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Eicosanoids are produced by microglia, not by astrocytes, in rat glial cell cultures
Abstract
To determine principal sources of eicosanoid production in glial cells, we analyzed the metabolites of arachidonic acid in cultured rat glial cells by use of reversed-phase, high-performance liquid chromatography and an on-line radioisotope detector. Prostaglandin D2, leukotriene B4, leukotriene C4, and 5-hydroxyeicosatetraenoic acid were present in cultures in which microglia appeared on a monolayer astrocytes. None were detected in culture dishes that contained only astrocytes, although astrocytes have been believed to be a main source of eicosanoid production in brain.
... Eicosanoids produced in the CNS play an important role in neuropathogenesis. Several different studies, mainly based on murine models, have shown that microglial cells produce many COX and 5-LO products, namely PGE 2 , PGD 2 , LTB 4 , LTC 4 , 5-HETE, 11-HETE and 15- HETE123124125126127128. Brain eicosanoids may also be secreted by cell types other than microglia, such as astrocytes, oligodendrocytes, neurons and endothelial cells, which play an important role in the propagation and maintenance of neuroinflammation [129,130]. ...
... These data clearly showed the protective role of LTs during viral infections in the CNS. We now know that LTs are present in the CNS of HIV-infected individuals, where they can be produced by different cell types, including monocytes and micro- glia [125,126,149,168]. Moreover, recent studies have shown that high concentrations of eicosanoids, including LTB 4 , and several other AA cascade markers are found in the brain of HIV-transgenic rats [169,170]. ...
In HIV-1-infected individuals, there is often discordance between viremia in peripheral blood and viral load found in the central nervous system (CNS). Although the viral burden is often lower in the CNS compartment than in the plasma, neuroinflammation is present in most infected individuals, albeit attenuated by the current combined antiretroviral therapy. The HIV-1-associated neurological complications are thought to result not only from direct viral replication, but also from the subsequent neuroinflammatory processes. The eicosanoids - prostanoids and leukotrienes - are known as potent inflammatory lipid mediators. They are often present in neuroinflammatory diseases, notably HIV-1 infection. Their exact modulatory role in HIV-1 infection is, however, still poorly understood, especially in the CNS compartment. Nonetheless, a handful of studies have provided evidence as to how these lipid mediators can modulate HIV-1 infection. This review summarizes findings indicating how eicosanoids may influence the progression of neuroAIDS.
... Furthermore, the increase of LT synthesis in microglial cells following LT-receptor activation requires the intracellular second messenger p38 MAP kinase (MAPK) [27]. Several studies have reported that LTs, along with their cognate receptors, are expressed in the brain [22,26,2829303132333435 . Moreover, in vitro cocultures of HIV-1- infected monocytes and astroglia secrete high levels of eicosanoids such as LTB 4 and LTD 4 , which largely contribute to neuroinflammation and neuronotoxicity [36]. ...
... Moreover, recent studies have shown that increased levels of 5-LO and high concentrations of eicosanoids, including LTB 4 , are found in the brain of HIV-1 transgenic rats [38,39]. Moreover, it has been demonstrated that activated microglia can produce several proinflammatory cytokines, chemokines and certain eicosanoid metabolites [29,404142. In turn, chemotactic molecules secreted in neuropathologic conditions, such as LTs, contribute to the recruitment of potentially infected leukocytes (including monocytes and lymphocytes ) into the CNS [43]. ...
Microglia are one of the main cell types to be productively infected by HIV-1 in the central nervous system (CNS). Leukotriene B4 (LTB4) and cysteinyl-leukotrienes such as LTC4 are some of the proinflammatory molecules produced in infected individuals that contribute to neuroinflammation. We therefore sought to investigate the role of leukotrienes (LTs) in HIV-1 infection of microglial cells.
To evaluate the role of LTs on HIV-1 infection in the CNS, monocyte-derived microglial-like cells (MDMis) were utilized in this study. Leukotriene-treated MDMis were infected with either fully replicative brain-derived HIV-1 isolates (YU2) or R5-tropic luciferase-encoding particles in order to assess viral production and expression. The efficacy of various steps of the replication cycle was evaluated by means of p24 quantification by ELISA, luciferase activity determination and quantitative real-time polymerase chain reaction (RT-PCR).
We report in this study that virus replication is reduced upon treatment of MDMis with LTB4 and LTC4. Additional experiments indicate that these proinflammatory molecules alter the pH-independent entry and early post-fusion events of the viral life cycle. Indeed, LT treatment induced a diminution in integrated proviral DNA while reverse-transcribed viral products remained unaffected. Furthermore, decreased C-C chemokine receptor type 5 (CCR5) surface expression was observed in LT-treated MDMis. Finally, the effect of LTs on HIV-1 infection in MDMis appears to be mediated partly via a signal transduction pathway involving protein kinase C.
These data show for the first time that LTs influence microglial cell infection by HIV-1, and may be a factor in the control of viral load in the CNS.
... It is a potent leukocyte chemoattractant and activator that plays an important role in modulating immune responses [4]. LTB 4 can be synthesized by various cell types both in the periphery and in the central nervous system (CNS) -for example mast cells, neutrophils [5], alveolar macrophages [6,7] and epithelial lens cells [8] in the periphery; and cultured primary rat astrocytes [9], neuronal cells [10], and glial cells [11] of the CNS -upon challenge with a variety of stimuli including LTB 4 itself. LTB 4 serves as a potent inflammatory mediator through the high affinity LTB 4 receptor-1 (BLT1) on target cells. ...
... Interestingly, particularly high levels of neuronal 5-LO expression and LTB 4 content have been identified in CNS upon challenge with a variety of stimuli [10,11,39]. The gene encoding 5-LO appears to be subject to hormonal regulation [40,41], and its neuronal expression is remarkably upregulated during aging [42], while the glucocorticoid dexamethasone inhibits 5-LO and LTA4-H mRNA expression in cerebral cortex of asthmatic rats [22]. ...
