Stimulation of prostaglandin E2 and thromboxane B2 production by human monocytes in response to interleukin-2

Interleukin 2 (IL-2) is a potent lymphokine involved in the regulation of immune responses and is classically regarded as a stimulus for the activation and growth of T-cells. Recent reports have demonstrated the IL-2 dependent activation of human peripheral blood lymphocytes into lymphokine activated killer cells capable of lysing tumor cells both in vitro and in vivo. In this study we report data which clearly show IL-2 may also act to down-regulate the immune response by inducing the synthesis of arachidonic acid metabolites with known immunosuppressive actions. Stimulation of peripheral human blood monocytes with IL-2 caused an increased production of prostaglandin E2 (PGE2) and thromboxane (TXB2) in a dose-dependent manner. Kinetic analysis showed no increase above controls after 6 hours and maximal levels by 10 hours; elevated levels were maintained after 45 hours of incubation. After 20 hours of stimulation with 2000 U/ml IL-2, the level of PGE2 and TXB2 were greater than three-fold above controls, 0.7 and 19 ng/10(6) cells, respectively. The stimulation was relatively specific in that neither prostacyclin nor leukotrienes were produced in response to IL-2. These data demonstrate that IL-2 acts on human monocytes to induce the secretion of PGE2 and TXB2.     

Bradykinin stimulates prostaglandin E2 formation in isolated human osteoblast-like cells

The effect of bradykinin on prostaglandin E₂ formation in cells from human trabecular bone has been studied. The cells responded to parathyroid hormone with enhanced cyclic AMP formation and were growing as cuboidal-shaped, osteoblast-like cells. In these isolated human osteoblast-like cells, bradykinin (1 mol/l) caused a rapid (5 min) stimulation of prostaglandin E₂ formation. This finding indicates that human osteoblasts are equipped with receptors for bradykinin linked to an increase in prostaglandin formation.     

The stimulation of macrophage prostaglandin E2 and thromboxane B2 secretion by Streptococcus mutans insoluble glucans

The effect of insoluble glucan synthesized by Streptococcus mutans on [3H]arachidonate metabolites secretion from peritoneal macrophages was studied. Insoluble glucans stimulated [3H]arachidonate release and secretion of prostaglandin E2 and thromboxane B2 from macrophages. In contrast, commercial soluble glucan (dextran) did not induce [3H]arachidonate release.     

Inhibition of human B cell responsiveness by prostaglandin E2

The capacity of prostaglandin E2 (PCE2) to modulate human peripheral blood B cell proliferation and the generation of immunoglobulin-secreting cells (ISC) stimulated by Cowan 1 strain Staphylococcus aureus and mitogen-stimulated T cell supernatant was examined. PGE2 significantly inhibited both responses, whereas PGF2 alpha had no inhibitory effect. Responses of highly purified B cells obtained from spleen, lymph node, and tonsil were also inhibited. In addition PGE2 suppressed B cell responses supported by recombinant interleukin 2 rather than T cell supernatant. PGE2-mediated inhibition was mimicked by forskolin, a direct activator of adenylate cyclase. Kinetic studies indicated that PGE2 inhibited B cell responses by a progressively greater increment as cultures were prolonged. Evaluation by flow cytometry after staining with acridine orange or mithramycin indicated that PGE2 had no effect on initial B cell entry into the G1 phase of the cell cycle, passage through G1, and entry into S, G2, and M. Rather, PGE2 inhibited responses of postdivisional daughter cells. PGE2 inhibited responses in cultures stimulated by the calcium ionophore ionomycin and T cell supernatant but had minimal effects in cultures stimulated by the combination of ionomycin and phorbol myristate acetate. Moreover, phorbol myristate acetate reversed PGE2-mediated inhibition of proliferation stimulated by S. aureus or S. aureus + T cell supernatant. These results indicate that PGE2 suppresses the continued growth and differentiation of human B cells, although it has no effect on initial B cell activation and suggest that PGE2 may play a role in regulating human B cell responses in vivo.     

