Differential effects of phorbol myristate acetate and dexamethasone on protein kinase C activity and eicosanoids production in cultured rat astrocytes

The effects of phorbol myristate acetate (PMA) and dexamethasone on protein kinase C (PK-C) activity and eicosanoid production were characterized in primary cultures of rat glial cells. PMA (1,000 ng/ml) treatment for 2 hr resulted in a maximal effect (a 4-fold increase in PGE₂ production). Longer exposure to PMA (up to 96 hr) resulted in attenuation of PGE₂ production. Down-regulation of PK-C activity was assessed in glial cell homogenates under these conditions. Although a 70% inhibition of PK-C activity was measured upon staurosporine treatment, PGE₂ production was not affected both under basal conditions and following PMA activation. The production of thromboxane B₂ did not change following exposure to PMA. Pretreatment of the cultures with dexamethasone markedly inhibited the PMA-stimulated production of PGE₂ but had only a moderate (approximately 26%) inhibitory effect on PGE₂ production under basal conditions. Dexamethasone had no effect on basal or PMA-stimulated PK-C activity. Forskolin, which activates adenylate cyclase, did not affect PGE₂ production. These data may suggest that activation of PGE₂ production by PMA in glial cells is not unequivocally mediated by PK-C activation. The inhibitory effect of dexamethasone on the PMA-stimulated synthesis of PGE₂ supports previous findings that glucocorticoids are more effective in inhibiting stimulated rather than basal PGE₂ production. © 1993 Wiley-Liss, Inc.     

Lipopolysaccharides enhance the action of bradykinin in enteric neurons via secretion of interleukin‐1β from enteric glial cells

Functional changes of the enteric nervous system have been observed under inflammatory states of inflammatory bowel disease increasing the endotoxin level. The aim of the present study was to determine the effect of lipopolysaccharides (LPS) on myenteric neuron–glia interaction in vitro. We examined the increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the release of interleukin‐1β (IL‐1β) or prostaglandin E₂ (PGE₂) and COX‐2 expression in myenteric plexus cells from the rat intestine induced by LPS. LPS potentiated BK‐induced [Ca²⁺]_i increases in both myenteric neurons and enteric glial cells, which were suppressed by a B1R antagonist. Only in enteric glial cells, a B1R agonist increased [Ca²⁺]_i. The effects of LPS were blocked by pretreatment with an interleukin‐1 receptor antagonist or by reducing the density of enteric glial cells in culture. LPS prompted the release of IL‐1β from enteric glial cells. The augmenting effects of IL‐1β on the BK‐induced neural [Ca²⁺]_i increase and PGE₂ release from enteric glial cells were abolished by a phospholipase A₂ (PLA₂) inhibitor and a COX inhibitor, and partly suppressed by a COX‐2 inhibitor. IL‐1β up‐regulated the COX‐2 expression in enteric glial cells. LPS promotes IL‐1β secretion from enteric glial cells, resulting in augmentation of the neural response to BK through PGE₂ release via glial PLA₂ and COX‐2. The alteration of the regulatory effect of glial cells may be the cause of the changes in neural function in the enteric nervous system in inflammatory bowel disease. © 2009 Wiley‐Liss, Inc.     