Despite intensive studies focused on the pathophysiology of asthmatic inflammation, little is known about how cross-talk between neuroendocrine and immune systems regulates the inflammatory response during an asthmatic attack. We recently showed corresponding changes of cytokines and leukotriene B4 (LTB4) in brain and lung tissues of antigen-challenged asthmatic rats. Here, we investigated how LTB4 interacts with the neuroendocrine-immune system in regulating antigen-induced asthmatic responses in sensitized guinea pigs.
Ovalbumin-sensitized guinea pigs were challenged by inhalation of antigen. Vehicle, LTB4 or U75302 (a selective LTB4 BLT1 receptor inhibitor) was given via intracerebroventricular injection (i.c.v.) 30 min before challenge. Airway contraction response was evaluated using Penh values before and after antigen challenge. The inflammatory response in lung tissue was evaluated 24 h after challenge. The LTB4 content of lung and brain homogenate preparations was detected by reversed phase high-performance liquid chromatography (RP-HPLC). Plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were measured using ELISA kits.
Antigen challenge impaired pulmonary function and increased inflammatory cell infiltration in lung tissue. These responses could be significantly suppressed by LTB4, 30 ng i.c.v., in ovalbumin-sensitized guinea pigs. LTB4 content of lung and brain homogenates from antigen-challenged guinea pigs was significantly increased. In addition, administration of LTB4 via i.c.v. markedly increased CORT and ACTH level in plasma before antigen challenge, and there were further increases in CORT and ACTH levels in plasma after antigen challenge. U75302, 100 ng i.c.v., completely blocked the effects of LTB4. In addition, U75302, 100 ng via i.c.v. injection, markedly decreased LTB4 content in lung homogenates, but not in brain homogenates.
Increased LTB4 levels in brain during asthmatic attacks down-regulates airway contraction response and inflammation through the BLT1 receptor. Stimulation of the hypothalamic-pituitary-adrenal axis by LTB4 may result in an increase in systemic glucocorticoids which, in turn, would feed back to suppress the asthmatic response.
... Surprisingly, reactive microglial sensors for pathological events in the CNS [14], which are essential for tissue remodeling, and represent one of the major sources of prostaglandins in the CNS [21,23], are not the main source of the pathogen-associated COX-2 expression [10,31]. However, the role of microglia in lipid oxygenation is supported by its capacity to produce PLA 2 , COX-1 and the vasoactive PGE 2 and TXA [21,23,40]. ...
... Surprisingly, reactive microglial sensors for pathological events in the CNS [14], which are essential for tissue remodeling, and represent one of the major sources of prostaglandins in the CNS [21,23], are not the main source of the pathogen-associated COX-2 expression [10,31]. However, the role of microglia in lipid oxygenation is supported by its capacity to produce PLA 2 , COX-1 and the vasoactive PGE 2 and TXA [21,23,40]. This supports a distinct regulation of COX-1 and -2 in the brain and peripheral organs, which might relate to the organ-specific differences in the use of arachidonic acid as a substrate [35]. ...
Cyclooxygenases (COX; prostaglandin endoperoxide H synthases) are key enzymes in the conversion of arachidonic acid into prostanoids which mediate inflammation, immunomodulation, mitogenesis, ovulation, fewer, apoptosis and blood flow. Here, we report COX-1 expression following focal cerebral infarctions (FCI). In healthy control brains, COX-1 was localized by immunohistochemistry to a few endothelial cells, single neurons and rare, evenly distributed brain microglia/macrophages. In infarctioned brains, COX-1+ cells accumulated highly significantly (P < 0.0001) in peri-infarctional areas and in the developing necrotic core early after infarction. Here, cell numbers remained persistently elevated up to several months post infarction. Further, clusters of COX-1+ cells were located in perivascular regions related to the Virchow-Robin space. Double-labeling experiments confirmed co-expression of COX-1 by CD68+ microglia/macrophages. Co-expression of the activation antigens HLA-DR, -DP, -DQ (MHC class II) or the macrophage inhibitor factor-related protein MRP-8 (S100A8) by most COX-1+ microglia/macrophages was only seen early after infarction. Thus, COX-1 appeared to be expressed in microglial cells regardless of their activation state. However, the prolonged accumulation of COX-1+ microglia/macrophages restricted to peri-infarctional areas enduring the acute post-ischemic inflammatory response points to a role of COX-1 in tissue remodeling or in the pathophysiology of secondary injury. We have identified localized, accumulated COX-1 expression as a potential pharmacological target following FCI. Therefore we suggest that therapeutic approaches based on selective COX-2 blocking might not be sufficient for suppressing the local synthesis of prostanoids.
... Reactive microglia, a sensor for pathological events in the CNS (Kreutzberg, 1996), is essential to tissue remodeling and one of the major sources of prostaglandins in the CNS (Matsuo et al., 1995;Minghetti and Levi, 1998). The role of microglia in lipid oxygenation is supported by its capacity to produce PLA 2 , the vasoactive PGE 2 , TXA, and COX-1 (Matsuo et al., 1995;Mingheti and Levi, 1998;Yermakova et al., 1999). ...
