Mononuclear cells enhance prostaglandin E2 production of polymorphonuclear leukocytes via tumor necrosis factor alpha

To clarify the interactions between mononuclear cells and polymorphonuclear leukocytes, and to identify the cytokine(s) that mediate the interaction, the effects of a culture supernatant of LPS-stimulated mononuclear cells on production of arachidonic acid metabolites of polymorphonuclear cells were studied. The culture supernatant of LPS-stimulated mononuclear cells increased production of prostaglandin E2 of polymorphonuclear cells. TNF alpha, but not IL-1, IL-2, IL-6, or IFN gamma, enhanced the prostaglandin E2 production when added in vitro. Additionally, an anti-rTNF alpha monoclonal antibody inhibited the stimulating activity of the culture supernatants. TNF alpha, produced by mononuclear cells, appears to play an important role in the development of inflammation, such as rheumatoid arthritis, by enhancing the arachidonic acid metabolism of the polymorphonuclear cells.     

Macrolide antibiotics inhibit prostaglandin E2 synthesis and mRNA expression of prostaglandin synthetic enzymes in human leukocytes

We investigated the action of macrolide antibiotics, which are considered to have anti-inflammatory activity, on lipopolysaccharide (LPS)-stimulated prostaglandin (PG) E2 synthesis and the expression of mRNAs for cytosolic phospholipase A2 (cPLA2), cyclooxygenase (COX)-1, and COX-2 in human leukocytes. The production of LPS-stimulated PGE2 was significantly increased in peripheral polymorphonuclear leukocytes (PMNLs) and in mononuclear leukocytes (MNLs). Amounts of mRNAs for COX-2 and cPLA2, but not for COX-1, were enhanced by LPS in PMNLs and MNLs. The LPS-enhanced PGE2 synthesis and the expression of cPLA2 and COX-2 mRNAs were inhibited by clarithromycin, azithromycin and dexamethasone in PMNLs and MNLs. The mRNA expression of COX-1 in PMNLs was decreased by clarithromycin and azithromycin. Macrolide antibiotics inhibited PGE2 synthesis in human leukocytes by suppressing cPLA2, COX-1, and COX-2 mRNA expression. These data indicate one mechanism of macrolide anti-inflammatory activity.     

Actions of endothelin-1 on prostaglandin production by gestational tissues

Endothelin-1 (10(-11)M-10(-7)M) was incubated with human umbilical vein endothelial cells and cells derived from amnion and decidua and prostaglandin production was determined. The rates of biosynthesis of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2) by endothelial cells were increased significantly by treatment with endothelin-1. Amnion cell PGE2 production was reduced significantly by endothelin-1 treatment whereas decidual PGE2 and prostaglandin F2 alpha production was unaffected by this treatment. Thus, it is possible that endothelins may play a part in the regulation of uteroplacental hemodynamics and the mechanisms of parturition.     

Signal pathways involved in the regulation of prostaglandin E synthase-1 in human gingival fibroblasts

Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme regulating the synthesis of prostaglandin E2 (PGE2) in inflammatory conditions. In this study we investigated the regulation of mPGES-1 in gingival fibroblasts stimulated with the inflammatory mediators interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNFalpha). The results showed that IL-1beta and TNFalpha induce the expression of mPGES-1 without inducing the expression of early growth response factor-1 (Egr-1). Treatment of the cells with the PLA2 inhibitor 4-bromophenacyl bromide (BPB) decreased the cytokine-induced mPGES-1 expression accompanied by decreased PGE2 production whereas the addition of arachidonic acid (AA) upregulated mPGES-1 expression and PGE2 production. The protein kinase C (PKC) activator PMA did not upregulate the expression of mPGES-1 in contrast to COX-2 expression and PGE2 production. In addition, inhibitors of PKC, tyrosine and p38 MAP kinase markedly decreased the cytokine-induced PGE2 production but not mPGES-1 expression. Moreover, the prostaglandin metabolites PGE2 and PGF2alpha induced mPGES-1 expression as well as upregulated the cytokine-induced mPGES-1 expression indicating positive feedback regulation of mPGES-1 by prostaglandin metabolites. The peroxisome proliferator-activated receptor-gamma (PPARgamma) ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), decreased mPGES-1 expression but not COX-2 expression or PGE2 production. The results indicate that the inflammatory-induced mPGES-1 expression is regulated by PLA2 and 15d-PGJ2 but not by PKC, tyrosine kinase or p38 MAP kinase providing new insights into the regulation of mPGES-1.     

