Evidence that prednisolone is inhibitory to the cyclooxygenase activity of human rectal mucosa

The effect of prednisolone on prostaglandin (PG) synthesis by rectal biopsies in organ culture was investigated using laminary flow bioassay and radioimmunoassay (RIA) or PGE2. Prednisolone was consistently found to inhibit basal synthesis in cultures whose duration ranged from 2-40 hours. This appeared to be both time and dose dependent. The ability of biopsies homogenised at the end of culture to transform exogenous arachidonic acid into PGE2 under defined conditions was also investigated and operationally designated cyclooxygenase activity. Prior treatment with prednisolone resulted in a reduction in cyclooxygenase activity. This inhibition occurred with a longer latency and to a lesser extent than inhibition of overall basal synthesis. These results suggest that corticosteroids, in addition to their know (indirect) inhibitory action on phospholipase activity, also affect cycloooxygenase activity. The most likely mechanism are either a repression of synthesis of fresh cyclooxygenase enzyme of induction of an endogenous inhibitor of cyclooxygenase activity.     

PGE2 secretion from organ cultured gastric mucosa: Correlation with cyclooxygenase activity and endogenous substrate release

In gastrointestinal research the in vitro release of prostaglandins from incubated or cultured biopsies is a widely used method to estimate prostaglandin synthesis. We therefore investigated the rate limiting mechanisms of PGE₂ release in organ cultured gastric mucosa of the rabbit, determining PGE₂ secretion from organ cultured mucosal biopsies by radioimmunoassay and prostaglandin synthesizing capacity by in vitro incubation of mucosal homogenate or microsomes with [¹⁴C]-arachidonic acid.Freshly taken biopsies secreted PGE₂ at an initial high rate, that decreased during the following 4 hrs of culture. This PGE₂ release was dose dependently reduced by inhibitors of the prostaglandin cyclooxygenase. 5mM acetylsalicylic acid (ASA) maximally suppressed PGE₂ secretion to 7% of controls, and the inhibition by ASA was quantitatively similar at every given culture period. PGE₂ release was markedly increased by carbenoxolone but was only slightly activated by extracellular calcium and the Ca⁺⁺-ionophore A23187. However, Ca⁺⁺/A23187 were unable to maintain PGE₂ secretion at the initial rate.PGE₂ secretion was undisturbed in calcium-free medium but was reduced to 50–60% of controls by excess EDTA. The intracellular calcium chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,N′,N′,-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) similarly inhibited PGE₂ release to 72% of controls. In contrast, PGE₂ release was unaffected by the intracellular calcium antagonist 3,4,5-trimethylene-bis(4-formylpyridinium bromide) dioxime (TMB-8), the calmodulin antagonists N-(6-aminohexyl)-1-5-chloro-1-naphthalenesulfonamide (W-7) and calmidazolium (compound R24571) or various direct inhibitors of endogenous arachidonic acid release like tetracaine, bromophenacyl bromid, neomycine or low dose quinacrine, indicating that the reduction of PGE₂ release by EDTA or BAPTA may be mediated by mechanisms different from substrate release. In contrast, an inhibition of PGE₂ secretion by quinacrine at high concentrations (≥ 0.8mM) was attributed to a direct inhibition of the prostaglandin cyclooxygenase, similar to ASA. Finally, the reduction of the prostaglandin synthesizing capacity by ASA was strongly correlated with the inhibition of PGE₂ secretion, also at low concentrations and minor degrees of inhibition.From these data we conclude, that the activity of the prostaglandin cyclooxygenase is rate limiting for PGE₂ secretion from organ cultured mucosal biopsies rather than arachidonic acid release by a phospholipase A₂. This should be considered for interpretation of studies based on prostaglandin release from cultured mucosa.     

