Prostaglandin E2 Induces Interleukin-6 Synthesis in Human Astrocytoma Cells

Abstract: Prostaglandins (PGs) and cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), have been implicated in the etiopathology of various inflammatory and degenerative disorders, including Alzheimer's disease (AD) and prion diseases. Nonsteroidal antiinflammatory drugs (NSAIDs), potent inhibitors of PG synthesis, appear to be beneficial in the treatment of AD. To assess whether PGs are able to induce IL-6 synthesis in cells of the CNS, IL-6 mRNA and protein syntheses were measured in a human astrocytoma cell line after stimulation with different PGs. PGE₁ and PGE₂, but not PGD₂ and PGF_2α, led to a rapid and transient induction of IL-6 mRNA, followed by IL-6 protein synthesis. Furthermore, PGE₂ potentiated IL-1β-induced IL-6 mRNA synthesis. These results are discussed with respect to the participation of PGs in neurodegenerative diseases (and its inhibition by NSAIDs) by affecting cytokine expression.     

Possible Involvement of Interleukin-1 in Cyclooxygenase-2Induction After Spinal Cord Injury in Rats

Abstract : A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A₂ (detected as thromboxane B₂) and prostaglandin I₂ (detected as 6-ketoprostaglandin F_1α. Thromboxane B₂ was predominant during the first 1 h, whereas the 6-ketoprostaglandin F_1α level exceeded that of thromboxane B₂ at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1α and -1β detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1 α into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F_1α resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1α injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1 α.     

Roles of Prostaglandins D2 and E2 in Interleukin-1-Induced Activation of Norepinephrine Turnover in the Brain and Peripheral Organs of Rats

Abstract: Possible roles of prostaglandins (PGs) in interleukin-1 (IL-1)-induced activation of noradrenergic neurons were examined by assessing norepinephrine (NE) turnover in the brain and peripheral organs of rats. An intraperitoneal injection of human recombinant IL-1β accelerated NE turnover in the hypothalamus, spleen, lung, diaphragm, and pancreas. A similar increase in NE turnover was also observed after intracerebroventricular injection of corticotropin-releasing hormone (CRH). Pretreatment with indomethacin (cyclooxygenase inhibitor) abolished the IL-1-induced, but not the CRH-induced, increase in hypothalamic and splenic NE turnover. To elucidate which eicosanoid-cyclooxygenase product(s) is responsible for accelerating NE turnover, PGD₂, PGE₂, PGF_2α, U-46619 (stable thromboxane A₂ analogue), or carbacyclin (stable prostacyclin analogue) was administered intracerebroventricularly. Among them, PGE₂ was the only eicosanoid effective in increasing NE turnover in spleen, whereas PGD₂ was effective in the hypothalamus. The stimulative effect of PGD₂ was abolished by pretreatment with intracerebroventricular injection of a CRH antiserum. These results suggest that the action of IL-1 is mediated through PGD₂ production to activate the noradrenergic neurons in the hypothalamus, and through PGE₂ production to increase sympathetic nerve activity in spleen.     

The role of inositol lipids in the activation of monocytes by interleukin-1 and bacterial endotoxin

Abstract The effect of interleukin-1 (IL-1) and bacterial endotoxin (lipopolysaccharide, LPS) on the activation of phosphoinositidase C (PIC) and on prostaglandin E₂ release was studied in monocytes (Mø). Both IL-1α and IL-1β increased the release of PGE₂ in a concentration-dependent manner, with EC₅₀s of 0.48 nM and 0.12 nM, respectively. Intact Mø were prelabelled with [³H]inositol and the formation of inositol phosphates (IPs) was estimated by ion exchange chromatography. PIC activity was estimated directly by measuring the conversion of [³H]phosphatidylinositol-4,5,-bisphosphate to aqueous soluble radioactivity by Mø homogenates. IL-1α (5.8 nM) increased the accumulation of IPs within 1–4 minutes and increases in IP₃ and IP₄ occured before the increase in IP₁₊₂ whereas LPS only increased the IPs level after at least 30 min. IL-1α increased PIC activity in Mø homogenates within 15 min with an EC₅₀ of 0.58 nM and IL-1β (0.1 nM) also increased activity. Neither IL-1α nor IL-1β affected the PIC activity of membrane or cytosolic fractions. LPS decreased activity in all fractions. These data indicate that IL-1, but not LPS, can directly lead to an increased activity of PIC which may be involved in eicosanoid formation in Mø.     