Potential mediation of prostaglandin E2 release from isolated human parietal cells by protein kinase C

Parietal cells are a major source of gastric mucosal prostaglandins in various species. We examined cholinergic stimulation of prostaglandin E2 (PGE2) release from human parietal cells; using activators of the protein kinase C we attempted to get an indirect insight into cellular mechanisms which control PGE2 release. Gastric mucosal specimens were obtained at surgery and the cells were dispersed by collagenase and pronase E. Parietal cells were enriched to 65-80% by a Percoll gradient, and were incubated for 30 min. PGE2 release into the medium (radioimmunoassay) was 74-126 pg/10(6) cells/30 min under basal conditions and was 2.6-fold increased by carbachol (10(-5) and 10(-4) M). Similarly, PGE2 release was stimulated by phospholipase C (20-200 mU/ml, 364% above basal), 1-oleoyl-2-acetyl-sn-glycerol (10(-9)-10(-5) M, 229%), 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-9)-10(-5) M, 283%) and calcium ionophore A23187 (10(-7)-10(-5) M, 219%). Simultaneous presence of A23187 and TPA synergistically induced stimulation which was slightly higher than the sum of the individual responses. N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide W-7, a putative calmodulin antagonist, inhibited TPA-induced PGE2 release at concentrations regarded specific for blocking calmodulin (IC50 = 1.5 X 0(-6) M). We conclude that in human parietal cells PGE2 is released upon cholinergic stimulation and that phospholipase C and protein kinase C are involved in the control of PGE2 release. We speculate that calmodulin might interact with a protein phosphorylated by protein kinase C to cause PGE2 release.     

Prostaglandin E2, prostaglandin E1 and thromboxane B2 release from human monocytes treated with C3b or bacterial lipopolysaccharide

C3b or lipopolysaccharide treatment of human peripheral blood monocytes in culture stimulates an early release of thromboxane B₂ and a delayed release of prostaglandin E into culture supernatants. Immunoreactive thromboxane B₂ release is maximal from 2–8 h, whereas prostaglandin E release is maximal from 16–24 h after stimulation of monocytes in culture. We further examined this process by comparing the time course of labelled prostaglandin E₂, prostaglandin E₁ and thromboxane B₂ release from human monocytes which were pulse or continuously labelled with [³H]arachidonic acid and [¹⁴C]eicosatrienoic acid. The release of labelled eicosanoids was compared with the release of immunoreactive prostaglandin E and thromboxane B₂. The time course of prostaglandin E₂ release was virtually identical to the release of prostaglandin E₁ in all culture supernatants regardless of labelling conditions. However, release of immunoreactive prostaglandin E paralleled the release of labelled prostaglandin E₁ and E₂ only for continuously labelled cultures. The release of labelled prostaglandin E₁ and E₂ from pulse labelled cultures paralleled the release of thromboxane B₂ and not immunoreactive prostaglandin. In contrast, labelled and immunoreactive thromboxane B₂, quantitated in the same culture supernatants, demonstrated similar release patterns regardless of labelling conditions. These findings indicate that the differential pattern of prostaglandin E and thromboxane B₂ release from human monocytes is not related to a time-dependent shift in the release of prostaglandin E₁ relative to prostaglandin E₂. Because thromboxane B₂ and prostaglandin E₂ are produced through cyclooxygenase mediated conversion of arachidonic acid, these results further suggest that prostaglandin E₂ and thromboxane B₂ are independently metabolized in human monocyte populations.     

LPS-stimulated release of prostaglandin E and thromboxane B2 from the U937 cell line

Human monocytes are known to metabolize arachidonic acid (AA) and to release prostaglandins upon stimulation. Previous data indicate that in vitro maturation and differentiation of monocytes result in alteration of this property with greatly diminished response to stimulators of release of prostaglandin E (PGE) and thromboxane B2 (TxB2) occurring after cells have been cultured. To further study the effects of differentiation on human monocyte AA metabolism, a model system was established based upon the human histiocytic cell line U937. Among tested stimulants, which included opsonized zymosan, complement fragment C3b, phorbol myristate acetate (PMA), calcium ionophore A23187, and concanavalin A, it was found that Escherichia coli lipopolysaccharide (LPS) was unique in that it stimulated increased release of TxB2 from U937 cells. The effect of the phorbol ester PMA, a compound commonly used to induce differentiation of U937, on the ability of U937 to respond to LPS was examined. Following 48 hr of treatment with PMA, U937 became capable of releasing both PGE and TxB2 in response to small doses of LPS. As previously observed for human monocytes, the release of PGE was delayed for several hours following stimulation and failed to reach maximal cumulative levels in culture until 24-48 hr following stimulation. In contrast to human monocytes, PMA-induced U937 were capable of maintaining their responsiveness to LPS for several days. Thus, the U937 cell line provides a useful model for study of the effects of differentiation of human mononuclear phagocytes on their ability to metabolize AA, and for the effects of LPS on histiocytic tumor cell prostaglandin release.     

