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• W2004016651 abstract "The two cyclooxygenase isoforms, cyclooxygenase-1 and cyclooxygenase-2, both metabolize arachidonic acid to prostaglandin H2, which is subsequently processed by downstream enzymes to the various prostanoids. In the present study, we asked if the two isoforms differ in the profile of prostanoids that ultimately arise from their action on arachidonic acid. Resident peritoneal macrophages contained only cyclooxygenase-1 and synthesized (from either endogenous or exogenous arachidonic acid) a balance of four major prostanoids: prostacyclin, thromboxane A2, prostaglandin D2, and 12-hydroxyheptadecatrienoic acid. Prostaglandin E2 was a minor fifth product, although these cells efficiently converted exogenous prostaglandin H2 to prostaglandin E2. By contrast, induction of cyclooxygenase-2 with lipopol- ysaccharide resulted in the preferential production of prostacyclin and prostaglandin E2. This shift in product profile was accentuated if cyclooxygenase-1 was permanently inactivated with aspirin before cyclooxygenase-2 induction. The conversion of exogenous prostaglandin H2 to prostaglandin E2 was only modestly increased by lipopolysaccharide treatment. Thus, cyclooxygenase-2 induction leads to a shift in arachidonic acid metabolism from the production of several prostanoids with diverse effects as mediated by cyclooxygenase-1 to the preferential synthesis of two prostanoids, prostacyclin and prostaglandin E2, which evoke common effects at the cellular level. The two cyclooxygenase isoforms, cyclooxygenase-1 and cyclooxygenase-2, both metabolize arachidonic acid to prostaglandin H2, which is subsequently processed by downstream enzymes to the various prostanoids. In the present study, we asked if the two isoforms differ in the profile of prostanoids that ultimately arise from their action on arachidonic acid. Resident peritoneal macrophages contained only cyclooxygenase-1 and synthesized (from either endogenous or exogenous arachidonic acid) a balance of four major prostanoids: prostacyclin, thromboxane A2, prostaglandin D2, and 12-hydroxyheptadecatrienoic acid. Prostaglandin E2 was a minor fifth product, although these cells efficiently converted exogenous prostaglandin H2 to prostaglandin E2. By contrast, induction of cyclooxygenase-2 with lipopol- ysaccharide resulted in the preferential production of prostacyclin and prostaglandin E2. This shift in product profile was accentuated if cyclooxygenase-1 was permanently inactivated with aspirin before cyclooxygenase-2 induction. The conversion of exogenous prostaglandin H2 to prostaglandin E2 was only modestly increased by lipopolysaccharide treatment. Thus, cyclooxygenase-2 induction leads to a shift in arachidonic acid metabolism from the production of several prostanoids with diverse effects as mediated by cyclooxygenase-1 to the preferential synthesis of two prostanoids, prostacyclin and prostaglandin E2, which evoke common effects at the cellular level. prostaglandin arachidonic acid prostacyclin leukotriene cyclooxygenase hydroxyeicosatetraenoic acid 12-hydroxyheptadecatrienoic acid lipopolysaccharide peritoneal macrophage thromboxane phosphate-buffered saline acetylsalicylic acid tumor necrosis factor interleukin high pressure liquid chromatography enzyme immunoassay Prostaglandins (PGs)1are a family of intercellular and intracellular messengers derived from arachidonic acid (AA). These mediators exert a wide range of effects on processes such as smooth muscle tone, vascular permeability, cellular proliferation, and inflammatory/immune function. In many cases, different PGs will have opposing actions. For example, PGD2and thromboxane (TxA2) cause smooth muscle contraction, whereas PGE2 and prostacyclin (PGI2) cause relaxation (reviewed in Refs. 1Peters-Golden M. Lipscomb M. Russell S. Lung Macrophages and Dendritic Cells in Health and Disease. Marcel Dekker, Inc., New York1997: 151-182Google Scholar, 2Vane J. Bakhle Y. Botting R. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 97-120Crossref PubMed Scopus (2620) Google Scholar, 3Dubois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van De Putte L.B.A. Lipsky P.E. FASEB J. 1998; 12: 1063-1073Crossref PubMed Scopus (2231) Google Scholar, 4Bjorkman D. Am. J. Med. 1998; 105: 8S-12SAbstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Similarly, TxA2increases, but PGI2 inhibits, platelet aggregation. The net effect evoked by PGs may, ultimately, depend on the balance of these opposing forces. The initial step in the synthesis of PGs from AA is mediated by cyclooxygenase (COX, also known as prostaglandin H synthase or prostaglandin endoperoxide synthase), of which two isoforms are recognized (reviewed in Refs. 2Vane J. Bakhle Y. Botting R. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 97-120Crossref PubMed Scopus (2620) Google Scholar, 3Dubois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van De Putte L.B.A. Lipsky P.E. FASEB J. 1998; 12: 1063-1073Crossref PubMed Scopus (2231) Google Scholar, 4Bjorkman D. Am. J. Med. 1998; 105: 8S-12SAbstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). COX-1 is expressed constitutively in most cell types, and prostanoids derived from COX-1 are thought to be important in gastric and renal homeostasis. COX-2, on the other hand, is the product of an immediate early gene and is rapidly expressed only after exposure of cells to hormones, mitogenic stimuli, and inflammatory mediators, like bacterial lipopolysaccharide (LPS). The induction of COX-2, with the resultant production of prostanoids, can contribute to parturition, inflammation, pain, fever, and certain types of cancer. The ability of aspirin to permanently inactivate both COX isoforms indiscriminately explains both its analgesic and anti-inflammatory properties, through COX-2 inhibition, as well as its damaging effects on the gastric mucosa, through COX-1 inhibition. Both COX isoforms convert AA to PGH2, which is then acted upon by discrete PG synthases to give rise to the different PG species. The current model for the regulation of PG synthesis posits that the amount of each PG is determined by the availability of the substrate AA (through the action of phospholipases A2) and by the total mass of COX protein present. According to this model, COX-2 induction results in more COX protein, more processing of AA to PGH2, and more of each type of PG. The profile of different PGs produced by a given cell type is thought to be determined primarily by the relative amounts of the distal PG synthases present in that cell type. In simple cases, as when a single distal enzyme predominates, this model may be accurate. However, when several different PG synthases exist in a single cell, the opportunity exists for COX-1 and COX-2 to selectively deliver PGH2 to different distal enzymes and thus produce different PGs. If so, then COX-2 induction and action could result in a shift in the profile of PGs, rather than simply an increase in total PG production. This could then change the integrated prostanoid signal that is delivered to target cells. In the current study, we used intact rat peritoneal macrophages (PMs) to compare the products derived from either COX-1 or COX-2 action on AA. These primary cells were chosen because they contain several PG synthases and would thus allow detection of selective metabolism of PGH2, if it existed. Freshly isolated PMs, which lack COX-2, provide an indication of the prostanoids that are derived from COX-1 action. By contrast, cells treated first with aspirin to permanently inactivate COX-1 and then with LPS to induce COX-2 demonstrate those prostanoids that are derived from COX-2. Our results indicate that AA metabolized by COX-1 gives rise to balanced amounts of PGI2, TxA2, PGD2, and 12-hydroxyheptadecatrienoic acid (HHT) as well as smaller amounts of PGE2, whereas COX-2 preferentially metabolizes AA to PGI2 and PGE2. Because PGI2 and PGE2 can evoke similar effects on target cells and tissues, the result of COX-2 induction may be a dramatic shift in the balance of PG action. Resident PMs were obtained by peritoneal lavage of specific pathogen-free, female Wistar rats (Charles River Laboratories, Portage, MI) as described previously (5Peters-Golden M. McNish R.W. Hyzy R. Shelly C. Toews G.B. J. Immunol. 1990; 144: 263-270PubMed Google Scholar). Lavaged cells were centrifuged at 600 × g for 5 min at 4 °C, resuspended, and subjected to hypotonic lysis to remove contaminating erythrocytes. Cells were then centrifuged again and resuspended at 0.5 × 106 cells ml−1 in M199 and either maintained in suspension in Teflon tubes (“freshly isolated cells”) or plated on 24-well plates (for metabolic studies) or glass slides (for imaging). Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. As described previously (6Wilborn J. DeWitt D. Peters-Golden M. Am. J. Physiol. 1995; 268: L294-L301PubMed Google Scholar), cells were disrupted by sonication (10 bursts at 20% duty cycle) in ice-cold homogenizing buffer (50 mmTris-HCl, 25 mm KCl, 5 mm MgCl2, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 1 mm leupeptin, pH 7.4) and separated into soluble and pelletable fractions by centrifugation (100,000 ×g, 30 min, 4 °C); and protein concentrations were determined by a modified Coomassie Blue dye binding assay (Pierce). Samples containing 10 μg of protein were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions and transferred to nitrocellulose membranes. Following blocking, membranes were probed with antibodies to COX-1 (rabbit polyclonal raised against sheep seminal vesicle COX (7Smith W. Rollins T. Methods Enzymol. 