Cytokine-Regulated Expression of Platelet-Derived Growth Factor Gene and Protein in Cultured Human Astrocytes

Abstract: To elucidate mechanisms regulating the production of platelet-derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte-enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF AB protein was detected in serum-free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor-β₁ (TGF-β₁) or tumor necrosis factor-α (TNF-α); the largest increase was detected after combined treatment with the two cytokines. Interleukin-1β (IL-1β) by itself had little or no effect but synergized with TGF-β₁ in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A- and PDGF B-chain mRNAs were detected in untreated astrocytes. PDGF B-chain mRNA levels were increased by TGF-β₁, TNF-α, TNF-α/TGF-β₁, or IL-1β/TGF-β₁, whereas PDGF A-chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF-β₁, TNF-α, and IL-1β are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation.     

The role of laccase in prostaglandin production by Cryptococcus neoformans

Recently, it has been demonstrated that the opportunistic fungal pathogen Cryptococcus neoformans can synthesize authentic immunomodulatory prostaglandins. The mechanism by which this takes place is unclear as there is no cyclooxygenase homologue in the cryptococcal genome. In this study, we show that cryptococcal production of both PGE₂ and PGF_2α can be chemically inhibited by caffeic acid, resveratrol and nordihydroguaiaretic acid. These polyphenolic molecules are frequently used as inhibitors of lipoxygenase enzymes; however, blast searches of the cryptococcal genome were unable to identify any homologues of mammalian, plant or fungal lipoxygenases. Next we investigated cryptococcal laccase, an enzyme known to bind polyphenols, and found that either antibody depletion or genetic deletion of the primary cryptococcal laccase ( lac1 Δ) resulted in a loss of cryptococcal prostaglandin production. To determine how laccase is involved, we tested recombinant laccase activity on the prostaglandin precursors, arachidonic acid (AA), PGG₂ and PGH₂. Using mass spectroscopy we determined that recombinant Lac1 does not modify AA or PGH₂, but does have a marked activity toward PGG₂ converting it to PGE₂ and 15-keto-PGE₂. These data demonstrate a critical role for laccase in cryptococcal prostaglandin production, and provides insight into a new and unique fungal prostaglandin pathway.     

Prostaglandin E2 and 4-Aminopyridine Prevent the Lipopolysaccharide-Induced Outwardly Rectifying Potassium Current and Interleukin-1β Production in Cultured Rat Microglia

Abstract: Brain inflammation includes microglial activation and enhanced production of diffusible chemical mediators, including prostaglandin E₂. Prostaglandin E₂ is generally considered a proinflammatory molecule, but it also promotes neuronal survival and down-regulates some aspects of microglial activation. It remains unknown, however, if and how prostaglandin E₂ prevents microglial activation. In primary culture, microglial activation is predicted by a characteristic pattern of whole-cell potassium currents and interleukin-1β production. We investigated if prostaglandin E₂ could alter these currents and, if so, whether these currents are necessary for microglial activation. Microglia were isolated from mixed cell cultures prepared from neonatal rat brains and exposed to 0–10 µ M prostaglandin E₂ and lipopolysaccharide for 24 h. Currents were elicited by using standard patch-clamp technique, and interleukin-1β production was measured by ELISA. Peak outward current densities in microglia treated with lipopolysaccharide plus prostaglandin E₂ (10 n M ) were reduced significantly from those of cells treated with lipopolysaccharide alone. Prostaglandin E₂ and 4-aminopyridine (a blocker of outward potassium currents) also significantly reduced interleukin-1β production. Thus, although prostaglandin E₂ is classified generally as a proinflammatory chemical, it has complex roles in brain inflammation that include preventing microglial activation, perhaps by reducing the outward potassium current.     