PGE2 reduces arachidonic acid release in murine podocytes: evidence for an autocrine feedback loop

Increased glomerularprostaglandin E₂ (PGE₂) production isassociated with the progression of diseases such as membranous nephropathy, nephrotic syndrome, and anti-Thy1 nephritis. Weinvestigated the signaling pathways that regulate the synthesis andactions of PGE₂ in glomerular podocytes. To study itsactions, we assessed the ability of PGE₂ to regulate theproduction of its own precursor, arachidonic acid (AA), in a mousepodocyte cell line. PGE₂ dose-dependently reduced phorbolester (PMA)-mediated AA release. Inhibition of PMA-stimulated AArelease by PGE₂ was found to be cAMP/PKA-dependent, becausePGE₂ significantly increased levels of this secondmessenger, whereas the inhibitory actions of PGE₂ werereversed by PKA inhibition and reproduced by the cAMP-elevating agentsforskolin and IBMX. PGE₂ synthesis in this podocyte cellline increased fourfold at 60 min in response to PMA, coinciding withupregulation of cyclooxygenase (COX)-2 but not COX-1 levels. However,PGE₂ synthesis was significantly reduced by COX-1-selectiveinhibition, yet to a lesser extent by COX-2-selective inhibition. Ourfindings suggest that PMA-stimulated PGE₂ synthesis inmouse podocytes requires both basal COX-1 activity and induced COX-2expression, and that PGE₂ reduces PMA-stimulated AA releasein a cAMP/PKA-dependent manner. Such an autocrine regulatory loop mighthave important consequences for podocyte and glomerular function in thecontext of renal diseases involving PGE₂ synthesis.

     

Antiproliferative activity of guava leaf extract via inhibition of prostaglandin endoperoxide H synthase isoforms

Prostaglandin endoperoxide H synthase (PGHS) is a key enzyme for the synthesis of prostaglandins (PGs) which play important roles in inflammation and carcinogenesis. Because the extract from Psidium guajava is known to have a variety of beneficial effects on our body including the anti-inflammatory, antioxidative and antiproliferative activities, we investigated whether the extract inhibited the catalytic activity of the two PGHS isoforms using linoleic acid as an alternative substrate. The guava leaf extract inhibited the cyclooxygenase reaction of recombinant human PGHS-1 and PGHS-2 as assessed by conversion of linoleic acid to 9- and 13-hydroxyoctadecadienoic acids (HODEs). The guava leaf extract also inhibited the PG hydroperoxidase activity of PGHS-1, which was not affected by nonsteroidal anti-inflammatory drugs (NSAIDs). Quercetin which was one of the major components not only inhibited the cyclooxygenase activity of both isoforms but also partially inhibited the PG hydroperoxidase activity. Overexpression of human PGHS-1 and PGHS-2 in the human colon carcinoma cells increased the DNA synthesis rate as compared with mock-transfected cells which did not express any isoforms. The guava leaf extract not only inhibited the PGE₂ synthesis but also suppressed the DNA synthesis rate in the PGHS-1- and PGHS-2-expressing cells to the same level as mock-transfected cells. These results demonstrate the antiproliferative activity of the guava leaf extract which is at least in part caused by inhibition of the catalytic activity of PGHS isoforms.     

Prostaglandin E2 Receptors of Monocytes/Macrophages: Regulation by Insulin and Interleukin-1α

The regulation of receptors for prostaglandin E₂ (PGE₂) in monocyte/macrophage-like cells, P388D₁, by interleukin-1α (IL-1α) and insulin has been investigated. Many of the effects of IL-1, such as fever and other inflammatory activities, are linked to the stimulation of PGE₂ synthesis. On the other hand, PGE₂ exhibits suppressive effects on many steps in the immune response, including IL-1 production. The binding of PGE₂ to monocytes is reported to be essential for the inhibition of IL-1 production and activity. This inhibition occurs through the stimulation of cyclic AMP synthesis by the activation of PGE₂ receptor-linked adenylate cyclase. Although IL-1α stimulates PGE₂ synthesis in monocytes/macrophages during immunoactivation, it inhibits the binding of PGE₂ to these cells and may thereby exert a countervailing effect on the immunosuppressive action of this prostanoid. By contrast, insulin at physiological concentrations enhances the PGE₂ binding to these cells. This suggests that insulin at physiological concentrations may enhance the immunosuppressive action of PGE₂. Since the stimulation of cAMP synthesis in cells is regulated by PGE₂ binding, it is possible that these hormonal factors may control the immune response by modulating the PGE₂ receptor activity of monocytes/macrophages. This article focuses on the interactions of insulin and IL-1 with PGE₂ receptors of monocytes/macrophages.     