1982; 86: 213-222Crossref PubMed Scopus (22) Google Scholar)) and COX-2 (rabbit polyclonal raised against murine COX-2, from Cayman Chemical Co., Ann Arbor, MI; 1:10,000) and then peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000) with ECL detection (Amersham Pharmacia Biotech). Cells were prepared for indirect immunofluorescent microscopy as described previously (8Brock T.G. Paine III, R. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Abstract Full Text PDF PubMed Google Scholar). Following mounting on glass slides, cells were fixed using methanol (−20 °C, 30 min), permeabilized with acetone (−20 °C, 3 min), air-dried, rehydrated with sterile PBS, and blocked for 30 min using 0.1% (w/v) bovine serum albumin in PBS containing 1% (v/v) nonimmune goat serum. Preparations were then probed with antibodies to either COX-1 or COX-2, each at 1:200 in PBS supplemented with 0.1% fatty acid-free bovine serum albumin, followed by rhodamine-conjugated goat anti-rabbit antibody (1:200, Sigma). Preparations were examined and photographed using a Zeiss Aristoplan microscope equipped for epifluorescence. For overnight incubations, cells were adhered for 1 h, washed twice with sterile PBS, and cultured overnight in M199 containing 10% heat-inactivated newborn calf serum (Life Technologies, Inc.). In some experiments, aspirin (acetylsalicylic acid (ASA); 200 μm) was included during the 1 h adherence phase; this treatment did not affect cell adherence or survival, as determined by trypan blue staining and visualization of cells. During overnight incubation, some cells were cultured with LPS (1 μg/ml), TNF-α (100 ng/ml), IL-1 (5 ng/ml), or polymyxin B sulfate (10 μg/ml). For prelabeling of cellular lipids, 0.5 μCi of [3H]AA (specific activity, 76–100 Ci/mmol, NEN Life Science Products) was included in the incubation medium. Before cell stimulation, the unincorporated label was removed by washing three times with PBS. Cell stimulation was with 1 μm A23187 for 15 min at 37 °C. Inhibitors added during cell stimulation in designated experiments included NS-398 (3 μm) and indomethacin (1 μm). Metabolism of exogenous AA was evaluated by the addition of 0.5 μCi of [3H]AA (∼5 pm) to unlabeled cells during stimulation with A23187; the addition of additional cold, carrier AA did not alter results. Metabolism of exogenous PGH2 was evaluated by the addition of 0.1 μm[3H]PGH2 (for HPLC analysis) or unlabeled PGH2 (for enzyme immunoassay analysis) to unlabeled cells during stimulation with A23187. Radiolabeled eicosanoids were assayed as described (5Peters-Golden M. McNish R.W. Hyzy R. Shelly C. Toews G.B. J. Immunol. 1990; 144: 263-270PubMed Google Scholar). Briefly, eicosanoids were extracted from culture medium using Sep-Pak C18 cartridges (Waters Associates, Milford, MA), dried under nitrogen, resuspended in water:acetonitrile (2:1), and separated by reverse-phase HPLC using a Waters μBondapak C18 column with a mobile phase of acetonitrile/water/trifluoroacetic acid. Radiolabeled products were identified by their coelution with authentic standards and quantitated by on-line radiodetection. Enzyme immunoassay (EIA) of conditioned media was performed according to the supplier's instructions (Cayman Chemicals). Statistical significance was evaluated by paired Student's t test using p < 0.05. Freshly isolated PMs were evaluated for the presence of the two COX isoforms. By immunoblot analysis, fresh PMs contained abundant COX-1 in membrane-containing fractions but lacked detectable COX-2 (Fig. 1 A). Similarly, COX-1, but not COX-2, was detectable in individual PMs by immunofluorescent microscopy (Fig. 1 B). By this technique, COX-1 appeared to extensively decorate membranes throughout the cell, including perinuclear membranes and the endoplasmic reticulum. To determine the profile of prostanoids synthesized by COX-1 alone, freshly isolated PMs were prelabeled with [3H]AA for 2 h, maintained in suspension in Teflon tubes, washed to remove the unincorporated label, and stimulated with the calcium ionophore A23187 (1 μm, 15 min, 37 °C) to release cellular AA from endogenous membrane phospholipids. By this method, several products were identified after HPLC separation with approximately 60% of the released AA converted to COX metabolites, including PGI2 (detected as 6-keto-PGF1α), TxA2 (detected as TxB2), PGE2, PGD2, and HHT and 30% metabolized to products of the 5-lipoxygenase pathway (Fig.2 A). This profile is similar to that reported previously (5Peters-Golden M. McNish R.W. Hyzy R. Shelly C. Toews G.B. J. Immunol. 