Effects of adrenaline in human colon adenocarcinoma HT-29 cells

AimsStress has been implicated in the development of cancers. Adrenaline levels are increased in response to stress. The effects of adrenaline on colon cancer are largely unknown. The aims of the study are to determine the effects of adrenaline in human colon adenocarcinoma HT-29 cells and the possible underlying mechanisms involved.Main methodsThe effect of adrenaline on HT-29 cell proliferation was determined by [³H] thymidine incorporation assay. Expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) were detected by Western blot. Matrix metalloproteinase-9 (MMP-9) activity and prostaglandin E₂ (PGE₂) release were determined by zymography and enzyme immunoassay, respectively.Key findingsAdrenaline stimulated HT-29 cell proliferation. This was accompanied by the enhanced expression of COX-2 and VEGF in HT-29 cells. Adrenaline also upregulated MMP-9 activity and PGE₂ release. Adrenaline stimulated HT-29 cell proliferation which was reversed by COX-2 inhibitor sc-236. COX-2 inhibitor also reverted the action of adrenaline on VEGF expression and MMP-9 activity. Further study was performed to determine the involvement of β-adrenoceptors. The stimulatory action of adrenaline on colon cancer growth was blocked by atenolol and ICI 118,551, a β₁- and β₂-selective antagonist, respectively. This signified the role of β-adrenoceptors in this process. In addition, both antagonists also abrogated the stimulating actions of adrenaline on COX-2, VEGF expression, MMP-9 activity and PGE₂ release in HT-29 cells.SignificanceThese results suggest that adrenaline stimulates cell proliferation of HT-29 cells via both β₁- and β₂-adrenoceptors by a COX-2 dependent pathway.     

Neuropathogenesis of Japanese Encephalitis in a Primate Model

Background

Japanese encephalitis (JE) is a major cause of mortality and morbidity for which there is no treatment. In addition to direct viral cytopathology, the inflammatory response is postulated to contribute to the pathogenesis. Our goal was to determine the contribution of bystander effects and inflammatory mediators to neuronal cell death.

Methodology/Principal Findings

Material from a macaque model was used to characterize the inflammatory response and cytopathic effects of JE virus (JEV). Intranasal JEV infection induced a non-suppurative encephalitis, dominated by perivascular, infiltrates of mostly T cells, alongside endothelial cell activation, vascular damage and blood brain barrier (BBB) leakage; in the adjacent parenchyma there was macrophage infiltration, astrocyte and microglia activation. JEV antigen was mostly in neurons, but there was no correlation between intensity of viral infection and degree of inflammatory response. Apoptotic cell death occurred in both infected and non-infected neurons. Interferon-α, which is a microglial activator, was also expressed by both. Tumour Necrosis Factor-α, inducible nitric oxide synthase and nitrotyrosine were expressed by microglial cells, astrocytes and macrophages. The same cells expressed matrix metalloproteinase (MMP)-2 whilst MMP-9 was expressed by neurons.

Conclusions/Significance

The results are consistent with JEV inducing neuronal apoptotic death and release of cytokines that initiate microglial activation and release of pro-inflammatory and apoptotic mediators with subsequent apoptotic death of both infected and uninfected neurons. Activation of astrocytes, microglial and endothelial cells likely contributes to inflammatory cell recruitment and BBB breakdown. It appears that neuronal apoptotic death and activation of microglial cells and astrocytes play a crucial role in the pathogenesis of JE.     

Dose-dependent changes of the ratio of apoptosis to proliferation by norethisterone and medroxyprogesterone acetate in human breast epithelial cells.