Effects of cyclooxygenase inhibitors and prostaglandin E2 on macrophage activation in vitro

The effect of the cyclooxygenase inhibitors, indomethacin and diclofenac, and of PGE₂ on either resting or stimulated macrophages was investigated. Peritoneal macrophages were obtained from untreated mice and cultured for 10 days. Macrophage activation was induced by zymosan phagocytosis and was monitored by testing for plasminogen activator secretion and the cellular levels of lactate dehydrogenase, β-glucoronidase and alkaline phosphodiesterase I.It was found that cyclooxygenase inhibitors activate resting macrophages and enhance the degree of activation obtained after zymosan phagocytosis. Addition of exogenous PGE₂, on the other hand, had the opposite effect, it suppressed activation induced either by cyclooxygenase inhibitors, phagocytosis or a combination of both. Cyclooxygenase inhibitors and PGE₂ did not affect the hexose monophosphate shunt activity of resting macrophages and had only a minor effect on the respiratory on the respiratory macrophages and had only a minor effect on the respiratory burst occuring during zymosan phagocytosis. It appears, therefore, that the observed changes in the state of activation of the machrophages are not related to hexose monophosphate shunt activity.The described effects suggest that PGE₂ and possibly other cyclooxygenase products may function as inhibitory feed-back regulators of macrophage activation.     

Endogenous prostaglandin E2 enhances polyclonal immunoglobulin production by tonically inhibiting T suppressor cell activity

The immunoregulatory effect of endogenous prostaglandin E₂ (PGE₂) on immunoglobulin production was studied in an in vitro culture system of human peripheral blood mononuclear cells, stimulated with pokeweed mitogen (PWM). Three different cyclooxygenase inhibitors (indomethacin, carprofen, and piroxicam) suppressed Ig synthesis by ~50%. This inhibitory effect could be reversed by adding low doses of exogenous PGE₂ (3 × 10^?9 to 3 × 10^?8M). These doses are endogenously produced in PWM-stimulated cultures, and a concentration of 2 × 10^?8M is reached after 48 hr of culture. When B cells were directly stimulated with helper factor, PGE₂ did not enhance Ig production and doses of 3 × 10^?7M to 3 × 10^?6M were inhibitory. The effects of indomethacin and PGE₂ were eliminated when T cells were irradiated or treated with mitomycin prior to culture. The enhancing effects of PGE₂ were substantially reduced after OKT8(+) T cells were removed from the system. PWM-stimulated cultures of lymphocytes from healthy subjects over age 70 were more sensitive to inhibition by indomethacin and to stimulation by PGE₂ than were cultures of lymphocytes from young controls. Thus the major role of endogenous PGE in polyclonal Ig production in vitro is to tonically inhibit a radiosensitive, OKT8(+) suppressor T cell. This tonic inhibition is increased in subjects over 70, which provides one explanation for decreased suppressor cell function in elderly subjects.     

Sphingosine 1-phosphate (S1P) induces COX-2 expression and PGE2 formation via S1P receptor 2 in renal mesangial cells

Understanding the mechanisms of sphingosine 1-phosphate (S1P)-induced cyclooxygenase (COX)-2 expression and prostaglandin E₂ (PGE₂) formation in renal mesangial cells may provide potential therapeutic targets to treat inflammatory glomerular diseases. Thus, we evaluated the S1P-dependent signaling mechanisms which are responsible for enhanced COX-2 expression and PGE₂ formation in rat mesangial cells under basal conditions. Furthermore, we investigated whether these mechanisms are operative in the presence of angiotensin II (Ang II) and of the pro-inflammatory cytokine interleukin-1β (IL-1β).     