1990; 144: 263-270PubMed Google Scholar). The production of prostanoids was inhibited by indomethacin (1 μm), a nonselective COX inhibitor, resulting in increases in free AA and 12-HETE but not the 5-lipoxygenase products, leukotriene (LT)B4 and 5-HETE (Fig. 2 B). Pooled results from four independent experiments indicated that freshly isolated PMs generated a balance of four prostanoids from endogenous AA: PGI2, TxA2, PGD2, and HHT (Table I). PGE2 was a minor fifth product, accounting for less than 10% of all COX-1 products. Similar results were obtained when the AA was supplied exogenously; if trace levels of [3H]AA were provided to fresh PMs in the absence of an agonist, then prostanoid ratios similar to those generated by prelabeled, A23187-stimulated cells were produced (TableI). Acute stimulation of cells with A23187 in the presence of exogenously supplied radiolabeled AA increased the synthesis of radiolabeled 5-lipoxygenase products but did not significantly change the ratios of the COX-1 products (data not shown).Table IProducts from COX-1 in freshly isolated PMsProduct6-keto-PGF1αTxB2PGE2PGD2HHTFresh PMsFrom endogenous AA19.1 (8.7)25.5 (6.1)7.1 (5.1)20.3 (10.3)28.0 (8.4)From exogenous AA17.1 (6.4)17.8 (5.3)9.8 (4.1)24.5 (8.4)30.8 (7.6)Cells were either prelabeled with [3H]AA for 2 h, washed, and stimulated with A23187 (1 μm, 10 min, 37 °C) or given exogenous [3H]AA without A23187 (2 h, 37 °C). Radioactive metabolites in conditioned media were separated by HPLC; results indicate the disintegrations/min per product as a percentage of all prostanoid disintegrations/min. Values are the mean of four experiments; S.E. is in parentheses. Open table in a new tab Cells were either prelabeled with [3H]AA for 2 h, washed, and stimulated with A23187 (1 μm, 10 min, 37 °C) or given exogenous [3H]AA without A23187 (2 h, 37 °C). Radioactive metabolites in conditioned media were separated by HPLC; results indicate the disintegrations/min per product as a percentage of all prostanoid disintegrations/min. Values are the mean of four experiments; S.E. is in parentheses. COX-2 induction in rat PMs was pronounced in response to LPS (1 μg/ml, 24 h), appearing as two bands of approximately 69 and 70 kDa on immunoblots (Fig.3 A). By indirect immunofluorescent microscopy, the degree of expression of COX-2 protein varied greatly among cells within a culture treated with LPS for 24 h (Fig. 3 B). Some cells were largely negative for COX-2, some cells showed intermediate expression with COX-2 restricted to the nuclear envelope and perinuclear membranes, and others showed that COX-2 was concentrated on the nuclear envelope and was also abundant on the endoplasmic reticulum. By Nomarski optics, it appeared that cells staining greatest for COX-2 were spread and commonly binucleate, whereas those staining the least were rounded and mononucleate. In parallel experiments, all cells excluded trypan blue dye, indicating that the rounded cells were not dead. COX-2 protein was also induced to a lesser extent by TNF-α (100 ng/ml), appearing as a single 69-kDa band (Fig. 3 A). There was no detectable induction by IL-1 (5 ng/ml). COX-1 expression was unchanged following treatment with LPS, TNF-α, or IL-1 (Fig. 3 A), and it was not altered by overnight incubation alone, ASA pretreatment, or the combination of ASA pretreatment and LPS treatment (Fig. 3 C). Based on these results, subsequent experiments focused on LPS-induced COX-2. According to current models, the result of elevated mass of COX protein via COX-2 induction should be a nonselective increase in the synthesis of all PGs that the given cell type can make. However, when PMs were treated with LPS for 24 h to induce COX-2, washed, and stimulated with A23187 (1 μm, 15 min, 37 °C) to release AA from cell membranes, selective changes in PG production were readily apparent, as compared with that seen in freshly isolated cells. These PMs, which contained both active COX-1 and COX-2, synthesized the same products as freshly isolated PMs (Fig.4 A). However, following LPS treatment, the proportion of radiolabeled AA metabolized to PGE2 was increased, whereas that converted to TxA2, as well as the 5-lipoxygenase product 5-HETE, was reduced. One explanation for the altered PG profile following LPS treatment would be that COX-2 preferentially metabolizes AA to PGI2 and PGE2, whereas the other prostanoids were derived from COX-1 action. To address this, COX-1 was irreversibly inactivated in fresh