... Reactive microglia, a sensor for pathological events in the CNS (Kreutzberg, 1996), is essential to tissue remodeling and one of the major sources of prostaglandins in the CNS (Matsuo et al., 1995;Minghetti and Levi, 1998). The role of microglia in lipid oxygenation is supported by its capacity to produce PLA 2 , the vasoactive PGE 2 , TXA, and COX-1 (Matsuo et al., 1995;Mingheti and Levi, 1998;Yermakova et al., 1999). Since COX activity is important following disturbed CNS maintenance contributing to homeostatic conditions by modulation of blood homeostasis (vasoconstriction, platelet aggregation, angiogenesis, blood pressure; Crofford, 1997;DuBois, 1996;DuBois et al., 1998;Smith et al., 1996;Tsujii et al., 1998;Williams and;Yasojima et al., 1999), TBI AND PROLONGED ACCUMULATION OF COX-1 inflammation, and secondary injury (lipid peroxygenation; Dubois et al., 1998), we defined COX-1 expression following experimental brain injury. ...
Inflammatory cellular responses to brain injury are promoted by proinflammatory messengers. Cyclooxygenases (prostaglandin endoperoxide H synthases [PGH]) are key enzymes in the conversion of arachidonic acid into prostanoids, which mediate immunomodulation, mitogenesis, apoptosis, blood flow, secondary injury (lipid peroxygenation), and inflammation. Here, we report COX-1 expression following brain injury. In control brains, COX-1 expression was localized rarely to brain microglia/macrophages. One to 5 days after injury, we observed a highly significant (p < 0.0001) increase in COX-1+ microglia/macrophages at perilesional areas and in the developing core with a delayed culmination of cell accumulation at day 7, correlating with phagocytic activity. There, cell numbers remained persistently elevated up to 21 days following injury. Further, COX-1+ cells were located in perivascular Virchow-Robin spaces also reaching maximal numbers at day 7. Lesion-confined COX-1+ vessels increased in numbers from day 1, reaching the maximum at days 5-7. Double-labeling experiments confirmed coexpression of COX-1 by ED-1+ and OX-42+ microglia/ macrophages. Transiently after injury, most COX-1+ microglia/macrophages coexpress the activation antigen OX-6 (MHC class II). However, the prolonged accumulation of COX-1+, ED-1+ microglia/macrophages in lesional areas enduring the acute postinjury inflammatory response points to a role of COX-1 in the pathophysiology of secondary injury. We have identified localized, accumulated COX-1 expression as a potential pharmacological target in the treatment of brain injury. Our results suggest that therapeutic approaches based on long-term blocking including COX-1, might be superior to selective COX-2 blocking to suppress the local synthesis of prostanoids.
... Interestingly, the COX-2 selective drugs showed a marked increase in potency in microglia when compared to astrocytes which suggests that maybe the inducible isoform contributes to a greater extent to the TXB2 synthesis in microglia than it does in astrocytes. This would support the suggestions put forward by Matsuo et al. (1995) and Giulian et al. (1996) that the microglia in cultured glial cell populations are responsible for the COX-2 activity detected therein. ...
In this study, a panel of inhibitors with a range of selectivities for the two cyclooxygenase isoforms (COX-1 and -2) was used to probe the contribution of each to the synthesis of thromboxane B2 (TXB2) in cultured glia after stimulation with A23187 or arachidonic acid. Mixed glial cell cultures (~95% astrocytes, 5% microglia) were prepared from the cortices of newborn rat pups and maintained in vitro for 14 days. Controversy exists as to the contribution of astrocytes and microglia to the prostanoid synthetic capacity of mixed glial cell cultures. In order to investigate this, derivative cultures were made which were enriched in either astrocytes or microglia. Both astrocytes and microglia were found to express COX protein, and TXB2 production was inhibited in each by inhibitors preferential for the different COX isoforms suggesting that both COX-1 and COX-2 contributed to TXB2 synthesis in both cell types. Removal of serum from the culture medium for 4 days caused a decrease in the level of basal and stimulated TXB2 synthesis, with a concomitant decrease in the presence of COX protein. There was also a decrease in potency of COX-2 selective inhibitors suggesting that the removal of serum caused a down-regulation of COX-2 expression. COX protein expression and TXB2 synthesis were restored to control levels 7 days after serum re-addition, but the inhibitor profile subsequently obtained showed a pattern which differed considerably from either control or serum-deprived cells. Of particular note was an increase in the potency of paracetamol after serum re-addition. The effect of nitric oxide (NO) donors on COX activity in mixed glial cells under normal culture conditions was also investigated. Although inhibition of endogenous NO production was without effect, NO donors reduced stimulus-induced TXB2 release. This was found to be independent of the COX isoform present in the cells and was additive with that evoked by COX inhibitors.
... Eicosanoids families are modulated by neuroglia such as microglia and astrocyte. In particular, eicosanoids are produced by microglia [25]. ...
Evidence support that brain membrane fatty acids play a crucial role in psychopathologies such as depression and anxiety disorders. Although the pathogenesis of depression is not still defined, drugs commonly used to reduce arachidonic turnover in the brain can control mood disorders, such as depression. Both astrocytes and mast cells release arachidonic acid during silent inflammation. Here, we hypothesize that arachidonic acid freed from lipid droplets of mast cells, as well as the one released from activated astrocytes, could contribute to characterize a depressive condition, and the fatty acids profile of mast cells, astrocytes and microglia could also vary, reflecting the pathophysiological depressive state of the subject. Finally, there is evidence that gut microbiota is deeply implicated in mood and behavioral disorders. Human gut microbiota can control nervous system diseases through neuroimmune pathways.
... Together, these pathways augment cell survival and foster neuroprotection by activating calcium channels, increasing protein synthesis, mitochondrial biogenesis and inhibiting apoptosis and inflammation (Athauda andFoltynie, 2016, 2018;Kim et al., 2017;Li et al., 2010b;Salcedo et al., 2012). Activated microglia are a major source of a broad range of toxic compounds, including superoxide, nitric oxide, eicosanoids and cytokines (Kim et al., 2009;Matsuo et al., 1995) that, if elevated, can initiate and drive neuroinflammation (Frankola et al., 2011). A growing literature indicates that GLP-1R activation provides anti-inflammation, either directly or indirectly, which our study confirms. ...