Prostaglandin E2 stimulates insulin-like growth factor binding protein-4 expression and synthesis in cultured human articular chondrocytes: Possible mediation by Ca++ -calmodulin regulated processes

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism whose action is mediated by high affinity cell surface receptors and bioactivity and bioavailability regulated, in part, by IGF-1 binding proteins (IGFBPs). Prostaglandin E₂ (PGE₂) stimulates collagen and proteoglycan synthesis in cartilage via an autocrine feedback loop involving IGF-1. We determined whether the eicosanoid could regulate IGFBP-4, a major form expressed by chondrocytes and, as such, act as a modifier of IGF-1 action at another level. Using human articular chondrocytes in high-density primary culture, Western and Western ligand blotting to measure secreted IGFBP-4 protein, and Northern analysis to monitor IGFBP-4 mRNA levels, we demonstrated that PGE₂ provoked a 2.7 ± 0.3- and 3.8 ± 0.5- (n = 3) fold increase in IGFBP-4 mRNA and protein, respectively. This effect was reversed by the Ca⁺⁺ channel blocker, verapamil, and the Ca⁺⁺/calmodulin inhibitor, W-7. The Ca⁺⁺ ionophore, ionomycin, mimicked the effects of PGE₂. The phorbol ester, PMA, which activated phospholipid-dependent protein kinase C (PKC) in chondrocytes, had no effect on IGFBP-4 production. Cyclic AMP mimetics and PKA activators, IBMX, and Sp-cAMP, inhibited the expression of the binding protein as did the PGE₂ secretagogue, interleukin-1β (IL-β). The inhibitory effect of the latter cytokine was mediated by a erbstatin/genistein (tyrosine) sensitive kinase. Dexamethasone, an inhibitor of cyclooxygenase (COX-2) expression and PGE₂ synthesis, down-regulated control, constitute levels of IGFBP-4 mRNA and protein, eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE₂-receptor signalling pathways. The results suggest that extracellular signals control IGFBP-4 production by a number of different transducing networks with changes in Ca⁺⁺ and calmodulin activity exerting a strong positive influence, possibly maintaining the constitutivity of IGFBP-4 synthesis under basal conditions. PGE₂ activation of the IGF-1/IGFBP axis may play a pivotal role in the metabolism of cartilage and possibly connective tissues in general. Eicosanoid biosynthesis may be a rate-limiting step in cartilage repair processes. J. Cell. Biochem. 65:408–419. © 1997 Wiley-Liss, Inc.     

Reaction mechanism of prostaglandin E2 biosynthesis and its modeling

The reaction mechanism of PGE₂ biosynthesis was investigated by a detailed examination of the cyclo-oxygenase and PGE₂-isomerase activities in acetone-pentane powder (microsomal fraction of ram seminal vesicular glands). Two main types of inactivating process were recognized in the reaction system. One type was due to irreversible inactivation caused by the oxidizing agent [O]^·_X released through the reduction of PGG₂ to PGH₂, while the other type was due to reversible inhibition which was supposed to be derived from the precursor arachidonic acid (AA). This inhibitor was found to block the activities of both cyclooxygenase and PGE₂-isomerase, and to compete with the substrates AA and PGH₂. Although no significant substrate inhibition was observed, arachidonic acid was slightly inhibitory toward PGE₂-isomerase.     

Differential effects of ibuprofen and naproxen sodium on menstrual prostaglandin release and on prostaglandin production in the rat uterine homogenate

In two independent studies, ibuprofen and naproxen sodium were found to be equi-effective in alleviating dysmenorrheic symptoms. However, the effects of these drugs on menstrual PG release were found to be dissimilar. Ibuprofen primarily inhibited menstrual PGF_2α release with little effect on PGE release, whereas, naproxen sodium inhibited both PGF_2α and PGE₂ release equally. To verify these results, we determined the inhibitory potency, IC 50' of ibuprofen and naproxen sodium on PGF_2α and PGE₂ synthesis in the rat uterine homogenate system. The preferential PGF2a inhibitory activity of ibuprofen was confirmed.These findings suggest that ibuprofen may, in addition to inhibiting fatty acid cyclooxygenase, also inhibit PGF_2α reductase, or some other PG metabolic pathways which affect PGF a and PGE₂ synthesis differently. The significance of this differential PG synthesis inhibitory effect in dysmenorrheic therapy is discussed.     