Recognition of major histocompatibility complexantigens on murine glial cells

Recognition of autologous major histocompatibility complex (MHC) antigens by T cells is an essential step in the induction of an immunologic reaction to either endogenous or exogenous antigens. We investigated the ability of murine glial cells of different ages to stimulate clones of allospecific T lymphocytes. We also investigated the effects of supernatants from cultures of activated T cells on the immunologic recognition of MHC antigens on murine glial cells. Lymphocyte clones specific for Class I, Class II and non-MHC, background antigens were obtained from C57B1/6J-anti-DBA/2 mixed lymphocyte cultures. Glial cell cultures were prepared from newborn syngeneic (C57B1/6J) and allogeneic (DBA/2) mouse brains. Glial cultures 1-4 weeks of age were able to stimulate alpha-Class I-specific clones. No stimulation of alpha-Class II or alpha-background clones was noted. Incubation of glial cells with supernatants from cultures of alloantigen-activated spleen cells (C57B1/6J-anti-DBA/2) resulted in a decreased ability of glial cells to stimulate alpha-Class I responses. In contrast supernatant-treated cultures acquired the capacity to stimulate alpha-Class II-specific clones. No responses were noted in clones responsive to non-MHC antigens. The ability to stimulate alpha-Class II-specific clones was most prominent with one-week-old glial cultures and was lost by four weeks of culture. The increased susceptibility of younger glial cultures to the modulatory effects of lymphokines from activated T cells may be a factor in the increased susceptibility of the immature central nervous system to persistent viral infections and the development of autoimmune phenomena.     

Evidence for cyclooxygenase activation by nitric oxide in astrocytes

We have evaluated the role of nitric oxide (NO) on the cyclooxygenase pathway in mouse glial cells. Exposure of primary cultures of neonatal mouse cortical astrocytes to bacterial lipopolysaccharide (LPS; 1 μg/ml, 18 h) caused an increase in the release of both nitrite (NO⁻₂) and prostaglandin E₂ (PGE₂2), products of NO synthase (NOS) and cyclooxygenase, respectively. Production of both, NO⁻₂ and PGE₂ by astrocytes, was inhibited by the exposure of the NOS inhibitor Nw-nitro-L-arginine methyl ester (L-NAME: 1, 10, and 100 μM) in a dose related manner. Besides, other NOS inhibitors such as Nitro L-arginine (NNA: 10⁻³ M) prevented the increase in PGE₂ release from LPS-stimulated astrocytes. Sodium nitroprusside (SNP; 100–200 μM) used as a NO donor caused a dose-related enhancement in the accumulation of PGE₂ induced by LPS and the presence of hemoglobin blocked the SNP effects. The exposure to SNP counteracted the decrease of PGE₂ production in LPS-treated astrocytes in which NO synthesis was blocked by L-NAME. In addition, SNP also enhanced the synthesis of PGE₂ following exogenous arachidonic acid astrocytes exposure. Interestingly, this effect was blocked by indomethacin. Treatment of astrocytes cultures with dexamethasone (0.1, 1 μM) blocked dose-relatedly the LPS-induced release of both NO⁻₂ and PGE₂. As expected, the presence of indomethacin (1, 10, and 20 μM) prevented in a dose related fashion, PGE₂ production by astrocytes following exposure to LPS. These results strongly indicate that in astroglial cells, NO is able to activate the cyclooxygenase pathway. © 1995 Wiley-Liss, Inc.     

NSAIDS inhibit the IL-1β-induced IL-6 release from human post-mortem astrocytes: the involvement of prostaglandin E2

Epidemiological studies have shown that steroidal as well as non-steroidal anti-inflammatory drugs lower the risk of developing Alzheimer's Disease (AD). A suppressive effect of these anti-inflammatory drugs on local inflammatory events in AD brains has been suggested, however the mechanisms responsible are still unknown. In this study we investigated at cellular level the influence of two anti-inflammatory drugs—dexamethasone and indomethacin—and an experimental specific cyclooxygenase-2 inhibitor, BF389, on the production of the pro-inflammatory cytokine IL-6 and the inflammatory mediator PGE₂ by human astrocytes. Two human post-mortem astrocyte cultures (A157 and A295) and astroglioma cell lines (U251 and U373 MG) were found to secrete considerable amounts of IL-6 upon stimulation with IL-1β. The glucocorticoid dexamethasone inhibited the IL-1β-activated release of IL-6 from the postmortem astrocyte cultures A157 and A295 and from the astroglioma cell lines. The non-specific cyclooxygenase inhibitor indomethacin and BF389 only suppressed the IL-6 release by post-mortem astrocyte culture A157. This post-mortem astrocyte culture was found to produce large amounts of PGE₂ upon stimulation with IL-1β, whereas in the supernatants of the postmortem astrocyte culture A295 and the astroglioma cell lines, low PGE₂ concentrations were detected. Addition of exogenous PGE₂ prevented the inhibitory effect of indomethacin and BF389 on the IL-1β-activated IL-6 release from A157 astrocytes and largely potentiated the IL-1-induced release of IL-6 from all astrocytes/astroglioma cells tested. Dexamethasone also inhibited the PGE₂ release from the astrocytes and astroglioma cells, however the inhibitory effect of dexamethasone on the IL-1β-activated IL-6 release could not be prevented by the addition of PGE₂. The observed reduction of IL-6 and/or PGE₂ from astrocytes may be involved in the mechanism underlying the beneficial effects of these drugs in AD.     