Evidence for Two Distinct Forms of Fatty Acid Cyclooxygenase in Brain

Abstract: The enzymatic metabolism of [¹⁴C]arachidonic acid (AA) was studied with microsomes prepared from rabbit medulla. Prostaglandin E₂ (PGE₂) levels, measured either by radiochemistry or radioimmunoassay, rose rapidly and abruptly plateaued within 5 min, while prostaglandin F_2a (PGF_2a) levels continued to rise for 30 min. The rapid termination of PGE2 biosynthesis was not the result of limited cofactor, substrate, or product feedback inhibition, nor was it due to PGE₂-9-ketoreductase activity. Inhibition of the PGH₂→ PGE₂ isomerase by arachidonic acid or its metabolites could not explain the abrupt halt in PGE₂ biosynthesis. Proof for two separate cyclooxygenases comes from our observation that a preincubation of the brain microsomes with unlabeled AA eliminated PGE₂ biosynthesis while PGF2o production continued. Further evidence to suggest two cyclooxygenases in brain is derived from the observation that indomethacin inhibited PGE₂ production at concentrations that did not affect PGF_2a biosynthesis. These results suggest that one fatty acid cyclooxygenase is closely associated with PGH₂→ PGE₂ isomerase and readily undergoes autodestruction and the second cyclooxygenase is associated with a PGH₂→ PGF_2a reductase and is somewhat resistant to arachidonate-induced destruction and to nonsteroidal antiinflammatory agents.     

MODULATION OF CYCLIC AMP LEVELS IN THE BOVINE SUPERIOR CERVICAL GANGLION BY PROSTAGLANDIN E, AND DOPAMINE

Abstract— Dopamine, norepinephrine, carbamylcholine and PGE₁ (prostaglandin E₁). increased cyclic AMP concentrations in slices of bovine superior cervical ganglia. PGF_1α was less effective and neither PGE₂ nor PGF_2α had any effect. Dopamine and PGE, alone or in combination, did not modify low K _m cyclic AMP phosphodiesterase activity. Combinations of dopamine and PGE, showed a marked synergistic effect, increasing ganglionic cyclic AMP to a much greater extent than that observed when the two compounds were tested alone. Norepinephrine (10 μ M) , which increased cyclic AMP as much as 10 μ m -dopamine, showed no synergistic effect when tested in the presence of PGE₁ or other PGs. Phentolamine, fluphenazine and triflupromazine blocked the dopamine effect without suppressing its synergism with PGE₁ Adenylate cyclase of synaptosomes isolated from the ganglia under a variety of experimental conditions appeared to be as responsive to PGE₁ as the slices, but it was poorly stimulated by dopamine and was not synergistically modulated by dopamine in the presence of PGE₁
These and other data are interpreted as indicating the presence of both a PGE₁-sensitive and a PGE₁-modulated dopamine-sensitive adenylate cyclase in the cervical ganglion. These adenylate cyclases are tentatively assigned to pre- and post-synaptic structures respectively.     