Two different mechanisms for modulation of bronchoconstriction in guinea-pigs by cyclooxygenase metabolites

Leukotriene D₄ (LTD₄)-induced bronchoconstriction in guinea-pig airways has a cyclooxygenase (COX)-dependent component. The main objective of this study was to establish if prostaglandin (PG) D₂-induced bronchoconstriction also was modulated by COX products. The effects of non-selective and selective COX-1 and COX-2 inhibitors on bronchoconstriction induced by LTD₄ and PGD₂ were investigated in the perfused and ventilated guinea-pig lung (IPL). Both LTD₄-induced bronchoconstriction and thromboxane (TX) A₂ release was suppressed by COX inhibitors or by TX synthesis inhibition. The release of additional COX products following CysLT₁ receptor activation by LTD₄ was established by measurements of immunoreactive 6-keto PGF_1α (a stable metabolite of PGI₂) and PGE₂. In contrast, TP receptor-mediated bronchoconstriction by PGD₂ was somewhat enhanced by COX inhibitors, and there was no measurable release of COX products after TP receptor activation with U-46619. PGE₂ was bronchoprotective in IPL as it inhibited the histamine-induced bronchoconstriction. In the isolated guinea-pig trachea, neither PGD₂ nor U-46619 actively released PGE₂, but continuous production of PGE₂ and PGI₂ was established, and the response to PGD₂ was enhanced also in the trachea by COX inhibition. The study documented that bronchoconstriction induced by LTD₄ and PGD₂ in IPL was modulated differently by COX products. Whereas bronchoconstriction induced by LTD₄ was amplified predominantly by secondarily released TXA₂, that induced by PGD₂ was attenuated by bronchoprotective PGE₂ and PGI₂, presumably tonically produced in the airways.     

Limited effect of anti-rheumatic treatment on 15-prostaglandin dehydrogenase in rheumatoid arthritis synovial tissue

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which prostaglandin E₂ (PGE₂) displays an important pathogenic role. The enzymes involved in its synthesis are highly expressed in the inflamed synovium, while little is known about 15- prostaglandin dehydrogenase (15-PGDH) that metabolizes PGE₂. Here we aimed to evaluate the localization of 15-PGDH in the synovial tissue of healthy individuals or patients with inflammatory arthritis and determine the influence of common RA therapy on its expression.

Methods

Synovial tissue specimens from healthy individuals, psoriatic arthritis, ostheoarthritis and RA patients were immunohistochemically stained to describe the expression pattern of 15-PGDH. In addition, the degree of enzyme staining was evaluated by computer analysis on stained synovial biopsies from two groups of RA patients, before and after RA specific treatment with either intra-articular glucocorticoids or oral methotrexate therapy. Prostaglandins derived from the cyclooxygenase (COX) pathway were determined by liquid-chromatography mass spectrometry in supernatants from interleukin (IL) 1β-activated fibroblast-like synoviocytes (FLS) treated with methotrexate.

Results

15-PGDH was present in healthy and inflamed synovial tissue, mainly in lining macrophages, fibroblasts and vessels. Intra-articular glucocorticoids showed a trend towards reduced 15-PGDH expression in RA synovium (p = 0.08) while methotrexate treatment left the PGE₂ pathway unaltered both in biopsies ex vivo and in cultured FLS.

Conclusions

Early methotrexate therapy has little influence on the expression of 15-PGDH and on any of the PGE₂ synthesizing enzymes or COX-derived metabolites. Thus therapeutic strategies involving blocking induced PGE₂ synthesis may find a rationale in additionally reducing local inflammatory mediators.     

In vivo metabolism of dihomo-γ-linolenic acid to bronchoactive products in the canine lung

We investigated the effects of dihomo-γ-linolenic acid (DGLA), the fatty acid precursor to the monoenoic prostaglandins, on pulmonary mechanics in the intact chest, artificially ventilated dog. Under conditions of normal airway tone, intravenously administered DGLA produced a modest, dose-related increase in lung resistance and decrease in dynamic lung compliance. These responses were approximately 30–100 times less than those produced by arachidonic acid. The bronchoconstrictive responses to DGLA were abolished by prior inhibition of the cyclooxygenase with indomethacin. PGD₁ was equal in activity to PGF_1α in causing constriction of central and peripheral airways of the dog. PGE₁ was without significant airway effects in these animals under conditions of resting airway tone. We conclude that DGLA is a moderately good substrate for the cyclooxygenase pathway enzymes of the canine lung, producing products having a predominantly bronchoconstrictive effect. This bronchoconstriction is most likely due to the synthesis of PGD₁ and PGF_1α and is not due to the synthesis of lipoxygenase pathway products.     