OBJECTIVE: There is increasing evidence that adding progestogens to estrogen replacement therapy may do more harm than good; however, whether all progestogens act equally on breast cells is debatable. Apart from estrogens, mitogenic growth factors from stromal breast tissue are important in growth-regulation of breast cells, and may modify the response to progestogens. We investigated the effects of medroxyprogesterone acetate (MPA) as well as norethisterone (NET) in the presence of a growth factor mixture and/or estradiol in normal and cancerous human epithelial breast cells. METHODS: MCF10A cells (human epithelial, estrogen- and progesterone-receptor negative, normal breast cells), HCC1500 (human estrogen and progesterone receptor-positive primary breast cancer cells) and MCF-7 cells (human estrogen and progesterone receptor-positive metastatic breast cancer cell line) were used in the experiments. The cells were incubated with progestogens at concentrations of 10(-10) to 10(-6) M for 7 days and growth factors (GFs), estradiol (E2) alone and a combination of GFs + E2. Cell proliferation rate was measured by ATP assay. Apoptosis was measured by cell death assay. Ratios of cell death : proliferation were calculated from these results. RESULTS: In MCF10A cells growth factors elicited a decrease in the ratio of apoptosis to proliferation. This effect was further stimulated by the addition of MPA, whereas NET had no effect. In HCC cells growth factors and estradiol alone and in combination led to a reduction in the ratio. This effect could be partly reversed dose-dependently by the addition of MPA and NET, being more pronounced for MPA. Similar results were found for MCF-7 cells stimulated by estradiol. CONCLUSION: The results of our investigations demonstrate that there are differences between the two progestogens NET and MPA investigated with respect to their effects on normal and cancerous cells. By increasing the mitotic rate of normal epithelial cells, MPA may increase breast cancer risk in women when used in long-term treatment. In this respect NET reacts neutral. The mitosis of pre-existing cancerous cells may be partly inhibited by the addition of both progestogens. Thus, our results indicate that it is necessary to differentiate between normal and malignant breast cells concerning the assessment of progestogens as a risk factor for breast carcinogenesis.     

Urokinase-Type Plasminogen Activator Induces BV-2 Microglial Cell Migration Through Activation of Matrix Metalloproteinase-9

In response to brain injury, microglia migrate and accumulate in the affected sites, which is an important step in the regulation of inflammation and neuronal degeneration/regeneration. In this study, we investigated the effect of urokinase-type plasminogen activator (uPA) on the BV-2 microglial cell migration. At resting state, BV-2 microglial cells secreted uPA and the release of uPA was increased by ATP, a chemoattractant released from injured neuron. The migration of BV-2 cell was significantly induced by uPA and inhibited by uPA inhibitors. In this condition, uPA increased the activity of matrix metalloproteinase (MMP-9) and the inhibition of MMP activity with pharmacological inhibitors against either uPA (amiloride) or MMP (phenanthrolene and SB-3CT) effectively prevented BV2 cell migration. Interestingly, the level of MMP-9 protein and mRNA in the cell were not changed by uPA. These results suggest that the increase of MMP-9 activity by uPA is regulated at the post-translational level, possibly via increased activation of the enzyme. Unlike the uPA inhibitor, plasmin inhibitor PAI-1 only partially inhibited uPA-induced cell migration and MMP-9 activation. The incubation of recombinant MMP-9 with uPA resulted in the activation of MMP-9. These results suggest that uPA plays a critical role in BV-2 microglial cell migration by activating pro-MMP-9, in part by its direct action on MMP-9 and also in part by the activation of plasminogen/plasmin cascade.     

UP-regulaton of prostaglandin E2 binding and of prostanoid release in rabbits producing antibodies

German Giant rabbits successfully immunized agianst prostaglandin (PG) E₂ as shown by a rise in antibody titers developed gastric mucosal lesions. Enzymatically dispersed gatric mucosal cells of these animals had a significantly enhanced production of PG₂ and PG I₂ as measured by specific radioimmunoassays. This may be explained by an increased supply with endogenous arachidonic acid (as indicated by an enhanced phospholipase A₂/LAT ratio) and by a higher activity of the subsequent PG forming enzymes (as indicated by a more effectvie stimulation of PG production by exogenous arachidonic acid). Gastric mucosal plasma membranes of immunized rabbits had significantly high PG E₂ binding capacity (108 ± 9 fmol/mg protein) than those of nomimmunized rabbits (72 ± 5 fmol/mg protein). The ligand affinity was not afected by immunization. Neither histamine-stimulated ¹⁴C-aminopyrine uptake of isolated parietal cells as a marker for acid production nor its inhibition by PG E₂ were influenced by receptor up-regulation. The increased eicosanoid release can be regarded as an endogenous defense emchanism against increased mucosal vulnerability caused by PG E₂ scavenging. The potential role of PG E₂ receptor up-regullation in support of this process remains to be established.     

15-Hydroxyprostaglandin Dehydrogenase Generation of Electrophilic Lipid Signaling Mediators from Hydroxy Ω-3 Fatty Acids

15-Hydroxyprostaglandin dehydrogenase (15PGDH) is the primary enzyme catalyzing the conversion of hydroxylated arachidonic acid species to their corresponding oxidized metabolites. The oxidation of hydroxylated fatty acids, such as the conversion of prostaglandin (PG) E₂ to 15-ketoPGE₂, by 15PGDH is viewed to inactivate signaling responses. In contrast, the typically electrophilic products can also induce anti-inflammatory and anti-proliferative responses. This study determined that hydroxylated docosahexaenoic acid metabolites (HDoHEs) are substrates for 15PGDH. Examination of 15PGDH substrate specificity was conducted in cell culture (A549 and primary human airway epithelia and alveolar macrophages) using chemical inhibition and shRNA knockdown of 15PGDH. Substrate specificity is broad and relies on the carbon position of the acyl chain hydroxyl group. 14-HDoHE was determined to be the optimal DHA substrate for 15PGDH, resulting in the formation of its electrophilic metabolite, 14-oxoDHA. Consistent with this, 14-HDoHE was detected in bronchoalveolar lavage cells of mild to moderate asthmatics, and the exogenous addition of 14-oxoDHA to primary alveolar macrophages inhibited LPS-induced proinflammatory cytokine mRNA expression. These data reveal that 15PGDH-derived DHA metabolites are biologically active and can contribute to the salutary signaling actions of Ω-3 fatty acids.     

Angiotensin II Regulation of Intracellular Calcium in Astroglia Cultured from Rat Hypothalamus and Brainstem

Abstract: Several pieces of evidence suggest a major role for brain macrophages in the overproduction of neuroactive kynurenines, including quinolinic acid, in brain inflammatory conditions. In the present work, the regulation of kynurenine pathway enzymes by interferon-γ (IFN-γ) was studied in immortalized murine macrophages (MT2) and microglial (N11) cells. In both cell lines, IFN-γ induced the expression of indoleamine 2,3-dioxygenase (IDO) activity. Whereas tumor necrosis factor-α did not affect enzyme induction by IFN-γ, lipopolysaccharide modulated IDO activity differently in the two IFN-γ-activated cell lines, causing a reduction of IDO expression in MT2 cells and an enhancement of IDO activity in N11 cells. Kynurenine aminotransferase, kynurenine 3-hydroxylase, and 3-hydroxyanthranilic acid dioxygenase appeared to be constitutively expressed in both cell lines. Kynurenine 3-hydroxylase activity was stimulated by IFN-γ. It was notable that basal kynureninase activity was much higher in MT2 macrophages than in N11 microglial cells. In addition, IFN-γ markedly stimulated the activity of this enzyme only in MT2 cells. IFN-γ-treated MT2 cells, but not N11 cells, were able to produce detectable amounts of radiolabeled 3-hydroxyanthranilic acid quinolinic acids from l -[5-³H]tryptophan. These results support the notion that activated invading macrophages may constitute one of the major sources of cerebral quinolinic acid during inflammation.     

Nicotine promotes atherosclerosis development in apolipoprotein E-deficient mice through α1-nAChR

α1 Nicotinic acetylcholine receptor (α1nAChR) is an important nicotine receptor that is widely distributed in vascular smooth muscle cells, macrophages, and endothelial cells. However, the role of α1nAChR in nicotine-mediated atherosclerosis remains unclear. The administration of nicotine for 12 weeks increased the area of the atherosclerotic lesion, the number of macrophages infiltrating the plaques, and the circulating levels of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-α, in apolipoprotein E-deficient (ApoE^−/−) mice fed a high-fat diet. Nicotine also increased α1nAChR, calpain-1, matrix metalloproteinase-2 (MMP-2), and MMP-9 expression in the aortic tissue. Silencing of α1nAChR with an adenoassociated virus decreased the atherosclerotic size, lesion macrophage content, and circulating levels of inflammatory cytokines and suppressed α1nAChR, calpain-1, MMP-2, and MMP-9 expression in the nicotine group. In vitro, nicotine-induced α1nAChR, calpain-1, MMP-2, and MMP-9 expression in mouse vascular smooth muscle cells (MOVAS) and macrophages (RAW264.7), and enhanced the migration and proliferation of these cells. The silencing of α1nAChR inhibited these effects of nicotine MOVAS and RAW264.7 cells. Thus, we concluded that nicotine promoted the development of atherosclerosis partially by inducing the migration and proliferation of vascular smooth muscle cells and macrophages and inducing an inflammatory reaction. The effect of nicotine on atherogenesis may be mediated by α1nAChR-induced activation of the calpain-1/MMP-2/MMP-9 signaling pathway.     

Infection with human cytomegalovirus alters the MMP-9/TIMP-1 balance in human macrophages

Human cytomegalovirus (HCMV) has been suggested to contribute to the development of vascular diseases. Since matrix metalloproteinases (MMPs) have been implicated in atherosclerosis and plaque rupture, we investigated the effect of HCMV infection on MMP expression in human macrophages. We used quantitative real-time PCR, Western blotting, and gelatin zymography to study the expression and activity of MMP-2, -3, -7, -9, -12, -13, and -14 and of tissue inhibitor of metalloproteinase 1 (TIMP-1), -2, -3, and -4. HCMV infection reduced MMP-9 mRNA, protein, and activity levels but increased TIMP-1 mRNA and protein levels. Furthermore, a decrease in MMP-12, MMP-14, TIMP-2, and TIMP-3 mRNA levels could be detected. The MMP-9 and TIMP-1 mRNA alterations required viral replication. MMP-9 mRNA expression was affected by an immediate-early or early viral gene product, whereas TIMP-1 mRNA expression was affected by late viral gene products. We conclude that HCMV infection specifically alters the MMP-9/TIMP-1 balance in human macrophages, which in turn reduces MMP-9 activity in infected cells. Since MMP-9 prevents atherosclerotic plaque development in mice, these results suggest that HCMV may contribute to atherogenesis through specific effects on MMP-9 activity.     

Matrix Metalloproteinase 9 (MMP-9) Regulates Vein Wall Biomechanics in Murine Thrombus Resolution

Objective

Deep venous thrombosis is a common vascular problem with long-term complications including post-thrombotic syndrome. Post-thrombotic syndrome consists of leg pain, swelling and ulceration that is related to incomplete or maladaptive resolution of the venous thrombus as well as loss of compliance of the vein wall. We examine the role of metalloproteinase-9 (MMP-9), a gene important in extracellular remodeling in other vascular diseases, in mediating thrombus resolution and biomechanical changes of the vein wall.

Methods and Results

The effects of targeted deletion of MMP-9 were studied in an in vivo murine model of thrombus resolution using the FVB strain of mice. MMP-9 expression and activity significantly increased on day 3 after DVT. The lack of MMP-9 impaired thrombus resolution by 27% and this phenotype was rescued by the transplantation of wildtype bone marrow cells. Using novel biomechanical techniques, we demonstrated that the lack of MMP-9 significantly decreased thrombus-induced loss of vein wall compliance. Biomechanical analysis of the contribution of individual structural components showed that MMP-9 affected the elasticity of the extracellular matrix and collagen-elastin fibers. Biochemical and histological analyses correlated with these biomechanical effects as thrombi of mice lacking MMP-9 had significantly fewer macrophages and collagen as compared to those of wildtype mice.

Conclusions

MMP-9 mediates thrombus-induced loss of vein wall compliance by increasing stiffness of the extracellular matrix and collagen-elastin fibers during thrombus resolution. MMP-9 also mediates macrophage and collagen content of the resolving thrombus and bone-marrow derived MMP-9 plays a role in resolution of thrombus mass. These disparate effects of MMP-9 on various aspects of thrombus illustrate the complexity of individual protease function on biomechanical and morphometric aspects of thrombus resolution.     

Study of the PG E2 synthetase activity of different regions of dog and rabbit hearts

Prostaglandin E₂ synthetase activity of the microsomal fraction from different parts of dog and rabbit heart was tested with 3H-arachidonic acid as substrate. PG E₂ synthesized was separated and purified by TLC and determined by the radiometric method or by bioassay. In the experimental conditions adopted, it was shown that the heart tissue is endowed with an enzyme system capable of synthesizing PG E₂ but this PG E₂ synthetase activity is not uniformly distributed in the different parts of the heart. It is highest in the right atrium and the activity of the atria is higher than that of the ventricles. It is species-dependent. The closely similar repartition of PG E₂ synthetase activity and sympathetic nerve endings strongly suggests that PG E₂ modulates adrenergic neurotransmission in the heart.     