Prostanoid production in Saccharomyces cerevisiae provides a novel assay for nonsteroidal anti-inflammatory drugs

Prostanoids are a large family of lipid mediators originating from prostaglandin H synthase (PGHS) activity on the 20-carbon polyunsaturated fatty acids dihomo-γ-linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid. The two mouse PGHS isoforms, PGHS-1 and PGHS-2, were expressed in Saccharomyces cerevisiae (yeast), as was a signal-peptide-deleted version of PGHS-1 (PGHS-1MA). PGHS-1 showed high activity with both AA and DGLA as substrate, whereas PGHS-2 activity was high with DGLA but low with AA. Signal peptide removal reduced the activity of PGHS-1MA by >50% relative to PGHS-1, but the residual activity indicated that correct targeting to the lumen of the endoplasmic reticulum may not be necessary for enzyme function. Coexpression of PGHS-1 with cDNAs encoding mouse prostaglandin I synthase and thromboxane A synthase, and with Trypanosoma brucei genomic DNA encoding prostaglandin F synthase in AA-supplemented yeast cultures resulted in production of the corresponding prostanoids, prostaglandin I₂, thromboxane A₂ and prostaglandin F_2α. The inhibitory effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on prostanoid production were tested on yeast cells expressing PGHS-1 in AA-supplemented culture. Dose-dependent inhibition of prostaglandin H₂ production by aspirin, ibuprofen and indomethacin demonstrated the potential utility of this simple expression system in screening for novel NSAIDs.     

Elevation of Cyclic AMP Levels in Astrocytes Antagonizes Cytokine-Induced Adhesion Molecule Expression

Abstract: We have examined the effect of elevating cyclic AMP levels on cytokine-mediated enhancement of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) gene expression by astrocytes. Treatment of astrocytes with the cyclic AMP mimetic dibutyryl-cyclic AMP, or the agonists norepinephrine, forskolin, prostaglandin E₂, and cholera toxin alone had no effect on ICAM-1 or VCAM-1 mRNA gene expression. However, elevating cyclic AMP levels within the cells by these agents suppressed interleukin-1β- and tumor necrosis factor-α-induced adhesion molecule expression at both the mRNA and protein levels. The phosphodiesterase type IV inhibitor, rolipram, was able to potentiate the inhibitory effect of forskolin on ICAM-1 and VCAM-1 gene expression. Inhibition of tumor necrosis factor-α-induced VCAM-1 mRNA levels by forskolin was partially due to enhanced degradation of VCAM-1 message, whereas the decay rates of tumor necrosis factor-α-induced ICAM-1 message and interleukin-1β-induced ICAM-1/VCAM-1 message were not affected by forskolin treatment. These results demonstrate that the pathways used by interleukin-1β and tumor necrosis factor-α to induce adhesion molecule expression are antagonized by cyclic AMP-dependent protein kinase-mediated signaling pathways.     

Induction of interleukin-2 receptor α-chain gene expression by cytochalasin B and 12-O-tetradecanoylphorbol-13-acetate in T lymphocytes

Abstract. The high affinity form of interleukin-2 receptor (IL-2R) is composed of two subunits; the α (p55) and β (p75). The α chain, unlike the β, is expressed only on activated T lymphocytes. Therefore, high affinity binding of interleukin-2 (IL-2) is controlled by the expression of the IL-2R α-chain. In this study, we examined the effect of cytochalasin B (CB) plus 12-O-tetradecanoylphorbol-13-acetate (TPA) on expression of IL-2 and IL-2R. Northern blot and flow cytometric analysis showed that the IL-2R α-chain was expressed both at mRNA and protein levels. However, IL-2 gene expression was not induced by this treatment. Unlike the cells treated individually with CB or TPA, cells treated with CB plus TPA accumulated IL-2R mRNA at all the times examined. In order to determine the percentage of cells that incorporated tritiated thymidine ([³H]dT) in the presence of IL-2 after treatment with CB plus TPA, autoradiography was carried out. We found that about 11% of the cells were labelled. Because the percentage of labelled cells and cells expressing IL-2R α-chain was relatively low (11% and 9% respectively), perhaps CB plus TPA caused IL-2R expression in only a subset of T cells.     