Prostaglandin regulation of macrophage function: Effect of endogenous and exogenous prostaglandins

The expression of macrophage antitumor activity and the production of prostaglandins (PG) by operationally defined macrophage populations differed under varying culture conditions. Culture conditions that caused increased PGE₂ production by activated macrophages resulted in an inhibition of their tumoricidal activity. In contrast, production of high levels of PGE₂ by resident and elicited macrophages was associated with an increase in antitumor activity. The activation of resident or elicited cells by lipopolysaccharide (LPS) could be blocked by indomethacin. Treatment of these macrophages with PGE₂ alone also resulted in their activation and subsequent tumor cell destruction. Activation of resident and elicited macrophages by LPS appears to be mediated by PGE₂.     

Mechanism for leukotriene C4 stimulation of chloride transport in cornea

Summary The leukotriene, LTC₄, exerts a stimulatory effect on chloride transport in the frog cornea. In the work described here, the mechanism of action of LTC₄ to stimulate chloride transport was studied.In corneas pretreated with indomethacin, the effect of LTC₄ was abolished, suggesting the involvement of cyclo-oxygenase products in the response. Incubation of corneas with LTC₄ resulted in a significant stimulation in PGE₂ synthesis, as determined by TLC-autoradiography and radioimmunoassay. In addition, LTC₄ was found to stimulate cAMP synthesis in the cornea, and this stimulation was blocked with indomethacin. PGE₂ was previously shown by us to be the dominant cyclo-oxygenase product formed in the frog cornea, and is capable of stimulating cAMP and chloride transport. We suggest that LTC₄ stimulation of chloride transport is mediated via activation of the cyclooxygenase pathway, resulting in enhanced PGE₂ synthesis. Elevated PGE₂ levels induce cAMP synthesis, and ultimately, the stimulation of chloride transport. Further, the activation of cyclo-oxygenase was found to be dependent on phospholipase A₂ activity. This was shown by the inhibition of the LTC₄ effect in the presence of quinacrine. Similarly, inhibition of the LTC₄ effect in the presence of trifluoperazine suggests that cyclo-oxygenase activation by LTC₄ may be mediated via calmodulin. We have previously demonstrated that the frog cornea has the biosynthetic capacity to produce LTC₄. Therefore LTC₄ may function as an endogenous regulator of chloride transport in this tissue.     

Prostaglandin E2 mediated effects on the synthesis of fetal and adult hemoglobin in blood erythroid bursts

The effects of prostaglandin E₂ (PGE₂) in association with erythropoietin on the synthesis of fetal and adult hemoglobin in peripheral blood-derived erythroid burst colonies from normal adults and from patients with sickle cell anemia were investigated. The synthesized hemoglobin at the end of 8, 14 or 18 days in culture was separated by DEAE-cellulose chromatography of ³⁵S-methione labelled hemoglobin. Quantitative estimation of the synthesized hemoglobin phenotypes, for the three indicated culture periods, showed preferential synthesis of Hb F in addition to an overall increase in hemoglobin synthesis in PGE₂ treated colonies. Furthermore, the reactivation of fetal hemoglobin production by PGE₂ was more pronounced when the adherent cells were included in the culture dishes. These results indicate that the addition of PGE₂ to culture dishes presumably constitutes an environmental change to promote the functional seen in the blood erythroid bursts in terms of Hb synthesis and switching.     