Traumatic brain injury (TBI) is a neurodegenerative disorder for which no effective pharmacological treatment is available. Glucagon-like peptide 1 (GLP-1) analogues such as Exenatide have previously demonstrated neurotrophic and neuroprotective effects in cellular and animal models of TBI. However, chronic or repeated administration was needed for efficacy. In this study, the pharmacokinetics and efficacy of PT302, a clinically available sustained-release Exenatide formulation (SR-Exenatide) were evaluated in a concussive mild (m)TBI mouse model. A single subcutaneous (s.c.) injection of PT302 (0.6, 0.12, and 0.024 mg/kg) was administered and plasma Exenatide concentrations were time-dependently measured over 3 weeks. An initial rapid regulated release of Exenatide in plasma was followed by a secondary phase of sustained-release in a dose-dependent manner. Short- and longer-term (7 and 30 day) cognitive impairments (visual and spatial deficits) induced by weight drop mTBI were mitigated by a single post-injury treatment with Exenatide delivered by s.c. injection of PT302 in clinically translatable doses. Immunohistochemical evaluation of neuronal cell death and inflammatory markers, likewise, cross-validated the neurotrophic and neuroprotective effects of SR-Exenatide in this mouse mTBI model. Exenatide central nervous system concentrations were 1.5% to 2.0% of concomitant plasma levels under steady-state conditions. These data demonstrate a positive beneficial action of PT302 in mTBI. This convenient single, sustained-release dosing regimen also has application for other neurological disorders, such as Alzheimer's disease, Parkinson's disease, multiple system atrophy and multiple sclerosis where prior preclinical studies, likewise, have demonstrated positive Exenatide actions.
... It is tempting to speculate that membrane modifications of these microglial cells may disrupt their phagocytosis capacity and antigenic presentation function. Moreover, because of the role of microglia in brain homeostasis and inflammation control, further lipid analysis should also focus on signaling molecules of lipid origin secreted by microglial cells, i.e. docosanoid and eicosanoids fatty acid derivatives, which are tightly connected to peroxisomal metabolism [62]. ...
Acyl-CoA oxidase 1 (ACOX1) deficiency is a rare and severe peroxisomal leukodystrophy associated with a very long-chain fatty acid (VLCFA) β–oxidation defect. This neurodegenerative disease lacks relevant cell models to further decipher the pathomechanisms in order to identify novel therapeutic targets. Since peroxisomal defects in microglia appear to be a key component of peroxisomal leukodystrophies, we targeted the Acox1 gene in the murine microglial BV-2 cell line. Using CRISPR/Cas9 gene editing, we generated an Acox1-deficient cell line and validated the allelic mutations, which lead to the absence of ACOX1 protein and enzymatic activity. The activity of catalase, the enzyme degrading H2O2, was increased, likely in response to the alteration of redox homeostasis. The mutant cell line grew more slowly than control cells without obvious morphological changes. However, ultrastructural analysis revealed an increased number of peroxisomes and mitochondria associated with size reduction of mitochondria. Changes in the distribution of lipid droplets containing neutral lipids have been observed in mutant cells; lipid analysis revealed the accumulation of saturated and monounsaturated VLCFA. Besides, expression levels of genes encoding interleukin-1 beta and 6 (IL-1β and IL-6), as well as triggering receptor expressed on myeloid cells 2 (Trem2) were found modified in the mutant cells suggesting modification of microglial polarization and phagocytosis ability. In summary, this Acox1-deficient cell line presents the main biochemical characteristics of the human disease and will serve as a promising model to further investigate the consequences of a specific microglial peroxisomal β–oxidation defect on oxidative stress, inflammation and cellular functions.
... Microglia in the CNS and resident macrophages in the PNS may also play a role in modulating some aspects of the glia-axon relationship; they certainly participate in metabolic events following demyelination. Interaction between microglia and oligodendroglia is an emerging field of research (Matsuo et al., 1995; see also a publication on microglia and prostaglandins). Knowledge of mechanisms of neurotoxicity is limited by the boundaries of understanding how the nervous system is developed and how it operates. ...
A compound may be neurotoxic by perturbing a metabolic, structural, or functional property characteristic of myelin or myelinating cells. Perturbations of these properties during nervous system development are more deleterious than those occurring after maturation of the nervous system. Myelin and myelination are described in this section, with emphasis on points relevant to neurotoxic mechanisms of chemicals that target myelin. More information is readily available in more specialized reviews of subtopics on myelin development and biology.
... LPS can induce eicosanoid signaling pathway and subsequent release of Cys-LTs and CysLT 1 R activation in different type of cell cultures, including astrocytes (40)(41)(42). LPS is also an activator of microglia (4,5) and microglial cells are known as a source of eicosanoids synthesis, including the Cys-LTs (43). In our study, LPS caused a great number of microglial activation, which was suppresses by inhibiting CysLT 1 R activation. ...