Regional Prostaglandin Levels in Cerebral Ischaemia

Abstract: Regional quantitative studies of brain prostaglandins (PG) in gerbils with bilateral carotid ligation have shown increases in PG2_α in all areas of ischaemia but with no increases in PGE₂. Pretreatment of animals with indomethacin (3 mg/kg) reduced this response, whereas dexamethasone (2.5 mg/kg) had no such effect. PGE₁ and thromboxane-B₂ were found not to be present in our preparation. The significance of these findings lies in the fact that both drugs, which are known to inhibit the development of ischaemic oedema, have now been shown to produce markedly different effects on prostaglandin levels. This may indicate either, that prostaglandins are not involved in is chaemic oedema formation, or, that the drugs have different mechanisms of action.     

Inhibition of Inducible Nitric Oxide Synthase Expression by Endothelin in Rat Glial Cells Prepared from the Neonatal Rat Brain

Abstract: In primary cultured rat glial cells, a combination of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) decreased iNOS expression and nitrite accumulation induced by TNF-α/IL-1β. The inhibitory effect of ET on TNF-α/IL-1β-stimulated iNOS expression appears to be mediated by ET_B receptors, because (1) both ET-1 and ET-3 inhibited the effects of TNF-α/IL-1β on iNOS expression and nitrite accumulation, (2) a selective ET_B receptor agonist, Suc-[Glu⁹,Ala^11,15]-ET-1 (8–21) (IRL1620), decreased the effects of TNF-α/IL-1β, and (3) a selective ET_B receptor antagonist, N-cis -2,6-dimethylpiperidinocarbonyl- l -γ-methylleucyl- d -1-methoxycarbonyltryptophanyl- d -norleucine, abolished the inhibitory effects of ETs and IRL1620. Incubation of glial cells with lipopolysaccharide (LPS) caused an increase in iNOS expression. Simultaneous addition of ET-3 decreased the effects of LPS (10 and 100 ng/ml) on iNOS expression. Furthermore, cyclic AMP-elevating agents (dibutyryl cyclic AMP and forskolin) inhibited TNF-α/IL-1β-induced and LPS-induced iNOS expression and nitrite accumulation. These findings suggest that ETs can decrease TNF-α/IL-1β-induced and LPS-induced iNOS expression via ET_B receptors and that cyclic AMP may be involved in this process.     

In Human Fetal Astrocytes Exposure to Interleukin-1β Stimulates Acquisition of the GD3+ Phenotype and Inhibits Cell Division

Abstract: In human astrocyte cultures established from second-trimester fetal brain tissue, ∼5–10% of total astrocyte population in unstimulated cultures were GD3⁺/glial fibrillary acidic protein (GFAP)⁺. The GD3⁺ cells were always GFAP⁺ and grew as flat, highly spread cells but changed to process-bearing cells after interleukin-1β (IL-1β) stimulation. It is interesting that IL-1β, a known mitogen for rat astrocytes, suppressed human fetal astrocyte proliferation as determined by [³H]thymidine incorporation, bromodeoxyuridine (BrdU) labeling, and cell counting. The GD3⁺ population, however, consistently increased in absolute number after IL-1β stimulation, in a dose- and time-dependent manner. The IL-1β-mediated increase in number of GD3⁺ astrocytes was independent of initial cell density or serum concentration. By flow cytometry, IL-1β enhanced both the mean fluorescence intensity and the percentage of GD3⁺ cells. To investigate whether the increase in GD3⁺ astrocyte cell number was due to proliferation of preexisting GD3⁺ astrocytes or due to conversion of GD3⁻ to GD3⁺ cells, we performed BrdU/GD3 double immunocytochemistry. BrdU/GD3 double-positive cells were extremely rare in both control and IL-1β-stimulated cultures. Moreover, an increase in number of GD3⁺ astrocytes was still observed in control and IL-1β-stimulated cultures where GD3⁺ cells had been initially eliminated by cell sorting. These results indicate that GD3⁺ astrocytes in human fetal culture may represent a postmitotic, differentiated, distinct phenotype.