PMs with ASA, the ASA was washed away, and COX-2 was induced with LPS for 24 h. The treatment with ASA (200 μm) alone for 1 h effectively eliminated COX activity, and continued culture (after washing to remove ASA) in LPS-free medium for 24 h did not result in significant new COX activity (Fig. 4 B). The cells treated with ASA that were washed and given LPS produced predominantly PGI2 and PGE2, as well as LTB4 (Fig. 4 C). These cells, with only COX-2 active, had approximately 85% of all released AA converted to PGs, as compared with 60% in COX-1-containing fresh PMs. Interestingly, there were only minor changes in LTB4 production. Finally, the production of PGs was inhibited by the selective COX-2 inhibitor NS-398; in cells pretreated with ASA and then given LPS to induce COX-2, PGE2 synthesis was completely inhibited by 1 μm NS-398, as determined by EIA, with an IC50 of 0.12 μm NS-398. In contrast, in freshly isolated PMs, 50% inhibition of PGE2synthesis required 8.3 μm NS-398 (approximately 70-fold more than the IC50 of ASA-pretreated, LPS-treated cells); PGE2 synthesis was inhibited only 75% by 30 μm NS-398, the highest dose tested. Pooled data from three independent experiments indicated that cells given LPS overnight and thus with both active COX isoforms produced predominantly PGI2, TxB2, and PGE2, with PGD2 and HHT being less pronounced (TableII). This pattern was also generally seen when the radiolabeled AA was supplied exogenously. Pretreatment of PMs with ASA to inactivate COX-1 and thus leave COX-2 as the only active isoform following LPS treatment resulted in PGI2 and PGE2 being the major products of stimulated cells, with TxB2 occasionally found in significant amounts (Table II). In contrast, PGD2 and HHT were barely detectable. Similar results were obtained if radiolabeled AA was supplied exogenously and cells were not acutely stimulated. The two products, PGI2and PGE2, constituted 85.8 ± 14.8% of all PGs released via COX-2, as compared with 26.2 ± 13.9% of all PGs released from cells containing only COX-1 (Table I). PMs incubated overnight in LPS-free medium with or without polymyxin B, which binds and inactivates LPS, generated the same profile of products as freshly isolated PMs, except that PGD2 tended to be reduced and LTB4 elevated with overnight culture (data not shown).Table IIProducts from LPS-treated PMsProduct6-keto-PGF1αTxB2PGE2PGD2HHTPMs given LPS for 24 hFrom endogenous AA32.9 (11.3)21.9 (8.0)30.4 (12)4.7 (3.4)10.1 (6.1)From exogenous AA31.5 (8.2)8.21 (5.7)36.7 (12)7.4 (4.8)16.2 (2.6)PMs ASA pretreated, then given LPS for 24 hFrom endogenous AA41.5 (9.9)7.6 (6.1)44.4 (11)1.8 (2.5)4.8 (6.7)From exogenous AA40.0 (2.7)13.6 (7.0)27.7 (8.0)1.0 (1.3)18.7 (9.2)Cells were adhered in media with or without ASA (200 μm) for 1 hr, washed, and incubated in media with LPS (1 μg/ml) for 24 h with [3H]AA. Cells were then washed and stimulated with A23187 (1 μm, 10 min, 37 °C). Radioactive metabolites in conditioned media were separated by HPLC; results indicate the disintegrations/min per product as a percentage of all prostanoid disintegrations/min. Values are the mean of three experiments; S.E. is in parentheses. Open table in a new tab Cells were adhered in media with or without ASA (200 μm) for 1 hr, washed, and incubated in media with LPS (1 μg/ml) for 24 h with [3H]AA. Cells were then washed and stimulated with A23187 (1 μm, 10 min, 37 °C). Radioactive metabolites in conditioned media were separated by HPLC; results indicate the disintegrations/min per product as a percentage of all prostanoid disintegrations/min. Values are the mean of three experiments; S.E. is in parentheses. A second possible explanation for the altered PG profile following LPS treatment would be that LPS selectively changed the amount or activity of all enzymes that act distal to the COX isozymes. Specifically, LPS might have had up-regulating effects on PGI2 and PGE2 synthases and down-regulatory effects on synthases giving TxA2, PGD2, and HHT. To address this possibility, we examined the effects of LPS on the processing of the COX product, PGH2. Exogenous PGH2 (0.1 μm) was efficiently metabolized to a variety of PGs by freshly isolated PMs (Fig.5 A). The most abundant product was PGE2 at about 24% of all recovered product. This was surprising because freshly isolated PMs released little PGE2 from either endogenous AA (following ionophore stimulation) or exogenous AA (Table I). A similar profile of products was obtained when PGH2 was given to unfractionated cell lysates (data not shown). When PMs were first treated with LPS for 24 h, the proportion of PGH2 converted to PGE2 increased from 24 to approximately 30% of