Previously we reported that cysteinyl leukotrienes (Cys-LTs) and the type 1 receptor for Cys-LTs (CysLT1R) are related to amyloid β (Aβ)-induced neurotoxicity. The aim of the current study was to find out the role of CysLT1R on lipopolysaccharide (LPS)-induced cognitive deficit and neurotoxicity. shRNA-mediated knockdown or pharmacological blockade (by pranlukast) of CysLT1R were performed in ICR mice for 21 days prior to systemic infusion of LPS. From day 22, LPS was administered for 7 days and then a set of behavioral, histopathological and biochemical tests were employed to test memory, neuroinflammation and neuronal apoptosis in mice. LPS (only)-treated mice showed poor performance in both Morris water maze (MWM) and Y-maze tests. However, shRNA-mediated knockdown or pranlukast-treated blockade of CysLT1R improved performance of the mice in these tests. To find out the possible underlying mechanisms, we assessed several parameters such as microglial activation (by immunohistochemistry), level of CysLT1R (by WB and qRT-PCR) and the inflammatory/apoptotic pathways (by ELISA or TUNEL or WB) in the mouse hippocampus. LPS-induced memory impairment was accompanied by activation of microglia, higher level of CysLT1R, IL-1β, TNF-α and nuclear NF-κB p65. LPS also caused apoptosis in the hippocampus as detected by TUNEL staining, further supplemented by detection of increased Caspase-3 and a reduced Bcl-2/Bax ratio. All of these adverse changes in the mouse hippocampus were inhibited in mice pretreated with CysLT1R-shRNA and pranlukast. Through this study we suggest that CysLT1R shares a strong correlation with LPS-associated memory deficit, neuroinflammation and apoptosis and CysLT1R could be a novel target for preventive measures to intervene the progression of Alzheimer’s disease (AD)-like phenotypes.
... As a chemoattractant for inflammatory cells, LTB 4 triggers adherence and aggregation of leukocytes to the endothelium and recruits granulocytes and macrophages to the site of inflammation. LTB 4 can also be released by brain microglial cells (Matsuo et al. 1995), which have been shown to be important for the neurogenic component of experimental hypertension. Moreover, LTB 4 can also act as a strong chemoattractant for T cells (Goodarzi et al. 2003;Medoff et al. 2005;Lone & Taskén, 2013), which are implicated in the pathogenesis of experimental and human hypertension (Waki et al. 2013;Trott & Harrison, 2014). ...
Key points:
Evidence indicates an association between hypertension and chronic systemic inflammation in both human hypertension and experimental animal models. Previous studies in the spontaneously hypertensive rat (SHR) support a role for leukotriene B4 (LTB4 ), a potent chemoattractant involved in the inflammatory response, but its mode of action is poorly understood. In the SHR, we observed an increase in T cells and macrophages in the brainstem; in addition, gene expression profiling data showed that LTB4 production, degradation and downstream signalling in the brainstem of the SHR are dynamically regulated during hypertension. When LTB4 receptor 1 (BLT1) receptors were blocked with CP-105,696, arterial pressure was reduced in the SHR compared to the normotensive control and this reduction was associated with a significant decrease in systolic blood pressure (BP) indicators. These data provide new evidence for the role of LTB4 as an important neuro-immune pathway in the development of hypertension and therefore may serve as a novel therapeutic target for the treatment of neurogenic hypertension.
Abstract:
Accumulating evidence indicates an association between hypertension and chronic systemic inflammation in both human hypertension and experimental animal models. Previous studies in the spontaneously hypertensive rat (SHR) support a role for leukotriene B4 (LTB4 ), a potent chemoattractant involved in the inflammatory response. However, the mechanism for LTB4 -mediated inflammation in hypertension is poorly understood. Here we report in the SHR, increased brainstem infiltration of T cells and macrophages plus gene expression profiling data showing that LTB4 production, degradation and downstream signalling in the brainstem of the SHR are dynamically regulated during hypertension. Chronic blockade of the LTB4 receptor 1 (BLT1) receptor with CP-105,696, reduced arterial pressure in the SHR compared to the normotensive control and this reduction was associated with a significant decrease in low and high frequency spectra of systolic blood pressure, and an increase in spontaneous baroreceptor reflex gain (sBRG). These data provide new evidence for the role of LTB4 as an important neuro-immune pathway in the development of hypertension and therefore may serve as a novel therapeutic target for the treatment of neurogenic hypertension.
... Once secreted, LTB 4 can engage BLT1 or BLT2 receptor, whereas cysLTs exert their effect on receptors such as cysLT1, cysLT2 and GPR17 (Haeggstrom and Funk, 2011). Previous studies have reported that LTs along with their cognate receptors are expressed in the brain, notably during HIV-1 infection (Basselin et al., 2011;Froldi et al., 1992;Matsuo et al., 1995). ...
Eicosanoids, including cysteinylleukotrienes (cysLTs), are found in the central nervous system (CNS) of individuals infected with HIV-1. Few studies have addressed the contribution of cysLTs in HIV-1-associated CNS disorders. We demonstrate that conditioned medium from human astrocytes treated with leukotriene C-4 (LTC4) increases the transmigration of HIV-1-infected CD4(+) T cells across an in vitro blood-brain barrier (BBB) model using cultured brain endothelial cells. Additional studies indicate that the higher cell migration is linked with secretion by astrocytes of CX3CL1/fractalkine, a chemokine that has chemoattractant activity for CD4(+) T cells. Moreover, we report that the enhanced cell migration across BBB leads to a more important CD4(+) T cell-mediated HIV-1 transfer toward macrophages. Altogether data presented in the present study reveal the important role that LTC4, a metabolite of arachidonic acid, may play in the HIV-1-induced neuroinvasion, neuropathogenesis and disease progression.
... AA-derived Eicosanoids families are modulated by neuroglia such as microglia and astrocyte. Eicosanoids are produced by microglia [27]. Microglia are a type of glial cell that are the resident macrophages of the brain and spinal cord, and thus act as the first and main form of have active immune defense in the central nervous system. ...
Arachidonic acid (AA)-derived lipid mediators are called eicosanoids. Eicosanoids have
emerged as key regulators of a wide variety of physiological responses and pathological processes,
and control important cellular processes. AA can be converted into biologically active compounds by metabolism by
cyclooxygenases (COX). Beneficial effect of COX-2 inhibitor celecoxib add-on therapy has been reported in early stage
of schizophrenia. Moreover, add-on treatment of celecoxib attenuated refractory depression and bipolar depression.