Metabolism of prostaglandins, prostaglandin analogs and thromboxane B2 by lung and liver microsomes from pregnant rabbits

Liver microsomes from pregnant rabbits converted prostaglandins F2 alpha, E1, and E2 to their 20-hydroxy metabolites along with smaller amounts of the corresponding 19-hydroxy compounds. Prostaglandins E1 and E2 were also reduced to prostaglandins F1 alpha and F2 alpha, respectively, and prostaglandin E1 was isomerized to 8-isoprostaglandin E1. The above products were also identified after incubation of prostaglandins with liver microsomes from non-pregnant rabbits. In this case, the yield of 20-hydroxy metabolites was much lower. Thromboxane B2 and a number of prostaglandin F2 alpha analogs were also hydroxylated by lung and liver microsomes from pregnant rabbits. The relative rates of hydroxylation by lung microsomes were: prostaglandin E2 approximately prostaglandin F2 alpha approximately 16,16-dimethylprostaglandin F2 alpha approximately 13,14-didehydroprostaglandin F2 alpha greater than thromboxane B2 greater than 15-methylprostaglandin F2 alpha approximately 17-phenyl-18,19,-20-trinorprostaglandin F2 alpha approximately ent-13,14-didehydro-15-epiprostaglandin F2 alpha. Similar results were obtained with liver microsomes except that thromboxane B2 was a relatively poorer substrate for hydroxylation.     

Captopril effect on prostaglandin E2, thromboxane B2 and proteinuria in lupus nephritis patients

OBJECTIVE: High urinary Prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) levels have been reported in lupus nephritis (LN). Captopril diminishes proteinuria and improves glomerular filtration rate (GFR), and may have effect on immune function. We evaluate captopril effect on urinary PGE2, and TxB2. METHODS: Eighteen LN patients were randomly assigned to two groups. Group 1 received only prednisone plus cyclophosphamide. Group 2 received also captopril. Serum creatinine, GFR, RPF, urinary proteins, PGE2 and TxB2, were assessed. RESULTS: There were no differences between the initial and final assessments in Group 1. Group 2 showed a significant decrement in proteinuria (p=0.003) and serum creatinine (p=0.01) at the end of the study. PGE2 decreased significantly when compared with the initial value (p=0.02). CONCLUSION: Captopril plus usual treatment, improved serum creatinine and decreased proteinuria in parallel with prostaglandin E2 reduction. This effect is not related to changes in GFR or RPF. Captopril may have an immunomodulatory effect on local inflammatory processes in lupus nephritis.     

Selective inhibition by antiflamrnin-2 of thromboxane B(2) release from isolated and perfused guinea-pig lung

Antiflammin-2 (AF2) is a nonapeptide corresponding to the amino acid residues 246-254 of lipocortin-1 showing anti-inflammatory activity both in vitro and in vivo. The effect of AF2 on the thromboxane B(2) (TXB(2)) and histamine release from isolated and perfused guinea-pig lungs has been studied. AF-2 (10-100 nM) inhibited leukotriene C(4)- (LTC(4)) (3 ng) and antigen-induced (ovalbumin, 1 mg) TXB(2) release in normal and sensitized lungs, respectively. In contrast AF-2 (100 nM) did not modify TXB(2) release induced by histamine (5 mug) or bradykinin (5 mug) in normal lungs. Antigen-induced histamine release was not affected by 100 nM AF-2 infusion. When tested in chopped lung fragments AF-2 (0.1-25 muM) did not modify the release of histamine and TXB(2) induced by antigen (ovalbumin, 10 mug ml(-1)) or calcium ionophore A 23187 (1 muM). Our results show that the inhibitory effect of AF-2 on TXB(2) release is selective and depends on the stimulus applied. In this respect AF-2 mimics, at least in part, the actions of both glucocorticoids and lipocortin-1.     