Secretory products of central nervous system glial cells induce Schwann cell proliferation and protect from cytokine-mediated death

There continues to be interest in Schwann cells (SC) as a possible source of myelinating cells for transplantation into the central nervous system (CNS) of patients with multiple sclerosis (MS) and spinal cord injury. It has been suggested that CNS glial cells interfere with SC migration, survival, maturation, and clinically significant remyelination in the CNS. To investigate the effects of CNS glial cells on SC, we examined the effects of serum-free supernatants obtained from rat mixed CNS glial cultures on rat neonatal SC cultures. Supernatants from 1-, 3-, and 5-day CNS glial cultures induced proliferation of SC assayed at 5 days in vitro but did not induce SC differentiation as measured by induction of surface expression of galactolipids (GalL). High concentrations of cAMP simulate many of the effects of axolemma on SC; CNS glial cell supernatants did not inhibit cAMP induction of SC differentiation. CNS glial cell supernatants had no apparent effect on SC viability at 48 hr as measured by trypan blue exclusion. We have previously demonstrated that incubation of SC with transforming growth factor-beta1 (TGF-beta1) + tumor necrosis factor-alpha (TNF-alpha) induces SC death via apoptosis. We now show that CNS glial supernatants inhibits TGF-beta1/TNF-alpha-induced SC death. Our data show that soluble products of CNS glial cells do not induce or inhibit SC differentiation or increase cell death but have the potential to increase proliferation of SC and their resistance to cytokine-mediated death, and thus may affect the outcome of SC transplantation into the CNS.     

Multiple sclerosis: effects of activated T-lymphocyte-derived products on organ cultures of nervous tissue

Supernatants from multiple sclerosis (MS) T-lymphocytes cause damage to both myelin and glial cells in cerebellar cultures assessed visually and by radiolabel release. Control T-lymphocytes, even after phytohaemagglutinin (PHA) stimulation, yielded supernatants which induced only slight damage, and at later times patients with other neurological diseases (OND) gave variable results. These differences suggest that MS T-lymphocytes are pre-activated in vivo to produce demyelinating factors while control T-lymphocytes are not pre-activated to the same extent. The visual evidence of activation of cerebellar macrophage-like cells was a common finding after MS T-lymphocyte supernatant treatment but there was no correlation with the severity of demyelination. There was a positive correlation between the percentage IL-2 receptor-bearing lymphocytes and the degree of supernatant-induced in vitro demyelination.     

Hypoxia: modulation of endothelial cell proliferation by soluble factors released by retinal cells.

A devastating complication of ischemic retinopathies is retinal neovascularization. We studied the impact on retinal endothelial cell proliferation of soluble factors released from cultured retinal glial (Müller) cells and from retinal explant cultures. Hypoxia strongly stimulated VEGF release by all types of cultures but endothelial cell growth was not further increased by the corresponding conditioned media if compared to supernatants obtained under normoxia. When the final concentration of the hypoxia-conditioned media was adjusted to the VEGF level of normoxia-conditioned media, they even inhibited endothelial cell proliferation. Inhibition may be exerted by TGF-beta 2 but TGF-beta 2 mRNA and protein expression in Müller cells were found to be down-regulated under hypoxia. We conclude that retinal endothelial cell proliferation is controlled by the balance of the amount and/or efficacy of several stimulatory and inhibitory factors.     

Differential regulation of glial cell line–derived neurotrophic factor (GDNF) expression in human neuroblastoma and glioblastoma cell lines

Human SK‐N‐AS neuroblastoma and U‐87MG glioblastoma cell lines were found to secrete relatively high levels of glial cell line–derived neurotrophic factor (GDNF). In response to growth factors, cytokines, and pharmacophores, the two cell lines differentially regulated GDNF release. A 24‐hr exposure to tumor necrosis factor‐α (TNFα; 10 ng/ml) or interleukin‐1β (IL‐1β; 10 ng/ml) induced GDNF release in U‐87MG cells, but repressed GDNF release from SK‐N‐AS cells. Fibroblast growth factors (FGF)‐1, ‐2, and ‐9 (50 ng/ml), the prostaglandins PGA₂, PGE₂, and PGI₂ (10 μM), phorbol 12,13‐didecanoate (PDD; 10 nM), okadaic acid (10 nM), dexamethasone (1 μM), and vitamin D₃ (1 μm) also differentially effected GDNF release from U‐87MG and SK‐N‐AS cells. A result shared by both cell lines, was a two‐ to threefold increase in GDNF release by db‐cAMP (1 mM), or forskolin (10 μM). In general, analysis of steady‐state GDNF mRNA levels correlated with changes in extracellular GDNF levels in U‐87MG cells but remained static in SK‐N‐AS cells. The data suggest that human GDNF synthesis/release can be regulated by numerous facto, signaling through multiple and diverse secondary messenger systems. Furthermore, we provide evidence of differential regulation of human GDNF synthesis/release in cells of glial (U‐87MG) and neuronal (SK‐N‐AS) origin. J. Neurosci. Res. 55:187–197, 1999. © 1999 Wiley‐Liss, Inc.     

Prostaglandin E2 promotes neural proliferation and differentiation and regulates Wnt target gene expression

Prostaglandin E₂ (PGE₂) is an endogenous lipid molecule that regulates important physiological functions, including calcium signaling, neuronal plasticity, and immune responses. Exogenous factors such as diet, exposure to immunological agents, toxic chemicals, and drugs can influence PGE₂ levels in the developing brain and have been associated with autism disorders. This study seeks to determine whether changes in PGE₂ level can alter the behavior of undifferentiated and differentiating neuroectodermal (NE‐4C) stem cells and whether PGE₂ signaling impinges on the Wnt/β‐catenin pathways. We show that PGE₂ increases proliferation of undifferentiated NE‐4C stem cells. PGE₂ also promotes the progression of NE‐4C stem cell differentiation into neuronal‐lineage cells, which is apparent by accelerated appearance of neuronal clusters (neurospheres) and earlier expression of the neuronal marker microtubule‐associated protein tau. Furthermore, PGE₂ alters the expression of downstream Wnt‐regulated genes previously associated with neurodevelopmental disorders. In undifferentiated stem cells, PGE₂ downregulates Ptgs2 expression and upregulates Mmp9 and Ccnd1 expression. In differentiating neuronal cells, PGE₂ causes upregulation of Wnt3, Tcf4, and Ccnd1. The convergence of the PGE₂ and the Wnt pathways is also apparent through increased expression of active β‐catenin, a key signaling component of the Wnt/β‐catenin pathways. This study provides novel evidence that PGE₂ influences progression of neuronal development and influences Wnt target gene expression. We discuss how these findings could have potential implications for neurodevelopmental disorders such as autism. © 2016 Wiley Periodicals, Inc.     

In vitro intercellular adhesion molecule-1 expression on brain endothelial cells in multiple sclerosis

To investigate the origin of intercellular adhesion molecule-1 (ICAM-1) and its expression on brain endothelial cells, we studied the expression in vitro of ICAM-1 on human brain endothelial cells after incubation of T cells from patients with multiple sclerosis (MS) using a histochemical technique and flow cytometry. We determined soluble forms of ICAM-1 (sICAM-1) in the supernatants after mixtures of brain endothelial cells and T cells from patients with MS using an enzyme-liked immunosorbent assay. Flow cytometric analysis showed that a number of ICAM-1-positive cells were significantly increased after incubation of brain endothelial cells with T cells from patients with acute relapsing MS during an exacerbation as compared with those of controls (P<0.01). Patients with acute relapsing MS during an exacerbation and chronic progressive MS exhibited higher levels of ICAM-1 in the supernatants of mixtures with brain endothelial cells and lymphocytes than those of controls (P<0.001 and P<0.01, respectively). These results suggest that lymphocytes from patients with acute relapsing MS during an exacerbation lead to an increased expression of ICAM-1 on the brain endothelial cells and add to evidence involving this adhesion molecule in the pathogenesis of MS.     

The effect of methylprednisolone on monocyte eicosanoid production in patients with multiple sclerosis

The in vitro effect of methylprednisolone on prostaglandin E₂ (PGE₂), leukotriene B₄ (LTB₄) and thromboxane B₂ (TXB₂) synthesis by adherent monocytes was examined using samples of peripheral blood from 15 patients with multiple sclerosis and 18 normal controls. Eicosanoid production by monocytes was reduced in patients compared with controls and there was a dose-dependent inhibitory effect of methylprednisolone on eicosanoid production in both groups. In vitro production of PGE₂ and TXB₂ but not LTB₄ was reduced in patients with multiple sclerosis following intravenous treatment with methyl prednisolone compared with pretreatment samples. In a separate cohort of 20 patients with multiple sclerosis and 15 controls, the in vitro inhibition of PGE₂ release by methylprednisolone was not associated with reduced pokeweed-mitogenstimulated immunoglobulin G synthesis by peripheral blood mononuclear cells. These results suggest that methylprednisolone inhibits monocyte-macrophage function, but this effect is not specific to patients with multiple sclerosis.     

Fever During Localized Inflammation in Mice Is Elicited by a Humoral Pathway and Depends on Brain Endothelial Interleukin-1 and Interleukin-6 Signaling and Central EP3 Receptors

We examined the signaling route for fever during localized inflammation in male and female mice, elicited by casein injection into a preformed air pouch. The localized inflammation gave rise to high concentrations of prostaglandins of the E species (PGE₂) and cytokines in the air pouch and elevated levels of these inflammatory mediators in plasma. There were also elevated levels of PGE₂ in the cerebrospinal fluid, although there was little evidence for PGE₂ synthesis in the brain. Global deletion of the PGE₂ prostaglandin E receptor 3 (EP₃) abolished the febrile response as did deletion of the EP₃ receptor in neural cells, whereas its deletion on peripheral nerves had no effect, implying that PGE₂ action on this receptor in the CNS elicited the fever. Global deletion of the interleukin-1 receptor type 1 (IL-1R1) also abolished the febrile response, whereas its deletion on neural cells or peripheral nerves had no effect. However, deletion of the IL-1R1 on brain endothelial cells, as well as deletion of the interleukin-6 receptor α on these cells, attenuated the febrile response. In contrast, deletion of the PGE₂ synthesizing enzymes cyclooxygenase-2 and microsomal prostaglandin synthase-1 in brain endothelial cells, known to attenuate fever evoked by systemic inflammation, had no effect. We conclude that fever during localized inflammation is not mediated by neural signaling from the inflamed site, as previously suggested, but is dependent on humoral signaling that involves interleukin actions on brain endothelial cells, probably facilitating PGE₂ entry into the brain from the circulation and hence representing a mechanism distinct from that at work during systemic inflammation.     

Malignant glioma-derived soluble factors regulate proliferation of normal adult human astrocytes.

Malignant gliomas are characteristically surrounded by marked gliosis. To assess whether glioma-derived products contribute to the proliferation of astrocytes, a feature of the gliosis response, we evaluated the influence of culture supernatants from malignant human glioma lines and tumor cyst fluids collected from two patients with glioblastoma multiforme on the proliferation of non-transformed adult human astrocytes. Both the culture supernatants and cyst fluids significantly increased DNA synthesis in astrocytes as assessed by a double immunofluorescence glial fibrillary acidic protein-bromodeoxyuridine technique. The net proliferative effect mediated by glioma cell line supernatants was tumor growth phase-dependent, being preferentially expressed during the logarithmic phase of glioma cell growth. Specific growth factor molecules and cytokines known to be secreted by gliomas (epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, interleukin-6, and tumor necrosis factor-alpha) could not reproduce the mitogenic effects of the glioma-derived soluble factors. Cytokines which can induce DNA synthesis by adult human astrocytes in vitro, gamma-interferon and interleukin-1, were not detected in the culture supernatant of glioma lines used in this study. In conjunction with the documented effects of glioma products on endothelial and lymphoid cells, the current study suggests that soluble glioma products can contribute to the production of surrounding gliosis observed in vivo.     

Heme oxygenase attenuated angiotensin II-mediated increase in cyclooxygenase activity and decreased isoprostane F2α in endothelial cells

Heme oxygenase (HO) regulates cellular heme levels and catalyzes the formation of bilirubin and carbon monoxide (CO). We hypothesized that the status of the endothelial HO system influences the angiotensin (Ang) II-induced increase in the endothelial cell (EC) production of PGE₂, eicosanoids which modulate the vascular actions of Ang II. In this study, we investigated the effect of interventions that suppress HO activity or induce HO-1 gene expression on Ang II-mediated increase in PGE₂ in cultures of human microvessel endothelial cells (EC). Incubation of EC with Ang II (100 ng/ml) for 24 h increased the levels of PGE₂ in the culture media. This effect of Ang II on prostaglandin production by EC was attenuated in cells treated with heme, but was magnified in cells treated with the HO inhibitor, Stannis mesoporphyrin (SnMP). Upregulation of HO-1 gene expression by retrovirus-mediated delivery of the human HO-1 gene attenuated heme and Ang II-induced prostaglandin synthesis. We also investigated the physiological significance of human HO-1 overexpression on attenuation of Ang II-mediated oxidative stress. Decreases in COMET levels were found in EC transduced with the HO-1 gene. These results indicate that overexpression of the HO system in EC exerts an inhibitory influence on Ang II-induced synthesis of prostaglandins and attenuates DNA damage caused by exposure to Ang II.     

α1-Adrenergic receptors in the brain: characterization in astrocytic glial cultures and comparison with neuronal cultures

Binding of [¹²⁵I]HEAT to membranes prepared from primary cultures of astrocytic glial cells was time-dependent and 70–85% specific. Various adrenergic agonists and antagonists competed for [¹²⁵I]HEAT binding to the potencies of prazosin >, yohimbine , clonidine, norepinephrine (NE), and propranolol. Scatchard analysis showedB_max of 209 fmol/mg protein and a K_d of 184 pM for [¹²⁵I]HEAT binding by astrocytic glial membranes. Pretreatment of astrocytes with NE resulted in a dose-dependent downregulation of [¹²⁵I]HEAT binding sites with a maximal response observed after 8 h at 100 μM NE. Removal of NE from cultures after pretreatment resulted in a time- and protein synthesis-dependent recovery of binding sites to control levels within 120 h. Incubation of astrocytic glial cultures with NE stimulated phosphoinositide (PI) hydrolysis in a time- and dose-dependent manner with a maximal stimulation of 2-fold observed in 60 min by 100 μM NE.Clonidine expressed differential effects on α₁-adrenergic receptors of the neuronal and astrocytic glial cultures. Pretreatment with 10 μM clonidine caused a 40% decrease in the B_max of [¹²⁵I]HEAT binding without influencing the K_d value in neuronal cultures. This downregulatory effect of clonidine was associated with a reduction in the ability of NE to stimulate PI hydrolysis in clonidine pretreated cells. In contrast to neuronal cultures, clonidine neither downregulated [¹²⁵I]HEAT binding sites nor stimulated PI hydrolysis in glial cultures.     