Simultaneous measurement of endothelial cell damage,elastase release and chemiluminescence response during interaction between polymorphonuclear leukocytes and endothelial cells

Using cultured human umbilical cord vein endothelial cells and human blood neutrophils, the interaction between neutrophils and endothelial cells, in vitro, was studied. The aim of the study was to examine whether a respiratory burst stimulation by neutrophils would be observed by neutrophil/endothelial cell interaction and whether the respiratory burst stimulation of neutrophils by endothelial cells could be enhanced by lipopolysaccharide stimulation of neutrophils. The second aim was whether such an effect, or secretion of elastase, could cause an endothelial cell damage in vitro. Chemiluminescence as an indicator of oxygen-derived metabolites produced by neutrophils, elastase release by neutrophils, and endothelial cell damage, based on¹¹¹ In-oxine release from labelled endothelial cells, were measured simultaneously. The present investigation demonstrates that neutrophils can be directly stimulated by endothelial cells. A further amplification of this process following lipopolysaccharide priming up to 10 ng/ml blood could be demonstrated. A slight endothelial cell damage occurs following neutrophil stimulation, although elastase secretion does not increase during interaction between neutrophils and endothelial cells. These results raise the possibility that oxygen-derived metabolites rather than elastase contribute to an endothelial cell damage which might occur in conditions such as endotoxin-induced adult respiratory distress syndrome.     

Cellular confluence determines injury-induced prostaglandin E2 synthesis by human keratinocyte cultures

When keratinocyte cultures become confluent, their prostaglandin E2 synthesis is suppressed. To determine whether the injury response is characterized by increased prostaglandin E2 synthesis, an in vitro injury model was developed. When confluent keratinocyte cultures were focally lethally irradiated using ultraviolet light B, a dose-dependent increase in prostaglandin E2 synthesis was induced by the injury. After irradiation, confluent cultures' prostaglandin E2 synthesis increased for 2 days to 8-fold more than controls, then decreased to control values by day 6. Increased prostaglandin E2 synthesis was first detected 8 h after injury. Focal irradiation of non-confluent cultures (killing isolated colonies) caused no change in prostaglandin E2 synthesis, indicating that culture continuity must be disrupted before synthesis increases. In addition, partial irradiations of petri dishes demonstrated that enhanced metabolism was confined to cells adjacent to the injury site and was not mediated by a soluble factor. When confluent and injured cultures were incubated with [14C]arachidonic acid, and the products formed analyzed by thin layer chromatography, 10-fold more prostaglandin E2 microgram protein was seen in irradiated cultures relative to confluent controls. The products formed by each group were the same, and no consistent increases in metabolites other than prostaglandin E2 were observed. The increased synthesis of prostaglandin E2 by injured cultures was apparently due to an increase in cyclooxygenase activity as determined by kinetic experiments. These data indicate that the pattern of metabolism of arachidonic acid seen in non-confluent cultures is similar to that seen in injury, and that cell-cell contact modulates enhanced prostaglandin E2 synthesis.     

The prostacyclin analogue iloprost and prostaglandin E1 suppress sterol synthesis in freshly isolated human mononuclear leukocytes

The effects of the stable prostacyclin analogue iloprost, prostaglandin E1 and prostaglandin F2 alpha on sterol synthesis were investigated in freshly isolated human mononuclear leukocytes. Incubation of cells for 6 h in a medium containing lipid-depleted serum led to a 3-fold rise in the rate of sterol synthesis from [14C]acetate or tritiated water. Iloprost and prostaglandin E1 added in increasing concentrations at zero time resulted in an inhibition of the synthesis of sterols, the suppression being 50 and 55% at a concentration of 1 mumol/1, respectively. Both prostaglandins yielded a sigmoidal log dose-effect curve. In contrast, prostaglandin F2 alpha had no influence on sterol synthesis up to a concentration of 1 mumol/1. The action of the prostacyclin analogue and prostaglandin E1 on the relative rate of sterol synthesis was not immediate, since the prostaglandins had no effect when given at 6 h to the incubation medium, and the incorporation of [14C]acetate into sterols was measured thereafter. The results suggest that prostacyclin and prostaglandin E1 affect cholesterol synthesis and therefore may play a role in the regulation of cellular cholesterol homeostasis and in the development of atherosclerosis.     

