Cyclooxygenase-1-Dependent Prostaglandin Synthesis Modulates Tumor Necrosis Factor Alpha Secretion in Lipopolysachharide-Challenged Murine Resident Peritoneal Macrophages

: Comprehensive studies of prostaglandin (PG) synthesis in murine resident peritoneal macrophages (RPM) responding to bacterial lipopolysaccharide (LPS) revealed that the primary PGs produced by RPM were prostacyclin and PGE 2 . Detectable increases in net PG formation occurred within the first hour, and maximal PG formation had occurred by 6 to 10 hr after LPS addition. Free arachidonic acid levels rose and peaked at 1-2 hr after LPS addition and then returned to baseline. Cyclooxygenase-2 (COX-2) and microsomal PGE synthase levels markedly increased upon exposure of RPM to LPS, with the most rapid increases in protein expression occurring 2 to 6 hr after addition of the stimulus. RPM constitutively expressed high levels of COX-1. Studies using isoform selective inhibitors and RPM from mice bearing targeted deletions of ptgs-1 and ptgs-2 demonstrated that COX-1 contributes significantly to PG synthesis in RPM, especially during the initial 1-2 hr after LPS addition. Selective inhibition of either COX isoform resulted in increased secretion of tumor necrosis factor-alpha (TNF- a ), however this effect was much greater with the COX-1 than with the COX-2 inhibitor. These results demonstrate autocrine regulation of TNF- a secretion by endogenous PGs synthesized primarily by COX-1 in RPM, and suggest that COX-1 may play a significant role in the regulation of the early response to endotoxemia. A used COX-2 chemiluminescence from shown using cells incubated both the presence (LPS) and absence (CON) of LPS. The quantitative results for each blot were normalized to the COX-2 signal obtained for cells at 6 of incubation. Experimental conditions were the same for & B), but the blots were analyzed for the presence mPGES-1. The quantitative data in are normalized to the mPGES-1 chemiluminescence signal in the 24 hr samples. (E & Experimental conditions were the same (A & but the were analyzed for the presence of cPLA . The quantitative data are the cPLA 2 Experimental conditions were same as for but the blots were analyzed for the presence COX-1. The quantitative are normalized in the All quantitative results are the mean ± three separate experiments in which duplicate samples were analyzed.

and 0.10 mg/mL streptomycin (Sigma) (a-MEM/FCS). The cell suspension was plated onto 35 mm tissue culture dishes at 2 mL per dish and incubated for 2 hours at 37 o C in a humidified 5% CO 2 atmosphere. Non-adherent cells were removed by washing the plates four times with PBS, and the cultures were then incubated overnight in 2 mL of fresh a-MEM/FCS. The mean protein content of RPM cultures was 100 ± 10 mg/dish (8.2 ± 0.8 x 10 5 cells/dish). Immunoblotting for Protein Expression -The protein concentrations of 20 mL aliquots of macrophage cell lysates were determined using a BCA Protein Assay kit (Pierce) according to manufacturer's directions. Macrophage lysate samples containing 15 mg of protein were then subjected to SDS polyacrylamide gel electrophoresis using an 8% gel (12% for mPGES-1) overlaid with a 3% stacking gel. Proteins were transferred to a polyvinylidene fluoride 9 Assay for TNF-a -The concentration of TNF-a in cell culture medium was determined by using an OptEIA assay kit (PharMingen) according to manufacturer's instructions.

Synthesis of PGs by RPM in
Response to LPS -Murine RPM from C3H/HeN mice were incubated in the presence or absence of 100 ng/mL of LPS for periods of up to 24 hr, and the culture medium was analyzed for PGs by GC/MS. RPM secreted primarily prostacyclin (detected as its hydrolysis product, 6-keto-PGF 1a ) and PGE 2 in response to LPS (along with minor amounts of TXA 2 , which was detected as its hydrolysis product, TXB 2 ) (Fig. 1A).