Further, the COX/prostaglandin E pathway play an important role in synaptic plasticity and may be included in
pathophysiology in autism spectrum disorders (ASD). In this regard, plasma transferrin, which is an iron mediator related
to eicosanoid signaling, may be related to social impairment of ASD. COX-2 is typically induced by inflammatory stimuli
in the majority of tissues, and the only isoform responsible for propagating the inflammatory response. Thus, COX-2
inhibitors considered as the best target for Alzheimer’s disease.
... OA also inhibits gap junction permeability in astrocytes (Lavado et al., 1997) and 2-OHOA could reduce the impact of glia activation on the maintenance of behavioural hypersensitivity after SNI (Wang et al., 2014). Microglia cells are also a major constitutive source of leukotriene B4 in vitro (Matsuo et al., 1995), while in vivo it mediates mechanical hypersensitivity after SNI (Okubo et al., 2010). Eicosatrienoic acid, an OA derivative, is also a known inhibitor of leukotriene B4 synthesis (James et al., 1993). ...
Background
Recently, fatty acids have been shown to modulate sensory function in animal models of neuropathic pain. In this study, the antinociceptive effect of 2-hydroxyoleic acid (2-OHOA) was assessed following spared nerve injury (SNI) with reflex and cerebrally mediated behavioural responses.Methods
Initial antinociceptive behavioural screening of daily administration of 2-OHOA (400 mg/kg, p.o.) was assessed in Wistar rats by measuring hindlimb reflex hypersensitivity to von Frey and thermal plate stimulation up to 7 days after SNI, while its modulatory effect on lumbar spinal dorsal horn microglia reactivity was assessed with OX-42 immunohistochemistry. In vitro the effect of 2-OHOA (120 μM) on cyclooxygenase protein expression (COX-2/COX-1 ratio) in lipopolysaccharide-activated macrophage cells was tested with Western blot analysis. Finally, the effects of 2-OHOA treatment on the place escape aversion paradigm (PEAP) and the open–field-induced anxiety test were tested at 21 days following nerve injury compared with vehicle-treated sham and pregabalin-SNI (30 mg/kg, p.o.) control groups.ResultsOral 2-OHOA significantly reduced ipsilateral mechanical and thermal hypersensitivity up to 7 days after SNI. Additionally 2-OHOA decreased the COX-2/COX-1 ratio in lipopolysaccharide-activated macrophage cells and OX-42 expression within the ipsilateral lumbar spinal dorsal horn 7 days after SNI. 2-OHOA significantly restored inner-zone exploration in the open-field test compared with the vehicle-treated sham group at 21 days after SNI.Conclusions
Oral administration of the modified omega 9 fatty acid, 2-OHOA, mediates antinociception and prevents open–field-induced anxiety in the SNI model in Wistar rats, which is mediated by an inhibition of spinal dorsal horn microglia activation.
... Once secreted, LTB 4 can engage BLT1 or BLT2 receptor, whereas cysLTs exert their effect on receptors such as cysLT1, cysLT2 and GPR17 (Haeggstrom and Funk, 2011). Previous studies have reported that LTs along with their cognate receptors are expressed in the brain, notably during HIV-1 infection (Basselin et al., 2011;Froldi et al., 1992;Matsuo et al., 1995). ...
Eicosanoids, including cysteinylleukotrienes (cysLTs), are found in the central nervous system (CNS) of individuals infected with HIV-1. Few studies have addressed the contribution of cysLTs in HIV-1-associated CNS disorders. We demonstrate that conditioned medium from human astrocytes treated with leukotriene C4 (LTC4) increases the transmigration of HIV-1-infected CD4+ T cells across an in vitro blood–brain barrier (BBB) model using cultured brain endothelial cells. Additional studies indicate that the higher cell migration is linked with secretion by astrocytes of CX3CL1/fractalkine, a chemokine that has chemoattractant activity for CD4+ T cells. Moreover, we report that the enhanced cell migration across BBB leads to a more important CD4+ T cell-mediated HIV-1 transfer toward macrophages. Altogether data presented in the present study reveal the important role that LTC4, a metabolite of arachidonic acid, may play in the HIV-1-induced neuroinvasion, neuropathogenesis and disease progression.
... Hypertrophied astrocytes and reactive microglia constitute the major inducers and sustainers of neuroinflammation involved in the kainic acid induced neurotoxicity. Previous studies on ischemia models indicate that activated microglia can also release leukotrienes (LTC4 and LTD4) (Matsuo et al., 1995). Moreover, the CysLT 1 receptor mRNA has been found to be up regulated in neurons, marcophages and proliferated astrocytes (Ciccarelli et al., 2004;Fang et al., 2006), suggesting a possible regulatory role in the mediation of different phases of neuro-inflammation. ...
There is an evolving consensus that mild cognitive impairment (MCI) serves as a prodrome to Alzheimer's disease. Antioxidants and COX-2 (cyclo-oxygenase-2) inhibitors have also been reported to have beneficial effects against conditions of memory impairment. Newer drugs like cysteinyl leukotriene inhibitors have shown neuroprotective effect in animal models of ischemia. Thus, the present study purports to explore the potential role of montelukast (a cysteinyl leukotriene inhibitor) in concert with rofecoxib (COX-2 inhibitor) and caffeic acid (a 5-LOX inhibitor and potent antioxidant) against kainic acid induced cognitive dysfunction in rats. In the experimental protocol, kainic acid (0.4μg/2μl) in artificial cerebrospinal fluid (ACSF) was given intrahippocampally (CA3 region) to induce a condition similar to MCI. Memory performance was measured on days 10-14 and the locomotor activity was measured on days 1, 7 and 14. For estimation of biochemical, mitochondrial and histopathological parameters, animals were sacrificed on day 14, stored at -80°C and the estimation was done on the 15th day. The treatment groups consisting of montelukast (0.5 and 1mg/kg), rofecoxib (5 and 10mg/kg) and caffeic acid (5 and 10mg/kg) showed significant improvement in memory performance, oxidative stress parameters and mitochondrial function as compared to that of control (kainic acid treated), however, combination of montelukast with rofecoxib showed significant improvement in their protective effect. Thus the present study emphasizes the positive modulation of cysteinyl leukotriene receptor inhibition on COX (cyclooxygenase) and LOX (lipoxygenase) pathways in the control of the neuroinflammation in kainic acid induced cognitive dysfunction in rats.