all recovered disintegrations/min (Fig. 5 B), consistent with a slight increase in PGE2 synthase activity. However, in three independent experiments using both whole cells and cell lysates, there were no significant differences between freshly isolated and LPS-treated PMs in the profiles of products generated from PGH2. The increase in PGE2 synthase activity was further assessed using EIAs to specifically measure PGE2 production. Following ionophore stimulation to liberate endogenous AA, freshly isolated PMs made significant amounts of PGE2, and PGE2 production following acute stimulation increased approximately 2.5-fold when PMs were treated with LPS for 6 or 24 h (Fig. 6 A). Exogenous PGH2 was efficiently converted to PGE2 by untreated, freshly isolated PMs, consistent with the HPLC analysis. After 6 or 24 h of LPS treatment, PGE2 production from PGH2 increased significantly. However, this increase, although statistically significant, was only by 30%. These effects were further extended to TNF-α, which also induced COX-2 synthesis in these cells (Fig. 3). The treatment of PMs with TNF-α for 24 h also increased PGE2 synthesis from AA by 3-fold, whereas it increased PGE2 production from PGH2 again by only 30% (Fig. 6 B). It should be noted that PGE2 production from PGH2, as presented in Fig.6, is measured on a different scale than PGE2 production from AA, with the former being 8 times the latter. In this study, we found that freshly isolated PMs, which have COX-1 but not COX-2, make a balance of four major prostanoids from AA. A fifth prostanoid, PGE2, is only a minor product, although these cells can readily convert PGH2 to PGE2. Similar results were obtained if PMs were maintained overnight in LPS-free medium. In stark contrast, when PMs were incubated with LPS to induce COX-2, the profile of PG products shifted to emphasize two PGs, PGI2 and PGE2. Pretreatment with ASA to inactivate COX-1, leaving COX-2 as the only active isoform, accentuated this change. These results were surprising because increasing COX mass through LPS treatment did not result in an across-the-board increase in production of all COX products. Instead, COX-2 induction caused a profile shift characterized by preferential synthesis of PGI2 and PGE2 with reduced production of TxB2, PGD2, and HHT. A shift in product profile may arise from a change in the amounts of each of the distal enzymes, which process the COX product, PGH2. Indeed, Oh-ishi and colleagues (9Matsumoto H. Naraba H. Murakami M. Kudo I. Yamaki K. Ueno A. Oh-ishi S. Biochem. Biophys. Res. Commun. 1997; 230: 110-114Crossref PubMed Scopus (145) Google Scholar, 10Naraba H. Maurakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar) have shown that PGE2 synthase activity was induced by LPS in inflammatory PMs, whereas PGD2 synthase and TxA2 synthase activities were unaffected. In their studies, cell lysates from these cells lacked any detectable PGE2synthase activity; this would serve to make this enzymatic step rate-limiting in the synthesis of PGE2. In this study, using resident PMs, we found significant PGE2 synthase activity in both whole cells and cell lysates before treatment with LPS as reported previously (6Wilborn J. DeWitt D. Peters-Golden M. Am. J. Physiol. 1995; 268: L294-L301PubMed Google Scholar). Treatment with LPS resulted in a small (30%) but significant increase in PGE2 synthase activity. This change certainly may contribute to the increase in PGE2 production in LPS-treated cells. However, two results suggest that changes in distal enzymes were not sufficient to account for the change in PG profile. First, the changes in product profile from distal enzymes alone (Fig. 5) were not comparable to the changes in products derived from COX isoforms plus distal enzymes (Figs. 2 and4). Second, the magnitude of increase in PGE2 production from PGH2 was much less than that from AA (Fig. 6). The difference in scale for PGE2 synthesis from PGH2 versus AA, noted in Fig. 6, argues that the activity of COX, not PGE2 synthase, is rate-limiting in the production of PGE2, and thus the small changes in PGE2 synthase activity cannot account for the increased production of PGE2 following LPS treatment. These results suggest that other factors contributed to the shift in product profile following COX-2 induction. Previous researchers have suggested that the COX-1 and COX-2 metabolic pathways are at least partially independent from one another, particularly in terms of when and where the pathways are expressed (11Reddy S. Herschman H. J. Biol. Chem. 1994; 269: 15473-15480Abstract Full Text PDF PubMed Google Scholar, 12Masferrer J. Zweifel B. Manning P. Hauser S. Leahy K. Smith