Increased levels of PGs were detectable at the earliest time point measured (1 hr), but the most rapid rate of PG synthesis occurred from 1 to 6 hr (Fig. 1B). It is notable that PGE 2 was not the major product secreted by LPS-challenged RPM, but as shown in Fig. 1C, the relative proportion of PGE 2 increased in RPM cultures during the course of the incubation. By 24 h, 6-keto-PGF 1a reached levels of 3,300 ± 300 pmol/10 7 cells, and PGE 2 reached levels of 2,100 ± 300 pmol/10 7 cells (Fig. 1A, and Table I). In the absence of LPS, 6-keto-PGF 1a and PGE 2 levels remained constant at 70-120 pmol/10 7 cells and 18-27 pmol/10 7 cells, respectively.
Changes in 20:4 Levels in RPM During the LPS Response -In order to assess the availability of substrate for PG synthesis by LPS-challenged RPM, we measured total 20:4 levels (medium plus cells) in RPM cultures at varying times after addition of the stimulus. An elevation of free 20:4 was observed by 1 to 2 hr after LPS addition with maximal values reaching 250-400 pmol/10 7 cells, (Fig. 1D and Table II). Levels returned to control values by 6 hr. In the absence of LPS, free 20:4 remained constant at 90-140 pmol/10 7 cells in RPM cultures.
The data in Fig.1D reflect total free 20:4 present in the combined medium and macrophage cell lysates. In separate experiments, RPM cultures were incubated for 0, 2, or 6 hr in the presence of LPS, and cells plus the medium were analyzed separately for free 20:4 content.
The results showed that approximately 73-77% of the free 20:4 was localized to the cells regardless of the period of incubation with LPS. These results indicate that the maximum intracellular 20:4 content in RPM during LPS incubation reached values of 180-300 pmol/10 7 cells.

Changes in PG Synthetic Enzyme Levels in LPS-Challenged RPM -Prior evidence has
indicated that PG synthesis by macrophages in response to LPS is due exclusively to COX-2, implying that it cannot occur prior to COX-2 induction. As noted in Fig. 1A & B, however, a significant increase in net PG synthesis occurs in RPM within the first hour of incubation with LPS, before one would expect de novo COX-2 protein expression to have occurred. We therefore performed immunoblot analysis of RPM cell lysates to determine whether these cells constitutively express COX-2, and to ascertain the rate of appearance of new COX-2 protein. As shown in Fig. 2A & B, COX-2 levels were undetectable in RPM prior to LPS treatment and only a trace amount of the protein was observed after 1 hr of incubation. Protein levels markedly increased between 2 and 6 hr, and maximal levels were reached at 6 to 10 hr. Thus the PG synthesis occurring in RPM cultures during the first hour of LPS incubation could not easily be attributable to constitutive COX-2 expression. PGE 2 synthesis is dependent on three isoforms of PGE synthase, one that is cytosolic (cPGES), and two that are microsomal (mPGES-1 and mPGES-2) (62,63). LPS-dependent induction of mPGES-1 has been described in a number of cell systems (64)(65)(66), and such induction should explain the change in relative PGE 2 synthesis observed in RPM during the LPS response in these cells. As shown in Fig. 2C & D mPGES-1 protein levels markedly increased in RPM cultures in the presence of LPS.
The primary enzyme responsible for the release of free 20:4 in response to LPS is cPLA 2 , and induction of cPLA 2 in macrophages by LPS treatment has been reported (67,68). As shown in Fig. 2E & F, cPLA 2 was constitutively expressed in RPM. A gradual increase in cPLA 2 expression was observed after LPS treatment, although this increase was small compared to those observed for COX-2 and mPGES.

Role for COX-1 in the Macrophage LPS Response -Although PG synthesis in response to
LPS is believed to be exclusively attributable to COX-2 in most systems, the relatively rapid PG secretion by RPM during the first hour of LPS incubation led us to consider the possibility that this early response could be attributable to COX-1. As seen in Fig. 2G & H, a strong chemiluminescence signal corresponding to COX-1 protein suggested high levels of constitutive expression of this enzyme in RPM. This observation is not surprising, considering the ability of these cells to rapidly produce very large quantities of PGs in response to stimuli such as zymosan, immune complexes, phorbol ester, and calcium ionophores without prior treatment with agents that induce COX-2 expression (69)(70)(71)(72)(73)(74)(75).
In order to test the hypothesis that COX-1 contributes to LPS-mediated PG synthesis, we incubated RPM cultures with LPS in the presence and absence of 100 nM concentrations of SC560 and/or SC236. SC560 is reported to be a selective COX-1 inhibitor (IC 50 COX-1 = 9 nM, IC 50 COX-2 = 6.3 mM (76)), whereas SC236 is considered selective for COX-2 inhibition (IC 50 COX-2 = 5-10 nM, IC 50 COX-1 = 17 mM (77,78)). RPM were incubated for 1 hr in the presence or absence of these inhibitors, alone or in combination, followed by the addition of LPS.
Cultures were incubated for 2 or 6 hr after LPS addition. As seen in Fig. 3A, after 2 hr of incubation with LPS, SC236 caused an approximately 35% reduction in the synthesis of 6-ketoPGF 1a , and an approximately 30% reduction in the synthesis of PGE 2 . In contrast, SC560 caused a 90% inhibition of the synthesis of both PGs. The presence of both inhibitors resulted in a slightly greater inhibition than that seen with SC560 alone. In contrast, after 6 hr of incubation, each of the inhibitors resulted in an 85 -90% inhibition of both PGE 2 and 6-ketoPGF 1a synthesis, and the presence of both inhibitors resulted in a nearly 100% inhibition of PG formation (Fig.   3B). The inhibitors had no effect on free 20:4 content in the cell cultures after 2 or 6 hr of incubation with LPS, indicating that they did not cause a marked alteration in the availability of substrate ( Fig 3C). A small (approximately 20%) decrease in COX-2 protein expression was observed in the presence of either or both inhibitors as judged by chemiluminescence signal on immunoblot analysis (Fig. 3D).
The greater degree of inhibition of PG synthesis by SC560 than SC236 after 2 hr of incubation with LPS supports the hypothesis that COX-1 plays a role in this process. Since COX-2 levels are still relatively low at the 2 hr time point in RPM, it is reasonable that the contribution of COX-1 should be relatively greater at this time point. However, the observation that both SC236 and SC560 caused a >85% inhibition of PG synthesis at the 6 hr time point is inconsistent with the assumption that each inhibitor affects only the activity of the COX isoform for which it is selective. This apparent lack of specificity, which will be discussed in greater detail below, makes it difficult to draw conclusions concerning the role of COX-1 in LPSmediated PG synthesis with complete confidence based solely on inhibitor data.
In order to further explore the possible role of COX-1 in LPS-mediated PG synthesis, we isolated RPM from WT mice and mice bearing a targeted deletion of the gene encoding either COX-1 or COX-2 on the CD-1 background. RPM cultures were incubated for 0, 2, or 6 hr in the presence of 100 ng/mL LPS. Cell lysates were then analyzed by immunoblot for COX-1, COX-2, and cPLA 2 protein expression. Constitutive COX-1 levels were similar in COX-2 -/and WT macrophages, whereas this protein was undetectable in COX-1 -/macrophages ( Fig. 4A & B).
The induced expression of COX-2 was slightly lower in COX-1 -/than in WT macrophages, and undetectable in COX-2 -/cells ( Fig. 4C & D). The expression of cPLA 2 was similar in macrophages of all three genotypes, with a tendency toward slightly increased expression in COX-2 -/cells ( Fig. 4E & F).
RPM from CD-1 COX-1 -/mice produced very low levels of PGs after 2 hr of incubation with LPS, and by 6 hr, the levels achieved were only approximately 20% as high as those produced by CD-1 WT cells (Table III). These results support the hypothesis that COX-1 contributes significantly to PG synthesis in RPM, and is relatively more important at the early time points. This conclusion was supported by the pattern of PG synthesis observed in CD-1 COX-2 -/-RPM. These cells produced large quantities of PGs after only 2 hr of incubation, reaching levels approximately 14-fold greater than those produced by WT cells in the same time period. Between 2 hr and 6 hr, COX-2 -/-RPM synthesized additional PGs, reaching levels approximately 83% as great as those produced by WT cells. Thus, COX-1 is clearly able to respond to the LPS stimulus with PG synthesis in CD-1 COX-2 -/-RPM. However, the time course of PG formation is markedly different from that seen in WT cells, suggesting that compensatory mechanisms actually promote COX-1 activity in this RPM population.