Binding of prostaglandin E1 to human erythrocyte membrane

Prostaglandin E1 is known to alter the structural and functional characteristics of red blood cells, yet, little is understood about the membrane receptors mediating this process. We therefore studied the binding of tritium-labeled prostaglandin E1 to the intact human erythrocyte membrane and demonstrated that the interaction is highly specific, rapid, saturable and reversible. Scatchard analysis of prostaglandin E1 binding to the membrane preparations showed the presence of two independent classes of prostaglandin E1 binding sites which differed in their affinity for the autacoid. The high-affinity class had Kd = 3.6 X 10(-9) M and the low-affinity class had Kd = 5.6 X 10(-5) M. The optimum pH for the binding of [3H]prostaglandin E1 to the erythrocyte membrane was found to be around 7.5 and maximum specific binding occurred at a concentration of 5 mM Mg2+ in the incubation mixture. [3H]Prostaglandin E1 bound to the membrane preparation could not be displaced by GTP or by its stable derivative Gpp[NH]p. However, prostaglandin E1 bound to the erythrocyte membrane preparation could be rapidly displaced by cyclic AMP. The IC50 (concentration of the nucleotide displacing 50% bound [3H]prostaglandin E1 from the membrane) was 75 nM. Other adenine nucleotides or cyclic GMP could not substitute for cyclic AMP. Unlike the right-side-out erythrocyte membrane, the inside-out membrane preparations do not bind [3H]prostaglandin E1. Treatment of right-side-out erythrocyte membrane preparation with neuraminidase markedly decreases the binding of prostaglandin E1. Incubation of the erythrocyte membrane preparation with trypsin resulted in total loss of the binding activity. These results indicate that the prostaglandin E1 binding sites located on the cell surface and sialic acid residues are required for prostaglandin E1 binding to the human erythrocytes. These results also indicated that the binding sites are glycoprotein in nature.     

Microtubule disruption enhances prostaglandin E2 production in osteoblastic cells

Prostaglandins have been implicated in the response of bone to mechanical stimuli. To explore the potential role of the cytoskeleton in the control of prostaglandin production, we examined the effect of cytoskeleton disrupting agents on arachidonic acid metabolism in rat calvaria osteoblastic cells. We found that microtubule disrupting agents increase prostaglandin E production 4-5-fold. Stimulation was first detectable at 4 h and rose sharply between 4 and 8 h. 2 h exposure to 1 microM colchicine was sufficient to produce the maximum effect. Cytochalasin B at concentrations which caused marked shape changes had no effect on prostaglandin E production or on its stimulation by colchicine. Taxol, a stabilizer of microtubules, reduced the colchicine effect. The increase in prostaglandin E production was associated with enhanced conversion of arachidonic acid to prostaglandin E2 rather than enhanced release of arachidonic acid from phospholipids. This increase in enzymatic activity was not abolished by cycloheximide treatment at concentrations which inhibited 90% of protein synthesis in the cells.     

Induction of refractoriness of cyclic AMP responses to prostaglandin E1 and epinephrine by prior exposure of guinea pig macrophages to lipopolysaccharide

Prior exposure of guinea pig macrophages to LPS (lipopolysaccharide) resulted in reduced cAMP-generating responses to prostaglandin E1 and epinephrine. LPS-induced refractoriness was diminished when LPS treatment was carried out in the presence of an inhibitor of prostaglandin synthesis, hydrocortisone, or indomethacin, or an inhibitor of protein synthesis, cycloheximide. The release of arachidonic acid and its metabolites, especially prostaglandin E2 and thromboxane B2, increased during incubation of macrophages with LPS. These increases were efficiently antagonized by hydrocortisone, indomethacin, or cycloheximide. Preincubation of macrophages with prostaglandin E1 greatly reduced the subsequent responses of cAMP generation to prostaglandin E1 and unexpectedly also to epinephrine. Thus, increased production of prostaglandins during the LPS treatment is likely to be responsible for decreased cAMP responses to subsequent addition of prostaglandin E1 and epinephrine.     

Actions of endothelin-1 on prostaglandin production by gestational tissues

Endothelin-1 (10⁻¹¹M-10⁻⁷M) was incubated with human umbilical vein endothelial cells and cells derived from amnion and decidua and prostaglandin production was determined. The rates of biosynthesis of 6-keto-prostaglandin F₁ (6-keto-PGF₁) and prostaglandin E₂ (PGE₂) by endothelial cells were increased significantly by treatment with endothelin-1. Amnion cell PGE₂ production was reduced significantly by endothelin-1 treatment whereas decidual PGE₂ and prostaglandin F₂ production was unaffected by this treatment. Thus, it is possible that endothelins may play a part in the regulation of uteroplacental hemodynamics and the mechanisms of parturition.     