W. Isakson P. Seibert K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3228-3236Crossref PubMed Scopus (1293) Google Scholar, 13Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar). However, we propose that the COX-1 and COX-2 pathways differ in the profile of their products. A model of this relationship in PMs, based on the results of this study, is given in Fig.7. According to this model, AA metabolism by COX-1 produces PGH2 that is directed in a balanced way to enzymes that give rise to PGI2, TxA2, PGD2, and HHT. In contrast, COX-2 metabolism is much more tightly coupled to the PGI2- and PGE2-synthesizing enzymes than to other downstream enzymes. Then the result of COX-2 induction by inflammatory mediators is not simply an increase in all PGs that a given cell type can produce. Instead, there is a shift in the balance of PGs toward the preferential production of PGI2 and PGE2. A simple explanation for the shift in product profile relates to the subcellular distribution of the metabolic enzymes. First, the two COX isoforms appear to differ in their subcellular distribution. COX-1 is dispersed throughout the intracellular membrane system, whereas COX-2 is preferentially accumulated on and around the nuclear envelope (14Morita I. Schindler M. Regier M.K. Otto J.C. Hori T. DeWitt D.L. Smith W.L. J. Biol. Chem. 1995; 270: 10902-10908Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar,15Zhang Y. Cao H. Graf B. Meekins H. Smith T. Phipps R. J. Immunol. 1997; 160: 1053-1057Google Scholar). It should be noted that a recent study has questioned whether this difference is real or artifactual (16Spencer A.G. Woods J.W. Arakawa T. Singer I.I. Smith W.L. J. Biol. Chem. 1998; 273: 9886-9893Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar), but our results support distinct distributions for the two isoforms (Fig.8, see also Figs. 1 and 3). Second, the different PG synthases appear to differ in their subcellular distribution, as has been recently reviewed (17Brock T. Peters-Golden M. Szczeklik A. Gryglewski R. Vane J. Eicosanoids, Aspirin and Asthma. Marcel Dekker, Inc., New York1997: 91-109Google Scholar). For example, the glutathione-requiring form of PGD2 synthase is found in the cytoplasm of a variety of cell types (18Ujihara M. Urade Y. Eguchi N. Hayashi H. Ikai K. Hayaishi O. Arch. Biochem. Biophys. 1988; 260: 521-531Crossref PubMed Scopus (149) Google Scholar, 19Urade Y. Ujihara M. Horiguchi Y. Ikai K. Hayaishi O. J. Immunol. 1989; 143: 2982-2989PubMed Google Scholar), whereas PGE2synthase activity has been localized to the endoplasmic reticulum and nuclear envelope (20Mukherjee E. Ghosh D. Prostaglandins. 1989; 38: 557-563Crossref PubMed Scopus (6) Google Scholar). Finally, different pools of AA, liberated by distinct phospholipase A2 isoforms, may feed into the different COX pathways (21Murakami M. Matsumoto R. Urade Y. Austen K.F. Arm J.P. J. Biol. Chem. 1995; 270: 3239-3246Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 22Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 23Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Thus, COX-1, because of its diffuse subcellular distribution, may deliver PGH2 relatively nonselectively to downstream enzymes. On the other hand, COX-2 may monopolize AA liberated from the nuclear envelope, e.g. by the action of the 85-kDa phospholipase A2 acting at that site (24Glover S. Bayburt T. Jonas M. Chi E. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 25Schievella A.R. Regier M.K. Smith W.L. Lin L.-L. J. Biol. Chem. 1995; 270: 30749-30754Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 26Peters-Golden M. Song K. Marshall T. Brock T. Biochem. J. 1996; 318: 797-803Crossref PubMed Scopus (100) Google Scholar), and preferentially deliver PGH2 to distal enzymes located on and around the nuclear envelope, such as PGE2 synthase and, perhaps, PGI2 synthase. The paired production of PGI2 and PGE2 is a common response to inflammatory mediators in a wide range of settings. Both PGI2 and PGE2 increased significantly in vascular endothelial cells in response to LPS or TNF-α (27Riedo F. Munford R. Campbell W. Reisch J. Chien K. Gerard R. J. Immunol. 1990; 144: 3506-3512PubMed Google Scholar), in ciliary epithelial cells in response to LPS (28Helbig H. Kittredge K. Gurley R. Thurau S. Palestine A. Nussen-blatt R. Curr. Eye Res. 1990; 9: 501-505Crossref PubMed Scopus (17) Google Scholar), in gingival fibroblasts in response to IL-1 or TNF-α (29Modeer T. Bengtsson A. Rolla G. J. Clin. Periodontol. 1996; 23: 927-933Crossref PubMed Scopus (56) Google Scholar), and in myometrial cells in response to IL-1 or TNF-α (30Hertelendy F. Romero R. Molnar M. Todd H. Baldassare J. Am. J. Reprod. Immunol. 