These experiments were repeated using COX-1 -/and COX-2 -/mice on a C57BL/6 background. Age and sex-matched WT littermates were used as controls. As observed in CD-1 mice, immunoblot analysis showed no difference in COX-1 expression between COX-2 -/and WT mice or in COX-2 expression between COX-1 -/and WT mice. The expression of cPLA 2 was similar irrespective of the genotypes. The immunoblots also confirmed the absence of the protein for which the gene had been deleted (data not shown).
RPM from C57BL/6 COX-1 -/mice demonstrated a very similar pattern of PG synthesis as had been observed in the case of RPM from CD-1 COX-1 -/mice. PG synthesis was very low 2 hr after LPS addition, and remained reduced as compared to littermate WT RPM (COX-1 +/+ ) after 6 hr of incubation (Table III). Notably, however, the COX-1 -/-RPM produced 45% as much PG as their WT controls, as compared to only 20% for the corresponding experiment using CD-1 COX-1 -/-RPM. In the case of RPM from C57BL/6 COX-2 -/mice, the pattern of PG synthesis was very different from that obtained from CD-1 COX-2 -/-RPM. Rather than rapid, high levels of PG production, C57BL/6 COX-2 -/produced very low levels of PGs throughout the 6 hr incubation period, reaching a maximum of only about 3% of the WT control RPM (COX-2 +/+ ). These results suggest that in RPM from C57BL/6 mice COX-1 contributes to LPSdependent PG production, since RPM from COX-1 -/mice synthesize lower quantities of PGs than their WT counterparts. However, the results also appear to indicate that COX-1 cannot function in the absence of COX-2 in C57BL/6 RPM, since COX-2 -/-RPM produce almost no PGs even though their expression of COX-1 is comparable to that in WT mice.
The marked difference observed in PG synthesis between C57BL/6 and CD-1 COX-2 -/-RPM suggests a fundamental difference between the two strains of mice in their adaptation to the COX-2 gene deletion. In this context, it is interesting to note that RPM from WT CD-1 mice produce higher levels of PGs in response to LPS than do RPM from C3H/HeN mice, which in turn, produce higher levels of PGs than do RPM from C57BL/6 mice (Table I). Furthermore, the increase in free 20:4 levels after 2 hr of incubation with LPS is similar in WT CD-1 and C3H/HeN RPM, but lower in WT C57BL/6 RPM (Table II). It is notable that this difference in 20:4 release was not due to a marked difference in cPLA 2 expression between the strains as demonstrated by comparative immunoblot analysis (data not shown). A direct comparison of free 20:4 in RPM used for the gene deletion experiments indicated that levels were similar in LPS-stimulated CD-1 COX-1 -/and WT RPM, but levels in COX-2 -/-RPM were significantly higher (p < 0.05, Table IV). In contrast, there was little to no increase in free 20:4 levels after 2 hr of LPS treatment in any of the C57BL/6 RPM cultures (Table IV). The apparent lower release of free 20:4 may correlate with the lower quantities of PGs synthesized in C57BL/6 RPM as compared to RPM from the other strains that were studied, and may contribute to the differences in PG synthesis observed between CD-1 and C57BL/6 COX-2 -/-RPM.

COX-1-Dependent Autocrine Suppression of TNF-a Secretion in LPS-Challenged RPM -
The suppression of TNF-a secretion by PGE 2 and prostacyclin has been widely reported. This suppression is based on an inhibition of TNF-a mRNA synthesis, which occurs during the first 1 -3 hr of LPS treatment (79)(80)(81)(82)(83)(84)(85)(86). Therefore it was reasonable to hypothesize that the ability of RPM to produce significant quantities of PGs during the first two hours after LPS addition might lead to autocrine suppression of the secretion of this cytokine. As shown in Fig. 5A, TNF-a was undetectable in the medium of RPM cultured in the absence of LPS, and in LPS-treated cells, increases in the level of the cytokine were detectable only after 2 hr of incubation. TNF-a concentrations increased rapidly between 2 hr and 4 hr after LPS addition, reached a maximum (51 ± 20 ng/10 7 cells) at 6 hr of incubation and then decreased thereafter. The fact that TNF-a levels drop after 6 hr in RPM culture medium indicates that the cytokine must be unstable under these conditions and that RPM no longer produce it at a rate sufficient to compensate for the rate of disappearance. Whether the decrease in TNF-a is due to enzymatic degradation by RPM or to nonenzymatic decomposition is not known.
To test the hypothesis that early, COX-1-dependent PG synthesis suppresses TNF-a secretion, we examined the effects of SC236 and SC560, on the levels of this cytokine in the medium of LPS-treated RPM. Cells were preincubated for 1 hr with 100 nM of SC236 and then incubated for an additional 6 hr with LPS. This treatment effected a small (1.8-fold) but significant increase in LPS-dependent TNF-a secretion. In contrast, when SC560 was used at the same concentration, a 5.9-fold increase in TNF-a secretion occurred. Incubation with both inhibitors produced results similar to those with SC560 alone (Fig. 5B).
The finding that COX inhibitors cause increased TNF-a secretion in LPS-treated RPM suggests that endogenous PGs suppress TNF-a secretion in those cells. Furthermore, the fact that this effect is much more striking with SC560 than SC236 implies that COX-1 is the primary source of the suppressive PGs. The latter conclusion is confounded by the fact that both SC236 and SC560 cause a similar (85-90%) inhibition of PG synthesis in RPM after a 6 hr incubation with LPS (Fig. 3B). In should be remembered, however, that after only 2 hr of LPS exposure, the differential inhibition of PG synthesis between the two inhibitors in RPM is striking, as SC236 produces a much lower suppression of PG synthesis (30-35%) than that observed with SC560 (90%, Fig. 3A). This observation is attributable to the inability of SC236 to suppress the relatively high proportion of COX-1-dependent PG synthesis that occurs early in the RPM LPS response when TNF-a mRNA is being synthesized.
The above findings suggest that SC560 has a greater effect on RPM TNF-a secretion than does SC236 as a result of its better ability to inhibit the formation of PGs resulting primarily from COX-1 activity early in the LPS response. However, it is also possible that the effects of SC560 on the response of RPM to LPS are unrelated to its suppression of PG synthesis. In order to investigate this possibility, we treated RPM with LPS in the presence of SC560 plus varying concentrations of PGE 2 and/or cPGI 2 , a stable prostacyclin analog. The concentrations of PGs ranged from 3.5 to 57 nM, corresponding roughly to the concentrations of the PGE 2 and prostacyclin appearing in the medium of RPM during 1 through 6 hr of incubation with LPS in the absence of SC560 (Table I). The results (Fig. 5C) demonstrate that the increased TNF-a formation observed in SC560-treated RPM incubated with LPS is reversed efficiently by the combination of PGE 2 and cPGI 2 . PGE 2 and cPGI 2 alone were not as effective as the combination of the two PGs. These results support the hypothesis that the effect of SC560 on TNF-a secretion in LPS-treated RPM is due to suppression of PG synthesis in these cells.
Our results using selective COX inhibitors suggest that COX-1-dependent PG synthesis inhibits TNF-a secretion in LPS-treated RPM. If this were the case, then one would expect that genetic mutation of the gene for COX-1 should lead to increased TNF-a secretion in RPM responding to LPS. We tested this hypothesis by comparing the LPS response in RPM from CD-1 WT mice with RPM from CD-1 COX-1 -/and COX-2 -/mice. CD-1 WT mice produced lower quantities of TNF-a (3.3 ± 0.4 ng/10 7 cells) than did RPM from C3H/HeN WT mice after a 6 hr incubation with LPS. However, as predicted, RPM from CD-1 COX-1 -/mice produced 2.4-fold higher levels of TNF-a than did RPM from CD-1 WT mice, a statistically significant increase (p < 0.05, Fig. 5D). COX-2 -/-RPM produced slightly more TNF-a than did WT RPM, but this difference was not statistically significant.  (71,87,88). They respond to a variety of stimuli with the release of large amounts of free 20:4, and efficiently metabolize the free acid to an array of COX and LOX products (69)(70)(71)(72)(89)(90)(91)(92) (73)(74)(75)(93)(94)(95). In this study, we have investigated LPS-mediated 20:4 metabolism in RPM using mass spectrometric assays to evaluate quantities of all major PGs and both intracellular and extracellular 20:4. These methods offer a distinct advantage over commonly used radiolabel techniques in that they allow for absolute rather than relative quantitation of 20:4 and its metabolites. They also offer advantages over ELISA assays in that multiple metabolites may be monitored simultaneously. The results of our studies demonstrate that RPM produce predominantly prostacyclin and PGE 2 in response to LPS, and that net increases in PG levels occur within the first hour after LPS addition. Marked induction of COX-2 and mPGES protein expression occurs in LPS-treated RPM, but detectable increases in protein levels are not observed until 2 hr of LPS treatment.

LPS-Stimulated PG Synthesis in RPM Cultures
Role for COX-1 in the Macrophage LPS Response -Our finding that significant PG synthesis occurs in LPS-treated RPM prior to increased COX-2 expression led us to question whether COX-1 plays a significant role in the macrophage LPS response. We initially investigated this hypothesis using the selective COX inhibitors, SC560 and SC236. The concentration of inhibitor used in these studies (100 nM) was chosen to be well above the reported IC 50 of each compound for the isoform for which it is selective, but well below the reported IC 50 for the opposite isoform. Others have used this concentration in efforts to achieve selective inhibition in cultured macrophage populations under conditions similar to ours (96).
We chose to study the effects of the inhibitors at both 2 hr and 6 hr after LPS administration.
Based on the patterns of enzyme expression, COX-2 levels are still low after 2 hr of LPS exposure, so any contribution of COX-1 to PG synthesis would be relatively greater at that time.
Alternatively, by 6 hr COX-2 expression has reached near maximal levels so that its relative contribution to PG synthesis would probably have matched or exceeded that of COX-1. Thus, if COX-1 does contribute to PG synthesis, we predict that SC560 would be the more efficient inhibitor of PG synthesis at 2 hr, whereas by 6 hr, SC236 would be the most effective.
The results obtained with SC236 generally followed the predicted pattern, in that it inhibited PG synthesis less efficiently at 2 hr than at 6 hr. In contrast, SC560 caused a >80% inhibition of PG synthesis at both time points. The latter finding is inconsistent with inhibition of only COX-1 by SC560, leading us to conclude that SC560 is also inhibiting COX-2 to some degree, or that it has suppressive effects on PG synthesis by a mechanism other than direct COX inhibition. Relevant to the latter possibility, we found no change in free 20:4 levels in the presence of the inhibitors at either time point, indicating that they did not cause a marked reduction in gross availability of substrate. Both inhibitors caused a slight reduction in COX-2 expression, but this effect was too small to readily account for the levels of inhibition observed after 6 hr. It therefore seems highly likely that SC560 inhibited COX-2 directly. It should be noted that SC236 also might have exerted nonspecific effects, including COX-1 inhibition, in our experiments. However, the inhibitory effects of SC236 in RPM (30-35% inhibition at 2 hr and 85-90% inhibition at 6 hr) are consistent with expected contributions of COX-2 to LPSdependent PG synthesis in these cells.
Our finding that SC236 only causes partial inhibition of LPS-mediated PG synthesis in RPM, especially at the earlier time points, was consistent with the hypothesis that COX-1 contributes to this process. We also applied a genetic approach using RPM from mice bearing targeted deletions of the gene for COX-1 or COX-2. Prior reports have shown that thioglycollate-elicited peritoneal macrophages from COX-1 -/-C57BL/6 mice demonstrate reduced PG synthesis in response to exogenous 20:4, and that RPM from COX-2 -/-C57BL/6 mice show no LPS-dependent increase in PG synthesis in response to exogenous 20:4. These findings confirm that COX-1 expression is constitutive, and that LPS induces COX-2 expression in peritoneal macrophages. However since exogenous 20:4 was used in these experiments, they do not directly address the relative roles of the two isoforms in delayed PG synthesis from endogenous substrate (57,97). For our studies we used WT, COX-1 -/-, and COX-2 -/mice on CD-1 and C57Bl/6 backgrounds. When compared to WT RPM, RPM from mice bearing a targeted deletion of either COX isoform showed no difference in the expression of the opposite isoform as measured by immunoblot analysis. This was true for mice from both sources. In addition, similar expression of cPLA 2 was detected in RPM from mutant mice as compared to WT controls except for a trend to slightly higher expression in CD-1 COX-2 -/cells. Thus, we detected no evidence that deletion of the gene for either COX-1 or COX-2 leads to notably When RPM from COX-1 -/mice of either strain were compared to WT controls, a consistent pattern was observed. In both sets of experiments, these RPM produced very low levels of PGs during the first 2 hr of incubation with LPS. By 6 hr, considerable PG formation had occurred, but levels were still much lower than those observed with WT cells. These results are totally consistent with the hypothesis that COX-1 contributes significantly to PG synthesis in WT RPM, and that the contribution is proportionately larger at the earlier time points, before COX-2 expression has reached high levels.  (Table III). However, C57BL/6 WT RPM showed only a small increase in free 20:4 after 2 hr of incubation with LPS, and C57BL/6 COX-1 -/and COX-2 -/-RPM showed essentially no increase at all. In contrast, LPS induced a near doubling of free 20:4 in CD-1 WT RPM and COX-1 -/-RPM, while a four-fold increase was observed in COX-2 -/-RPM. Thus the ability of COX-1 to respond to LPS in COX-cyclooxygenase reaction, so that many enzyme turnovers can occur following a single activation event. However, environmental reducing agents may return the enzyme to its native state, necessitating reactivation (4,43,44). Thus, maintenance of COX in the active state will depend on the relative concentration of oxidants and reductants within the cell (45,99). The source of the initial activating agent for COX in the intracellular environment is not known, although the product, PGG 2 , can carry out this function (39,40). Therefore, the presence of high substrate concentrations would favor sustained activation through the rapid production of PGG 2 , a condition particularly important for COX-1, because it requires higher concentrations of oxidant for activation than does COX-2 (47,48). These considerations may explain why it appears that If it is true that COX-1 does not carry out LPS-dependent PG synthesis in COX-2 -/-C57BL/6 RPM due to low levels of 20:4 release, then it is reasonable to assume that COX-1 should also not contribute to PG synthesis in LPS-challenged C57BL/6 WT mice. However, results of experiments using C57BL/6 COX-1 -/-RPM indicate reduced PG synthesis as compared to WT cells, suggesting that COX-1 does contribute to this process in the WT RPM. A possible explanation for this apparent discrepancy is that the activation of COX-1 is dependent on newly expressed COX-2 in C57BL/6 WT RPM. COX-2 is readily activated at low concentrations of 20:4, and the PGG 2 that it generates would then be available to activate COX-1. If correct, this hypothesis implies that COX-1 can contribute to PG synthesis in LPS-treated macrophages as long as the cells respond to the LPS with a robust release of 20:4, or as long as sufficient COX-2 expression has occurred to provide adequate levels of oxidant for COX-1 activation. It should be noted again however, that other oxidants that have not been directly explored in these studies may also be involved in COX activation.

Studies of COX-2 -/-RPM gave very different results for cells obtained from
Regardless of the mechanism, our data clearly indicate a marked difference between CD-1 and C57BL/6 RPM in their ability to compensate for the deletion of the gene for COX-2.
Similar results have been reported recently by Wang et al. who observed that that female CD-1 COX-2 -/mice have dramatically improved fertility in terms of ovulation, fertilization, and implantation, giving rise to live births, as compared to female C57Bl/6 COX-2 -/mice, which are almost completely infertile. This improved fertility in CD-1 COX-2 -/mice is due to a compensatory upregulation of COX-1 in the uterus that does not occur in C57BL/6 COX-2 -/mice (59). Note, however, that a similar compensatory change in COX-1 expression was not observed in any of the COX-2 -/-RPM populations studied here. Thus compensatory mechanisms used to overcome the effects of COX-2 gene deletion must be tissue specific as well as strain specific.

Suppression of LPS-Mediated TNF-a Secretion in LPS-Treated RPM -Our data show
that LPS-mediated TNF-a secretion is suppressed by endogenous PGE 2 and prostacyclin in RPM. Since PGs suppress TNF-a secretion by blocking the LPS-mediated increase in TNF-a mRNA, it is clear that they must be present at significant concentrations during the first 1-2 hr after LPS addition when the major increase in mRNA transcription takes place. Therefore, the most likely source of endogenous PGs for suppression of TNF-a secretion is COX-1. Consistent with this hypothesis, the COX-2 selective inhibitor, SC236, had only a small effect (1.8-fold increase) on RPM TNF-a secretion over a 6 hr incubation with LPS, whereas the COX-1 selective inhibitor, SC560, had a much greater effect (5.6-fold increase). This finding correlates with the clear differential in PG synthesis inhibition, leading to much lower PG concentrations in the presence of SC560 than in the presence of SC236 during the critical initial 2 hr after LPS addition. Thus, during the period of maximal TNF-a mRNA transcription, SC560 has a much greater effect on PG synthesis than does SC236, and is therefore more effective than SC236 in attenuating the PG-mediated suppression of TNF-a secretion.
Since the discovery of COX-2, considerable effort has been made to define specific functions for each COX isoform. The definition of delayed PG synthesis as a solely COX-2dependent process implied a unique role for this isoform in response to the inflammatory agents and cytokines that also invoke its expression. Our finding that COX-1 contributes to PG synthesis in LPS-stimulated macrophages suggests that the processes of immediate and delayed PG synthesis may not be as biochemically distinct as is presently believed. In fact, considering the significant rate of PG synthesis observed during the first hour of LPS stimulation in RPM, the term "delayed" in this case is, perhaps, inappropriate. It is interesting to note that RPM are unusual cells in terms of their remarkable ability to synthesize exceptional quantities of PGs in response to a wide variety of both immediate and delayed PG stimuli. Apparently, a significant factor contributing to this ability is their high level of constitutive COX-1 expression. Because resident macrophages will be among the first to encounter a bacterial insult, and because COX-1-   were subjected to immunoblot analysis for COX-2 expression. The chemiluminescence signal from the immunoblots was quantified using the Fluor-S Max, and results were normalized to the signal obtained from control cultures. All results are the mean ± s.d. from three separate experiments in which duplicate samples were analyzed.

TABLE III
Comparison of LPS-induced PG synthesis of RPM from mice of different genetic backgrounds bearing targeted deletions of COX-1 and COX-2 genes. RPM cultures were prepared as described in Experimental Procedures and incubated for the indicated periods of time with LPS. PGs in culture medium were analyzed by LC/MS. CD-1 COX-1 -/and CD-1 COX-2 -/designate RPM from CD-1 mice bearing a targeted deletion for ptgs-1 or ptgs-2 respectively whereas CD-1 WT designates RPM from CD-1 wild-type mice. C57BL/6 COX-1 -/and COX-1 +/+ designate RPM from C57BL/6 mice bearing a targeted deletion of ptgs-1 and matched littermate WT controls, whereas C57BL/6 COX-2 -/and COX-2 +/+ designate RPM from C57BL/6 mice bearing a targeted deletion of ptgs-2 and matched littermate WT controls. Total PGs include PGE 2 plus 6-ketoPGF 1a . Percent PGE 2 represents the percent of indicated total PGs that was PGE 2 . Results are the combined mean ± s.d. for data from two experiments in which duplicate determinations were made (CD-1) or mean ± range from a single experiment in which duplicate determinations were made (C57BL/6).