The effects of norepinephrine and prostaglandin E1 on pharmacokinetics of lidocaine in isolated perfused rat liver

We hypothesized that depression of liver function by norepinephrine can be improved by prostaglandin E1. Isolated perfused rat liver was selected as an experimental model, since the flow rate can be regulated in it. Twenty-one rats were randomly allocated to three groups: control, norepinephrine, and norepinephrine and prostaglandin E1 groups. The liver was perfused in a recirculating system at a constant flow rate of 20 ml/min. After administration of two milligrams of lidocaine in each group, lidocaine and monoethylglycinexylidide concentrations in the recirculating system were measured. Lidocaine pharmacokinetics were analyzed using the SAAM II program, including metabolic rate from lidocaine to monoethylglycinexylidide using time-concentration curves. Norepinephrine significantly increased perfusion pressure and the area under the time-concentration curve for lidocaine. Norepinephrine decreased the clearance and the elimination rate constant of lidocaine compared with those in the control group. Although administration of prostaglandin E1 after infusion of norepinephrine did not significantly change perfusion pressure, it significantly (p < 0.05) improved metabolic rate, clearance and the elimination rate constant of lidocaine in the isolated rat liver model.     

Elimination of low steady-state concentrations of E15,6-3H2]prostaglandin E1 in the pulmonary and the systemic circulations of anaesthetized rats

The elimination of [3H]prostaglandin E1 in anaesthetized rats was studied by continuous intravenous or intraarterial infusions, producing steady-state concentrations at the level of endogenous prostaglandin E2 in mixed venous blood. Blood samples (0.5 ml) were collected from the carotid artery or the right atrium, respectively. The levels of [3H]prostaglandin E1 were measured at different infusion time intervals and the 3H-labeled hydrophobic metabolites characterized. Cardiac output was estimated by a modification of the dye injection method, using 125I-labelled albumin as the marker. From the cardiac output and the rate of infusion, the fractional clearance of the lung and the systemic beds in the steady-state situation were estimated to 88.3 +/- 3.2% and 54.1 +/- 15.2% (mean +/- S.D.), RESPECTIVELY. The hydrophobic metabolites were characterized chromatographically on Sephadez LH-20 columns, using synthetically prepared [14C]prostaglandin metabolites as internal standards and markers. The identities of some metabolites were further established by derivative formation to a constant [3H]/[14C] ratio. The major metabolite was 15-keto-13,14-dihydro-[3H]prostaglandin E1, while 15-keto-[3H]prostaglandin E1 and 13,14-dihydro-[3H]prostaglandin E1 could not be demonstrated.     

Cadmium stimulates prostaglandin E2 synthesis in osteoblast-like cells, MC3T3-E1

The effect of Cd on prostaglandin E2 production in osteoblasts was studied using cloned osteoblast-like cells, MC3T3-E1, which were established from new-born mouse calvaria. Treatment of the cells with Cd caused a dose- (0-10 microM) and time- (0-24 h) dependent increase in the release of prostaglandin E2 from the cells into the culture medium. A lag time of 4 h was required for the onset of the phenomenon. The release of [14C]arachidonic acid from prelabeled cell membrane was little influenced by the Cd treatment, while conversion of [14C]arachidonic acid to prostaglandin E2 by the homogenate of the cells treated with Cd was enhanced as compared to that by untreated cells. The stimulatory effect of Cd on prostaglandin E2 production was abolished in the presence of cycloheximide (100 ng/ml). By Western blot analysis with polyclonal rabbit anti-cyclooxygenase antibody, it was revealed that Cd treatment augmented the amount of immunoreactive cyclooxygenase in the cells. These results strongly suggest that Cd stimulates prostaglandin E2 production through the induction of cyclooxygenase in MC3T3-E1 cells. This effect of Cd may be involved in the mechanisms of Cd-induced bone injury.     

Effects of prostaglandin E1 on collagen production and degradation in human fetal lung fibroblasts

We examined the effects of prostaglandin E1 on the production and degradation of collagen in human fetal lung fibroblasts. Percentage collagen production was determined by incubating confluent cultures for 6 h with [3H]proline and either [14C]glycine or [14C]leucine and measuring the relative amounts of radioactivity incorporated into collagenase-sensitive and collagenase-insensitive material. Percentage collagen degradation was determined by measuring hydroxy[14C]proline in a low-molecular-weight fraction relative to total hydroxy[14C]proline. Prostaglandin E1, when present at a concentration as low as 0.25 micrograms/ml, reduced net collagen production by a factor of one-half, from 8 +/- 2 to 4 +/- 1% (P less than 0.05). In contrast, the change in percentage degradation was relatively gradual, rising steadily from the control value of 15 +/- 2 to 33 +/- 2% at 4 micrograms/ml (P less than 0.05). The increase in degradation, while significant, could not account for the total decrease in collagen production. We conclude that prostaglandin E1 exerts its inhibitory effect on collagen production in two essentially independent ways: lowering the rate of synthesis and increasing intracellular degradation. However, the decrease in synthesis is greater than the increase in degradation.     