... Although microglial activation is the brain's major defense against immune challenge, it has been widely described that activated microglia may contribute to neurodegeneration through the release of proinflammatory and/or cytotoxic factors as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), nitric oxide (NO) and reactive oxygen intermediates among others (Chao et al., 1992;Dickson et al., 1993;Lee et al., 1993;Brosnan et al., 1994;Matsuo et al., 1995;Espey et al., 1997;Minghetti and Levi, 1998). However, all these factors are necessary for normal function of microglia. ...
To elucidate whether density of cells could contribute to the extent of microglial activation, we performed in vitro assays using three different densities of N13 microglia stimulated with LPS. Our results showed that induction of pro-inflammatory factors as TNF-α and iNOS was directly related to cell density, meanwhile the induction of the anti-inflammatory IL-10 was inversely related to cell density. Accordingly, in vivo assays showed that after LPS-injection, iNOS expression was more intense in substantia nigra, a brain area showing specific susceptibility to neurodegeneration after microglia activation, whereas IL-10 expression was more sustained in striatum, an area resistant to damage. These results support that microglia density is pivotal to control the balance between pro- and anti-inflammatory factors release.
... In vitro, the synthesis of 5-LOX metabolites of arachidonic acid has been demonstrated in microglia (Matsuo et al., 1995) and neuronal precursors (Wada et al., 2006). The CNS is capable of producing 5-LOX metabolites (Chinnici et al., 2007;Hynes et al., 1991), possibly via a transcellular synthesis of cysteinyl leukotrienes in neurons and glia (Farias et al., 2007). ...
Typically, cyclooxygenases (COXs) and 5-lipoxygenase (5-LOX), enzymes that generate biologically active lipid molecules termed eicosanoids, are considered inflammatory. Hence, their putative role in Alzheimer's disease (AD) has been explored in the framework of possible inflammatory mechanisms of AD pathobiology. More recent data indicate that these enzymes and the biologically active lipid molecules they generate could influence the functioning of the central nervous system and the pathobiology of neurodegenerative disorders such as AD via mechanisms different from classical inflammation. These mechanisms include the cell-specific localization of COXs and 5-LOX in the brain, the type of lipid molecules generated by the activity of these enzymes, the type and the localization of receptors selective for a type of lipid molecule, and the putative interactions of the COXs and 5-LOX pathways with intracellular components relevant for AD such as the gamma-secretase complex. Considering the importance of these multiple and not necessarily inflammatory mechanisms may help us delineate the exact nature of the involvement of the brain COXs and 5-LOX in AD and would reinvigorate the search for novel targets for AD therapy.
... Interestingly, particularly high levels of neuronal 5-LO expression and LTB 4 content have been identified in CNS upon challenge with a variety of stimuli [27,28]. Our previous study found that antigen challenge induces the expression of 5-LO and LTA 4 H mRNA and LTB 4 levels in brain after antigen challenge in sensitized rats. ...
Basic and clinical studies suggest that hypothalamic-pituitary-adrenal (HPA) axis is the neuroendocrine-immune pathway that functionally regulates the chronic inflammatory disease including asthma. Our previous studies showed corresponding changes of cytokines and leukotriene B4 (LTB4) between brain and lung tissues in antigen-challenged asthmatic rats. Here, we investigated how the increased LTB4 level in brain interacts with HPA axis in regulating antigen-induced asthmatic response in sensitized rats.
Ovalbumin-sensitized rats were challenged by inhalation of antigen. Rats received vehicle, LTB4 or U75302 (a selective LTB4 BLT1 receptor inhibitor) was given via intracerebroventricular injection (i.c.v) 30 min before challenge. Lung resistance (RL) and dynamic lung compliance (Cdyn) were measured before and after antigen challenge. Inflammatory response in lung tissue was assessed 24 h after challenge. Expression of CRH mRNA and protein in hypothalamus were evaluated by RT-PCR and Western Blot, and plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were measured using the ELISA kits.
Antigen challenge decreased pulmonary function and induced airway inflammation, evoked HPA axis response in sensitized rats. Administration of LTB4 via i.c.v markedly attenuated airway contraction and inflammation. Meanwhile, LTB4 via i.c.v markedly increased CORT and ACTH level in plasma before antigen challenge, and followed by further increases in CORT and ACTH levels in plasma after antigen challenge in sensitized rats. Expression of CRH mRNA and protein in hypothalamus were also significantly increased by LTB4 via i.c.v in sensitized rats after antigen challenge. These effect were completely blocked by pre-treatment with BLT1 receptor antagonist U75302 (10 ng), but not by BLT2 antagonist LY255283.
LTB4 administered via i.c.v down-regulates the airway contraction response and inflammation through activation of the HPA axis via its BLT1 receptor. This study expands our concept of the regulatory role of intracranial inflammatory mediators in inflammatory diseases including asthma. The favourable effects of LTB4 on the HPA axis may help to explain the phenomenon of self-relief after an asthmatic attack.