Prostaglandin profiles in nervous tissue and blood vessels of the brain of various animals

The endogenous formation of prostaglandin (PG) D₂, E₂, F_2α, and 6-keto-PGF_1α was determined in homogenates of mouse, rat, and rabbit brain, and of rat cerebral blood vessels, using gas chromatography mass spectrometry. In all species tested, 6-keto-PGF_1α could be identified in the brain homogenates, but was a minor component in relation to other PGs. In contrast 6-keto-PGF_1α was the most abundant PG in the blood vessels, being present in about 40-fold higher levels than in the brain tissue. PGD₂ was the most abundant PG in rat and mouse brains, but was below detection limits in the analyzed blood vessels. These studies indicating differential metabolism of PG endoperoxides in nervous and vascular tissue, provide a biochemical basis for further studies on the role of the PGs in brain circulation and neuronal activity.     

Effect of Thrombin on Human Amnion Mesenchymal Cells,Mouse Fetal Membranes,and Preterm Birth

Here, we investigated the effects of thrombin on matrix metalloproteinases (MMPs) and prostaglandin (PG) synthesis in fetal membranes. Thrombin activity was increased in human amnion from preterm deliveries. Treatment of mesenchymal, but not epithelial, cells with thrombin resulted in increased MMP-1 and MMP-9 mRNA and enzymatic activity. Thrombin also increased COX2 mRNA and PGE₂ in these cells. Protease-activated receptor-1 (PAR-1) was localized to amnion mesenchymal and decidual cells. PAR-1-specific inhibitors and activating peptides indicated that thrombin-induced up-regulation of MMP-9 was mediated via PAR-1. In contrast, thrombin-induced up-regulation of MMP-1 and COX-2 was mediated through Toll-like receptor-4, possibly through thrombin-induced release of soluble fetal fibronectin. In vivo, thrombin-injected pregnant mice delivered preterm. Mmp8, Mmp9, and Mmp13, and PGE₂ content was increased significantly in fetal membranes from thrombin-injected animals. These results indicate that thrombin acts through multiple mechanisms to activate MMPs and PGE2 synthesis in amnion.     

The novel estrogen receptor GPER regulates the migration and invasion of ovarian cancer cells

G protein-coupled estrogen receptor (GPER) was identified as a new member of the estrogen receptor family in recent years. It has become apparent that GPER mediates the non-genomic signaling of 17β-estradiol (E₂) in a variety of estrogen-related cancers. Our previous study has found that GPER was overexpressed in human epithelial ovarian cancer and was positively correlated with the expression of matrix metalloproteinase 9 (MMP-9), which suggested GPER might promote the metastasis of ovarian cancer. However, the mechanisms underlying GPER-dependent metastasis of ovarian cancer are still not clear. In the present study, estrogen receptor α (ERα)-negative/GPER-positive OVCAR5 ovarian cancer cell line was used to investigate the role of GPER in the migration and invasion of ovarian cancer. Wound healing assay and transwell matrigel invasion assay were performed to determine the potentials of cell migration and invasion, respectively. The production and activity of MMP-9 in OVCAR5 cells were examined by Western blot and gelatin zymography analysis. The results showed that E₂ and selective GPER agonist G-1 increased cell motility and invasiveness, and upregulated the production and proteolytic activity of MMP-9 in OVCAR5 cells. Small interfering RNA (siRNA) targeting GPER and G protein inhibitor pertussin toxin (PTX) inhibited the migration and invasion of OVCAR5 cells, and also reduced the expression and activity of MMP-9. Our data suggested that GPER promoted the migration and invasion of ovarian cancer cells by increasing the expression and activity of MMP-9. GPER might play an important role in the progression of ovarian cancer.     

Interactions of prostaglandins with subpopulations of ovine luteal cells. I. Stimulatory effects of prostaglandins E1, E2 and I21