1993; 30: 49-57Crossref PubMed Scopus (53) Google Scholar). Similarly, PGI2and PGE2 production by aortic endothelial cells was elevated in response to xenoreactive antibodies and complement (31Bustos M. Coffman T. Saadi S. Platt J. J. Clin. Invest. 1997; 100: 1150-1158Crossref PubMed Scopus (76) Google Scholar). Inex vivo culture, the saphenous vein released PGI2 and PGE2 in response to IL-1 (32Bishop-Bailey D. Pepper J. Haddad E. Newton R. Larkin S. Mitchell J. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1644-1648Crossref PubMed Scopus (58) Google Scholar). In whole animal models, plasma levels of PGI2 and PGE2 were elevated when TNF-α was administered intravenously to rats (33Fletcher J. Collins J. Graves E. Luterman A. Williams M. Izenberg S. Rodning C. Ann. Surg. 1993; 217: 668-674Crossref PubMed Scopus (34) Google Scholar) or sheep (34Wheeler A. Hardie W. Bernard G. Am. Rev. Respir. Dis. 1992; 145: 632-639Crossref PubMed Scopus (38) Google Scholar) and also when bleomycin was given intratracheally to hamsters (35Chandler D. Giri S. Am. Rev. Respir. Dis. 1983; 128: 71-76Crossref PubMed Scopus (34) Google Scholar). Because COX-2 induction is common to all these experimental systems, these results are consistent with the thesis that COX-2 preferentially metabolizes AA to PGI2 and PGE2. What is the impact of the paired production of PGI2 and PGE2? Both PGs evoke a number of common cellular responses, including smooth muscle relaxation, decreased fibroblast and smooth muscle proliferation, diminished collagen deposition, and decreased leukocyte function. Although other signaling pathways are also used, both PGs evoke many of these effects through receptor-mediated elevation of cAMP (reviewed in Refs. 34Wheeler A. Hardie W. Bernard G. Am. Rev. Respir. Dis. 1992; 145: 632-639Crossref PubMed Scopus (38) Google Scholar and 35Chandler D. Giri S. Am. Rev. Respir. Dis. 1983; 128: 71-76Crossref PubMed Scopus (34) Google Scholar). Also through cAMP signaling, both PGI2 and PGE2 inhibit activation-initiated responses of many leukocytes, including neutrophils, macrophages, mast cells, and some T lymphocytes (reviewed in Refs. 36Phipps R. Stein S. Roper R. Immunol. Today. 1991; 12: 349-352Abstract Full Text PDF PubMed Scopus (496) Google Scholar and 37Kunkel S. Chensue S. Adv. Inflamm. Res. 1984; 7: 93-102Google Scholar). Thus, PGI2 and PGE2inhibit cytokine production by Th1-type lymphocytes, resulting in decreased secretion of IL-2, interferon-γ, IL-8, IL-12, and TNF-α (38Betz M. Fox B. J. Immunol. 1991; 146: 108-113PubMed Google Scholar, 39Fedyk E. Brown D. Phipps R. Adv. Exp. Med. Biol. 1997; 407: 237-242Crossref PubMed Google Scholar). Signaling through cAMP has variable effects on Th2-type lymphocytes, and thus these PGs may either have no effect or actually increase the production of Th2-type cytokines, including IL-4, IL-5, IL-10, and IL-13 (40Hilkens C. Vermeulen H. van Neerven R. Snijdewint F. Wierenga E. Kapsenberg M. Eur. J. Immunol. 1995; 25: 59-63Crossref PubMed Scopus (178) Google Scholar). These results suggest that the induction of COX-2 may, by shifting the profile of PG secretion, serve to actually diminish the inflammatory response in some tissues. Thus, in the absence of inflammatory mediators, COX-1 serves to generate a balance of PGs that may maintain a level of system homeostasis. Inflammatory mediators, like bacterial LPS, would evoke a number of responses, including the synthesis of Th1-type cytokines and COX-2. Our data, as well as the studies cited above, indicate that COX-2 metabolism of AA will tip the balance of secreted PGs to emphasize PGI2 and PGE2. Via cAMP signaling, these PGs would subsequently begin the resolution of the inflammatory response through the suppression of the synthesis of Th1-type cytokines and, at least indirectly, favor the development of a Th2-type profile of cytokines. According to this model, a defect in the ability to induce COX-2 expression would both suppress the synthesis of PGI2 and PGE2 and augment inflammation in some circumstances. Indeed, fibroblasts from patients with idiopathic pulmonary fibrosis have a severely impaired capacity to express COX-2, but not COX-1, and make much less PGE2 than do control fibroblasts (41Wilborn J. Crofford L. Burdick M. Kunkel S. Strieter R. Peters-Golden M. J. Clin. Invest. 1995; 95: 1861-1868Crossref PubMed Scopus (346) Google Scholar). The failure to induce COX-2 and produce prostanoids that can suppress inflammation, fibroblast proliferation, and collagen synthesis can thus contribute to disease progression." @default.
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