Biosynthesis of prostaglandins and thromboxane B2 by fetal lung homogenates

The conversion of arachidonic acid to prostaglandins (PG's) and thromboxane B2 (TXB2) was investigated in homogenates from fetal and adult bovine and rabbit lungs. Adult bovine lungs were very active in converting arachidonic acid (100 microgram/g tissue) to both PGE2 (10.7 microgram/g tissue) and TXB2 (6.2 microgram/g tissue. Smaller amounts of PGF2alpha (0.9 microgram/g) and 6-oxoPGF1alpha were formed. Homogenates from fetal calf lungs during the third trimester of pregnancy were quite active in converting arachidonic acid to PGE2, but formed very little TXB2, PGF2alpha or 6-oxoPGF1alpha. Homogenates from rabbit lungs converted arachidonic acid (100 microgram/g) mainly to PGE2, both before and after birth. The amount of PGE2 formed increased during gestation to a maximum of about 6 microgram/g tissue at 28 days of gestation. It then decreased to a minimum (1.5 microgram/g) which was observed 8 days after birth, followed by an increase to about 4 microgram/g in older rabbits.     

Elevated levels of prostaglandin e2 and thromboxane B2 in depression

Plasma prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) levels were measured in 30 depressed outpatients. Plasma PGE2 was increased in all but one of these patients (p<0.001) and all had increased levels of TXB2 (p<0.001). Since monamine oxidase inhibitors inhibit brain prostaglandin synthesis, and tricyclic antidepressants may antagonize prostaglandin actions, these observations suggest new approaches to depression research.     

Differential suppression of menstrual fluid prostaglandin F2a, prostaglandin E2, 6-keto prostaglandin F1a and thromboxane B2 by suprofen in women with primary dysmenorrhea

Eleven women with primary dysmenorrhea completed a randomized, double-blind, placebo-controlled, three-way cross-over study comparing 200 and 400mg suprofen. Menstrual fluid volume did not change. Mean+/-S.E.M. menstrual fluid PGF2a was significantly suppressed from 18.9+/-1.9 microg (placebo) to 10.9+/-1.7 and 9.3+/-2.1 microg with 200 and 400 mg suprofen, respectively (p=<0.005). PGE2 dropped from 7.8+/-0.9 to 4.6+/-0.8 and 4.6+/-1.1 microg (p=<0.05) and TxB2 from 17.5+/-4.3 to 7.5+/-2.9 and 3.6+/-1.3 microg (p=<0.01), respectively. 6-Keto PGF1a was significantly suppressed (2.7+/-0.4 to 1.9+/-0.5 microg, p=<0.025) with only 400 mg suprofen. Six subjects rated placebo poor and five fair to very good. In contrast, nine rated suprofen excellent to fair while two rated poor. Thus, suprofen was clinically effective but the differential suppression of prostanoids favors 200mg which spares 6-keto PGF1a.     

Enhancement of human neutrophil migration by prostaglandin E2

Physiologic concentrations of prostaglandin E₂ but not A₁ or F₁ α enhance human neutrophil migration in response to a chemotactic stimulus while higher concentrations decrease the response. Enhanced migration requires the presence of PGE₂ and is not observed when PMNs are preincubated with optimal doses of PGE₂. Furthermore, maximal enhancement is only observed when low concentrations of chemotactic factor are utilized to stimulate PMNs. Experiments indicate that PGE₂ acts to enhance the chemokinetic response of human PMNs to chemotactic factors. These data support a role for physiologic concentrations of PGE₂ in the control of PMN function at an inflammatory site.     

Activated human dendritic cells express inducible cyclo-oxygenase and synthesize prostaglandin E2 but not prostaglandin D2

Prostaglandins (PG) are well known lipid mediators with important immunoregulatory properties. While exogenous PGE2 has the ability to modulate the function and maturation of antigen presenting cells, such as dendritic cells (DC), it is not clear whether human DC have the capacity to synthesize PGE2 and other prostaglandins themselves. We therefore examined the expression of inducible cyclo-oxygenase (COX-2) by monocyte derived DC and the production of PGE2 and PGD2. Both monocyte derived DC and freshly isolated blood myeloid DC expressed little COX-2 constitutively, though COX-2 expression was rapidly but transiently upregulated in response to lipopolysaccharide stimulation. COX-2 mRNA was detectable within 1 h of LPS exposure, peaked at 4-6 h, and rapidly declined thereafter. COX-2 expression was accompanied by DC synthesis of PGE2, with peak levels present at 6-18 h post-stimulation. In contrast, PGD2 synthesis was not detected at any time point. When DC were activated with LPS in the presence of nimesulide, a COX-2 selective inhibitor, IL-10 synthesis was inhibited, indicating that endogenous prostaglandins regulate DC cytokine production. PGE2 production by DC may therefore modulate DC and T-cell function, thereby shaping the character of the immune response.     