Preparations of small and large steroidogenic cells from enzymatically dispersed ovine corpora lutea were utilized to study the $\text{in}$$\text{vitro}$ effects of luteinizing hormone (LH) and prostaglandins (PG) E₁, E₂ and I₂. Cells were allowed to attach to culture dishes overnight and were incubated with either LH (100 ng/ml), PGE₂, PGE₂, or PGI₂ (250 ng/ml each). The secretion of progesterone by large cells was stimulated by all prostaglandins tested (P < 0.05) while the moderate stimulation observed after LH treatment was attributable to contamination of the large cell population with small cells. Prostaglandins E₁ and E₂ had no effect on progesterone secretion by small cells, while LH was stimulatory at all times (0.5 to 4 hr) and PGI₂ was stimulatory by 4 hr. Additional studies were conducted to determine if the effects of PGE₂ upon steroidogenesis in large cells were correlated with stimulated activity of adenylate cyclase. In both plated and suspended cells PGE₂ caused an increase (P < 0.05) in the rate of progesterone secretion but had no effect upon the activity of adenylate cyclase or cAMP concentrations within cells or in the incubation media. Exposure of luteal cells to forskolin, a nonhormonal stimulator of adenylate cyclase, resulted in marked increases in all parameters of cyclase activity but had no effect on progesterone secretion. These data suggest that the actions of prostaglandins E₁, E₂ and I₂ are directed primarily toward the large cells of the ovine corpus luteum and cast doubt upon the role of adenylate cyclase as the sole intermediary in regulation of progesterone secretion in this cell type.     

Microglial expression of prostaglandin EP3 receptor in excitotoxic lesions in the rat striatum

Prostaglandins (PG) are produced by the enzymatic activity of cyclooxygenase (COX). PGs and COX have been implicated in the pathophysiology of excitotoxicity and neurodegeneration in the central nervous system (CNS). The PGE2 receptor EP3 is the most abundantly expressed PGE2 receptor subtype in the brain. So far, in the innate rat brain EP3 receptors have been found exclusively in neurons. The aim of this study was to investigate whether EP3 expression in the brain changes under neurodegenerative circumstances such as an acute excitotoxic lesion. Intrastriatal injection of quinolinic acid (QUIN) resulted in a loss of EP3-positive striatal neurons, while simultaneously small glial-shaped EP3-positive cells appeared. Five days after lesioning, 63% of the glial-shaped EP3-positive cells could be identified as ED-1 expressing microglial cells. This percentage increased to 82% after 10 days, suggesting that most of the EP3-positive ED-1-negative cells on day 5 may be microglia which did not yet express ED-1. ED-1-positive microglia also expressed COX-1. These experiments show for the first time that activated microglial cells in excitotoxic lesions express in vivo the PGE2 receptor EP3 and the PGE2 synthesizing enzyme COX-1. Activation of EP3 receptor downregulates cAMP formation and may counteract the upregulation of cAMP formation via EP2 receptors, which has been linked to the anti-inflammatory effects of PGs. This change in EP3-receptor expression in microglia might participate in acute or chronic microglial activation in a variety of brain diseases such as ischemia or Alzheimer's disease (AD). Investigation of the expression of different PGE2 receptor subtypes might promote a better understanding of the pathophysiology of these diseases as well as leading to a modulation of microglial activation by a more specific interference with selective EP receptors than can be achieved by inhibiting global PG synthesis by selective or non-selective COX inhibitors.     

Pretreatment with interferon-γ protects microglia from oxidative stress via up-regulation of Mn-SOD

Microglial cells, resident macrophage-like immune cells in the brain, are exposed to intense oxidative stress under various pathophysiological conditions. For self-defense against oxidative injuries, microglial cells must be equipped with antioxidative mechanisms. In this study, we investigated the regulation of antioxidant enzyme systems in microglial cells by interferon-γ (IFN-γ) and found that pretreatment with IFN-γ for 20 h protected microglial cells from the toxicity of various reactive species such as hydrogen peroxide (H₂O₂), superoxide anion, 4-hydroxy-2(E)-nonenal, and peroxynitrite. The cytoprotective effect of IFN-γ pretreatment was abolished by the protein synthesis inhibitor cycloheximide. In addition, treatment of microglial cells with both IFN-γ and H₂O₂ together did not protect them from the H₂O₂-evoked toxicity. These results imply that protein synthesis is required for the protection by IFN-γ. Among various antioxidant enzymes such as manganese or copper/zinc superoxide dismutase (Mn-SOD or Cu/Zn-SOD), catalase, and glutathione peroxidase (GPx), only Mn-SOD was up-regulated in IFN-γ-pretreated microglial cells. Transfection with siRNA of Mn-SOD abolished both up-regulation of Mn-SOD expression and protection from H₂O₂ toxicity by IFN-γ pretreatment. Furthermore, whereas the activities of Mn-SOD and catalase were up-regulated by IFN-γ pretreatment, those of Cu/Zn-SOD and GPx were not. These results indicate that IFN-γ pretreatment protects microglial cells from oxidative stress via selective up-regulation of the level of Mn-SOD and activity of Mn-SOD and catalase.