... benachbarten Neuronen und zu anderen mikroglialen Zellen, (Streit et al., 1987; Sudo et al., 1998). Sie exprimieren auch zahlreiche verschiedene Integrine, wie z.B. α4β1, α5β1, α6β1, und αMβ2 (Kloss et al., 1999Kloss et al., , 2001 et al., 2002), Thrombin an Orten der lokalen Koagulation sowie durch lokale Synthese in geschädigtem Gewebe (Citron et al., 2000) und PGD2, das eigentliche im ZNS vorkommende Prostaglandin (Siren, 1982), hauptsächlich von aktivierten mikroglialen Zellen (Matsuo et al., 1995; Minghetti und Levi, 1995). Interessanterweise schien keines dieser Moleküle einen starken, die Ramifikation inhibierenden Effekt in der Kokultur zu haben, obgleich sie andere Parameter der mikroglialen Aktivierung beeinflussten (Walz et al., 1993; Norenberg et al., 1997; Möller et al., 2000). ...
Morphologic changes of the Mikroglia are an important accompaniment in nearly all forms of brain pathology. In the existing work, the molecular signals were examined, that these morphological changes in vivo, in the facial motor nucleus model of the mouse, and in vitro, in the microglia-astrocyte coculture drive. In vivo MCSF deficiency led to a failure of microglia to show a normal increase in early activation markers, to spread on the surface of axotomized motoneurons, and to proliferate after injury. Transgenic deletion of IL6 caused a massive decrease in the early microglial activation during the first 4 days after injury. In vitro the addition of brain cell membranes and their phagocytosis led to a rapid and reversible loss of ramification. The addition of agents causing intracellular elevation of calcium and cAMP or inhibition of Gi-proteins and phosphatases led to a rapid loss of microglial branching. Signaling cascades controlled by these molecules may play an important role in the regulation of this common physiological process in the injured brain.
... Interestingly, the substantia nigra (SN) exhibits the highest concentration of microglia in the brain (4,5) and contains exceptionally active microglia in the brains of patients with PD (6,7). Although microglial activation is the brainÕs major defense against immune challenges, the activation of microglia was believed to contribute to neurodegenerative processes through the release of pro-inflammatory and ⁄ or cytotoxic factors, including IL-1b, TNF-a, nitric oxide (NO), reactive oxygen intermediates, arachidonic acid metabolites and quinolinic acid (8)(9)(10)(11)(12)(13)(14)(15). ...
Interleukin (IL)-12 is a heterodimeric cytokine produced by activated blood monocytes, macrophages and glial cells. It enhances differentiation and proliferation of T cells and increases production of proinflammatory cytokines. IL-10 is a pleiotropic cytokine produced by both lymphocytes and mononuclear phagocytes including microglia. Recent studies demonstrated the neuroprotective effect of IL-10. There is little information about the involvement of IL-12 or IL-10 in the pathophysiology of Parkinson's disease (PD).
The objective of our study was to assess the role of IL-12 as a potential marker of immune reactions in patients with PD and to investigate whether IL-10, an immunosuppressive cytokine, may have a neuroprotective effect in the pathogenesis of PD.
We measured using immunoassay serum IL-12 and IL-10 levels in 41 patients with PD in comparison with serum levels in 19 healthy subjects (controls) age and sex matched. IL-12 and IL-10 levels were tested for correlation with sex, age, disease duration, Hoehn and Yahr stage and the UPDRS III score.
The PD group presented with significantly increased IL-10 levels when compared with the control group (P = 0.02). The increase observed was not affected by the treatment status. A strong and significant correlation between IL-10 and IL-12 levels was observed in patients with PD (R(S) = 0.7, P < 0.000001).
Our findings suggest that IL-10 may be involved in the pathogenetic mechanisms of PD. The elevation of IL-10 and the significant correlation between IL-10 and IL-12, a proinflammatory cytokine, may suggest that immunological disturbances and neuroprotective mechanisms are involved in patients with PD.
... Astrocytes have always been considered a significant source of arachidonic derivatives in brain. Recent evidence, however, suggests that some products may be from contaminating microglia [164]. ...
Cultured brain cells are capable of generating many molecules associated with inflammatory and immune functions. They constitute the endogenous immune response system of brain. They include complement proteins and their regulators, inflammatory cytokines, acute phase reactants and many proteases and protease inhibitors. Most of the proteins are made by microglia and astrocytes, but even neurons are producers. Many appear in association with Alzheimer disease lesions, indicating a state of chronic inflammation in Alzheimer disease brain. Such a state can apparently exist without stimulation by peripheral inflammatory mediators or the peripheral immune system. A strong inflammatory response may be autotoxic to neurons, exacerbating the fundamental pathology in Alzheimer disease and perhaps other neurological disorders. Autotoxic processes may contribute to cellular death in chronic inflammatory diseases affecting other parts of the body, suggesting the general therapeutic value of anti-inflammatory agents. With respect to Alzheimer disease, multiple epidemiological studies indicate that patients taking anti-inflammatory drugs or suffering from conditions in which such drugs are routinely used, have a decreased risk of developing Alzheimer disease. In one very preliminary clinical trial, the anti-inflammatory drug indomethacin arrested progress of the disease. New agents directed against the inflammatory processes revealed in studies of Alzheimer disease lesions may have broad therapeutic applications.
... In addition to progesterone, apoD can bind pregnenolone (Pearlman et al., 1973;Dilley et al., 1990) and bilirubin (Peitsch and Boguski, 1989). ApoD has also been shown to bind with high affinity arachidonic acid (Cabral et al., 1995), a precursor to prostaglandins, which are specific modulators in the brain (Matsuo et al., 1995). Whether any or all of these molecules represent physiological ligands of apoD remains to be proven. ...
Apolipoprotein D (apoD) is a member of the lipocalin family of proteins. