels did not change, suggesting that TNF acts selectively by inducing PGE₂ activity.

Taken together, our results demonstrate that PGE₂ synthase activity is increased in TNF-primed macrophages compared with unstimulated cells. The unchanged PGD₂ synthase activity suggests that TNF does not have any effect at this level. The TNF-induced switch from PGD₂ to PGE₂ observed in the absence of exogenously added PGH₂ may be the result of preferential use of the endogenous substrate PGH₂ by the PGE₂ synthase pathway.

In previous studies, we showed that in murine macrophages, PKA activators such as PGE₂ and dbcAMP stimulated the synthesis of IGF-1 by a process that was independent of, and additive with, that induced by TNF (13). As described herein, PKA activators had only a slight enhancing effect by themselves on eicosanoid synthesis in response to zymosan uptake. On the other hand, they did synergistically potentiate the TNF-induced increase in PGE₂ production. Synergism between cAMP elevating agents and TNF or interleukin-1 have also been reported in up-regulation of group II PLA₂ mRNA in rat mesangial cells (52-54). Agents that increase cellular levels of cAMP used alone have additionally been shown to increase secretion of PLA₂ from vascular smooth muscle cells (55) and in mesangial cells (54). However, when we examined free AA release by mass spectrometry, no significant change was observed in these macrophages after treatment with PKA activators used alone or in association with TNF.

However, the combination of TNF and dbcAMP together did markedly up-regulate COX2 protein and mRNA. Neither stimulus was effective by itself, although, as noted above, TNF decreased the steady state levels of mRNA for COX1. Incubation of cells with stimulus and cycloheximide or actinomycin D demonstrated that both protein and mRNA synthesis were necessary for the increase in PGE₂ production induced by TNF plus dbcAMP. Glucocorticoids such as dexamethasone inhibit the induction of COX2 in cultured cells with little effect on COX1 mRNA and protein levels (44, 45). In the current studies, dexamethasone inhibited TNF plus dbcAMP-induced COX2 mRNA levels as well as PGE₂ generation. The implication that COX2 was required for the PGE₂ synthesis was confirmed by use of COX2-selective inhibitors. Importantly, inhibitors of COX2 prevented PGE₂ production after TNF stimulation whether or not the process was further enhanced by dbcAMP. This suggested that COX2 was the critical enzyme involved even without its up-regulation by the combined stimuli. In fact, even the PGD₂ produced after zymosan stimulation of macrophages that had not been exposed to either TNF or PKA activators was prevented by COX2 inhibitors and not by agents more selective toward COX1. All together, our results suggest that TNF and dbcAMP synergistically up-regulate COX2 gene expression and, as a consequence, provide more substrate (PGH₂) for PGE₂ production in TNF-primed macrophages. The synergistic effect of the combination TNF+dbcAMP on COX2 expression might be explained by the up-regulation of TNF receptors by agents that increase cAMP levels as it has been described in vitro in human histiocytic lymphoma cell line U-937 (56). Interestingly, we showed herein that TNF alone leads to a slight increase in COX2 mRNA steady state levels (Fig. 5).

The observed switch from PGD₂ to PGE₂ synthesis after activation of murine macrophages is biologically relevant because, in these cells, PGD₂ did not increase PKA activation or intracellular levels of cAMP (data not shown), although others have reported that it can induce adenylate cyclase activity and PGE₂ production in endothelial cells (57). In contrast and as expected, PGE₂ was a potent activator of PKA and cAMP generation (13). Thus, PGE₂ might be considered to provide a positive feedback stimulus in TNF-primed macrophages for its own synthesis. Since TNF by itself induced the switch to PGE₂ production by increasing PGE₂ synthase activity, the PGE₂ so produced would be expected to further enhance its production via up-regulation of COX2 with subsequent positive (e.g. IGF-1 synthesis) or negative (e.g. cytocidal activity) autocrine/paracrine effects on the macrophages.