Prostaglandin E2 protects cultured cortical neurons against receptor-mediated glutamate cytotoxicity

The effects of prostaglandin (PG) E₂ on glutamate-induced cytotoxicity were examined using primary cultures of rat cortical neurons. The cell viability was significantly reduced when cultures were briefly exposed to either glutamate or (NMDA) then incubated with normal medium for 1 h. Similar cytotoxicity was observed with the brief application of ionomycin, a calcium ionophore, and S-nitrosocysteine, a nitric oxide (NO)-generating agent. PGE₂ at concentrations of 0.01–1 μM dose-dependently ameliorated the glutamate-induced cytotoxicity. PGE₁, butaprost, an EP₂ receptor agonist, and 8-bromo-cAMP were also effective in protecting cultures against glutamate cytotoxicity. By contrast, neither 17-phenyl-ω-trinor-PGE₂, an EP₁ receptor agonist, nor M & B 28767, an EP₃ receptor agonist, affected glutamate-induced cytotoxicity. NMDA-induced cytotoxicity was ameliorated by PGE₂, butaprost, MK-801, , a NO synthase inhibitor, and hemoglobin, which binds NO. These agents excluding MK-801 ameliorated the ionomycin-induced cytotoxicity. The cytotoxicity induced by S-nitrosocysteine was prevented only by hemoglobin but not by the other agents including PGE₂. These findings indicate that PGE₂ protects cultured cortical neurons against NMDA receptor-mediated glutamate neurotoxicity via EP₂ receptors. EP₂ receptor stimulation may suppress a step in NO formation triggered by Ca²⁺-influx through NMDA receptors.     

Reorientation of prostanoid production accompanies “activation” of adult microglial cells in culture

Using morphological, immunocytochemical, and functional parameters we have previously shown that highly purified adult rat microglial cells undergo a process of “activation” when cultured in a serum-containing medium in the absence of added proinflammatory substances or other factors (Slepko and Levi: Glia 16:241–246, 1996). Here we studied the lipopolysaccharide (LPS)-evoked production of two prostanoids, thromboxane A₂ (measured as thromboxane B₂) (TXB₂) and prostaglandin E₂ (PGE₂), as a function of microglial “activation.” LPS induced a greater time- and dose-dependent release of TXB₂, compared to PGE₂, in the less “activated” cells. Further “activation” led to amplified synthesis of PGE₂ and not of TXB₂, so that the TXB₂/PGE₂ ratio changed from 2.2 to 0.25 between the 2nd and 4th day in culture. Western blot experiments showed that the LPS-evoked expression of the inducible form of cyclooxygenase (COX) was markedly higher in cells exhibiting a more “activated” phenotype. The expression of the constitutive isoform of COX was low in all conditions, was slightly greater in more “activated” cells, and was not affected by LPS. Neither progression in microglial “activation” nor LPS treatment enhanced thromboxane synthase activity. We hypothesize that reorientation of prostanoid synthesis toward a major production of PGE₂ in the more “activated” cells can be largely attributed to an increased inducibility of cellular COX expression, combined with the inability of thromboxane synthase to cope with the increased availability of the COX product prostaglandin H₂ (PGH₂), the common precursor of TXA₂ and PGE₂. In view of the different, and at times opposite, functional activity of TXB₂ and PGE₂, the described change in prostanoid production pattern may contribute to the role of “activated” microglia in inflammation and host defense. J. Neurosci. Res. 49:292–300, 1997. © 1997 Wiley-Liss, Inc.