Decidual adenylate cyclase and prostaglandin production in vitro

Human decidua contains an active adenylate cyclase, and a number of studies indicate that adenylate cyclase is functionally linked to increased in vitro prostaglandin synthesis. Increased decidual prostaglandin synthesis is associated with parturition, and therefore activation of adenylate cyclase may be involved in the control of human parturition. In this study, third trimester human decidual cells were preincubated for no more than 24 h prior to stimulation with a number of reagents which increase cellular cyclic AMP levels. Forskolin rapidly increased intracellular and extracellular cyclic AMP levels, but there was no increase in prostaglandin E₂ biosynthesis during incubations ranging from 5 min up to 24 h. Dibutyryl cyclic AMP or 8-bromo-cyclic AMP were also without effect on PGE₂ production, which suggests that the adenylate cyclase was not linked to the mechanisms regulating prostaglandin production. Cholera toxin increased basal cyclic AMP and PGE₂ synthesis, and was without effect on IL-1β-stimulated PGE₂ levels. PGE₂ synthesis was increased by 24 h culture with IL-1β in all the cell preparations, indicating that the cells were biologically active, and that the lack of effect of changes in cyclic AMP synthesis on PGE₂ levels could not be attributed to a defect in the prostaglandin synthetic pathway. Our findings did not agree with earlier work which showed that changes in cyclic AMP were correlated with changes in PGE₂ production by human decidual cells. It is clear that in the previous studies the decidual cells were preincubated for 4–7 days prior to stimulation, in contrast with 24 h in our investigation. We suggest that the functional link between cyclic AMP and PGE₂ synthesis reported previously may develop during culture, and not be a part of normal decidual cell function, but further studies are needed to test this hypothesis.     

The effects of anti-inflammatory drugs on prostaglandin production by rheumatoid synovial tissue

The effect of anti-inflammatory drugs on prostaglandin production by rheumatoid synovial tissue has been investigated. Synovial explants were maintained in tissue culture for periods up to six days and PGE₂ concentrations in culture were determined by radioimmunoassay. The more potent nonsteroidal inhibitors of PGE₂ production and their IC₅₀ (μM) values were indomethacin 0.005, flufenamic acid 0.2, flurbiprofen 0.6, ibuprofen 2.0, naproxen 6.0, phenylbutazone 10.0, and aspirin 20.0. Drugs with weak or insignificant effects were hydroxychloroquin, acetaminophen, azathioprine, chloroquin, penicillamine, gold Na thiomalate, and Na salicylate. Glucocorticoids were potent inhibitors; dexamethasone 0.003, prednisolone 0.01, hydrocortisone 0.03; while mineralocorticoids deoxycorticosterone and aldosterone were inactive at 1.0 μM. There is a reasonably good correlation between the IC₅₀ concentrations of the nonsteroidal inhibitors and their peak free plasma concentration achieved during therapy in man. Inhibition of prostaglandin synthesis may contribute to the effects of many but not all anti-inflammatory drugs.     

Regression mechanisms of mouse fibrosarcoma cells after in vitro exposure to quercetin: Diminution of tumorigenicity with a corresponding decrease in the production of prostaglandin E2

Summary We have previously reported that both regressor (QR) and progressor (metastatic, QP) clones were obtained after the in vitro exposure of a mouse fibrosarcoma BMT-11 cl-9 to quercetin [17]. In this study, we investigated possible mechanisms of spontaneous regression of QR clones as compared with tumorigenic QP and BMT-11 cl-9 tumor clones. We observed that BMT-11 cl-9 cells produced relatively high amounts of prostaglandin E₂ (PGE₂) during in vitro culture. The average production by 11 subclones of BMT-11 cl-9 cells was 9236±2829 pg/ml whereas that by 9 QR clones was 3411±2213 pg/ml (P <0.02). Indomethacin not only inhibited in vitro PGE₂ synthesis by QP clones (high-PGE₂ producers) but also the s.c. growth of QP clones in mice. Chronological changes in host immune responses to tumor-associated antigen were measured by cytotoxic T lymphocyte (CTL) activity examined after mixed lymphocyte/tumor cell culture of spleen cells obtained from tumor-bearing mice. The CTL activity disappeared abruptly in the spleen of QP-clonebearing mice 21 days after the inoculation of tumors, whereas the spleen cells of QR-clone-inoculated mice retained their CTL activity. We determined that the mechanism responsible for the regression of these regressor clones is not due to any qualitative or quantitative increase in pre-existing membrane antigens, nor the emergence of new antigen(s) on the cell surface of the QR clones; nor was it due to enhanced susceptibility of QR clones to natural killer cells, lymphokine-activated killer cells and macrophages. These finding suggest that the regression mechanism of QR clones may be the diminished inhibition of host response to tumor-associated antigen caused by the reduced production of PGE₂ by QR clones.