A cyclooxygenase-2/prostaglandin E2 pathway augments activation-induced cytosine deaminase expression within replicating human B cells

Within inflammatory environments, B cells encountering foreign or self-Ag can develop tertiary lymphoid tissue expressing activation-induced cytosine deaminase (AID). Recently, this DNA-modifying enzyme was detected in nonlymphoid cells within several inflamed tissues and strongly implicated in malignant transformation. This study examines whether a cyclooxygenase 2 (COX-2) pathway, often linked to inflammation, influences AID expression in activated B lymphocytes. In this paper, we report that dividing human B cells responding to surrogate C3d-coated Ag, IL-4, and BAFF express AID, as well as COX-2. A progressive increase in AID with each division was paralleled by a division-related increase in a COX-2-linked enzyme, microsomal PGE(2) synthase-1, and the PGE(2)R, EP2. Cells with the greatest expression of AID expressed the highest levels of EP2. Although COX-2 inhibitors diminished both AID expression and IgG class switching, exogenous PGE(2) and butaprost, a selective EP2 agonist, augmented AID mRNA/protein and increased the numbers of IgG(+) progeny. Despite the latter, the proportion of IgG(+) cells within viable progeny generally declined with PGE(2) supplementation. This was not due to PGE(2)-promoted differentiation to plasma cells or to greater downstream switching. Rather, because phosphorylated ataxia telangiectasia mutated levels were increased in progeny of PGE(2)-supplemented cultures, it appears more likely that PGE(2) facilitates AID-dependent DNA double-strand breaks that block B cell cycle progression or promote activation-induced cell death, or both. Taken together, the results suggest that a PGE(2) feed-forward mechanism for augmenting COX-2 pathway proteins promotes progressively increased levels of AID mRNA, protein, and function.     

Decrease of prostaglandin E2 receptor binding is accompanied by reduced antilipolytic effects of prostaglandin E2 in isolated rat adipocytes

The effect of treatment of isolated rat adipocytes with prostaglandin E2 (PGE2) on subsequent [3H]PGE2 binding was studied. In addition, the antilipolytic effects of was studied. In addition, the antilipolytic effects of PGE2, adenosine, and insulin were studied in control and PGE2-treated adipocytes. Treatment of adipocytes with PGE2 (1 microM) decreased the binding of [3H]PGE2 by 61% (from 11.0 to 4.6 fmol/10(6) cells, P less than 0.005). Scatchard analysis of the binding data demonstrated that the decrease of PGE2 receptor binding was due to a decrease in the apparent number of PGE2 receptors while the apparent receptor affinity was unaltered. Reduction of the PGE2 receptor binding was specifically regulated inasmuch as structurally related compounds such as PGF2 alpha and arachidonic acid had only minor effects on subsequent [3H]PGE2 receptor binding. Reduction of the available receptor number was associated with a significant decrease in the antilipolytic effect of PGE2 on the isoproterenol-stimulated lipolysis (P less than 0.05). The maximal antilipolytic effect of PGE2 was decreased by 45%. Desensitization of the biological effect of PGE2 (antilipolysis) was only partially specifically regulated inasmuch as the antilipolytic compound phenylisopropyladenosine also had reduced antilipolytic effect in PGE2-treated cells. However, the antilipolytic effect of insulin was similar in control and PGE2-treated cells. It was found that the PGE2-induced decrease of [3H]PGE2 receptor binding may be due to a very tight coupling between the PGE2 molecule and its specific receptor. This tight coupling may then represent an occupancy of the receptor rather than a true loss of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of zinc deficiency and food restriction on prostaglandin E2 and thromboxane B2 in saliva and plasma of rats

Zinc has been implicated in the regulation of prostaglandins and other arachidonic acid derivatives. Studies of zinc-deficient animals, however, are compromised by concomitant reduction in food intake that may also alter eicosanoid levels in body tissues and fluids. In this study, three groups of rats, designated as zinc-deficient, pair-fed and control, were fed diets containing 1 ppm, 15 ppm (in amounts paired to deficient rats) and 15 ppm Zn ad libitum, respectively, for 6 weeks. Saliva and blood were analyzed for PGE2 and TXB2 by radioimmunoassay. Saliva concentrations of both eicosanoids were lower (p less than 0.05) in the pair-fed animals, but not significantly altered by zinc deficiency. Plasma levels of PGE2 and TXB2 were unchanged by either zinc deficiency or food restriction. The results of this study support the contention that the effect of zinc on these prostaglandins is not mediated by altered rates of synthesis or degradation but rather by effects on eicosanoid function.