Production of prostacyclin and prostaglandin E2 in resting and IL-1beta-stimulated A549, HUVEC and hybrid EA.HY 926 cells.

Production of arachidonic acid (AA) metabolites - prostacyclin (PGI(2)) in large vessels and prostaglandin E(2) (PGE(2)) in microcirculation is intrinsically involved in maintenance of vascular wall homeostasis. EA.hy 926 is a hybrid cell line, is derived by fusion of HUVEC with A549 cells. The aim of this study was to examine the production of prostacyclin and PGE2 in resting and IL-1beta-stimulated EA.ha 926 cells, in comparison with its progenitor cells. Non-stimulated EA.hy 926 cells has been found to produce much lower amounts of prostacyclin than resting HUVEC. Resting hybrid cells produced more PGE(2) than prostacyclin, despite they expressed high levels of COX-1 and PGI(2) synthase. On the contrary to HUVEC and A549, EA.hy 926 cells did not respond to IL-1beta with COX-2 induction and increase of prostaglandin production, however they did it in response to lysophosphatidylcholine (LPC). The characteristics of EA.hy 926 cells in terms of the pattern of prostanoid formation could facilitate studies on endothelial metabolism and role of these important lipid mediators.     

Specific production of prostaglandin E by human amnion in vitro

Prostaglandin production by intra-uterine human tissues has been investigated using a method of tissue superfusion. Tissues were obtained at elective Caesarean section and after spontaneous vaginal delivery. It was found that all the tissues studied (amnion, chorion, decidua and placenta) produced more prostaglandin E (PGE) and 13,14-dihydro-15-keto-prostaglandin F (PGFM — the major circulating metabolite of prostaglandin F) than prostaglandin F (PGF). Amnion produced significantly more PGE (but not PGF or PGFM) than any other tissue. Prostaglandin production by each tissue was similar whether it was taken at elective Caesarean section or after spontaneous vaginal delivery.     

Prostaglandin E1 and prostaglandin I2 modulation of superoxide production by human neutrophils

15(S)-15-methyl-prostaglandin E1 and prostaglandin I2 rapidly and reversibly inhibit formyl-methionyl-leucyl-phenylalanine induced superoxide production by human neutrophils. In contrast, 15(S)-15-methyl-prostaglandin E1 and prostaglandin I2 did not alter the rate or the total amount of superoxide production by human neutrophils stimulated with either phorbol myristate acetate or arachidonic acid. These data suggest that the production of superoxide anion by human neutrophils may be mediated by at least two mechanisms, one regulated by prostaglandins and intracellular cyclic adenosine monophosphate levels and a second independent of prostaglandin modulation.     

Exaggerated human vascular cell prostaglandin biosynthesis mediated by monocytes: role of monokines and interleukin 1

Incubation of cultured human umbilical vein endothelial cells with factors derived from human peripheral blood mononuclear cells (MNCF) or adherent monocytes (AMF) resulted in concentration-and time-dependent increases in prostacyclin and prostaglandin E2 (PGE2) production. MNCF and AMF also stimulated prostacyclin and PGE2 biosynthesis in cultured human arterial smooth muscle cells and human dermal fibroblasts. The effect of these monokines on endothelial cells and fibroblasts was mimicked by treatment with purified human interleukin 1 (IL 1). Mononuclear cell-conditioned medium subjected to gel filtration yielded fractions (Mr 12,000 to 18,000 daltons) which simultaneously contained endothelial cell and fibroblast prostaglandin-stimulating activity and IL 1 activity. Therefore, monokines, specifically IL 1, appear to serve as chemical mediators of the interaction between monocytes and vascular cells as would occur in blood vessel injury, inflammation, and atherosclerosis.     

Prostaglandin E2 and I2 production in isolated dog renal arteries in the absence or presence of vascular endothelial cells

The spontaneous prostaglandin I2 production was significantly reduced by the removal of endothelial cells from the isolated dog renal arteries compared with relative slight reduction of prostaglandin E2 production. The stimulation of prostaglandin I2 production induced with angiotensin II was also markedly reduced under the absence of endothelial cells, while its potentiation of prostaglandin E2 production was not inhibited. The results suggest that the vascular endothelial cells are the major sources of prostaglandin I2 in the dog renal arteries, while prostaglandin E2 is mainly produced in other cell types, perhaps vascular smooth muscle cells.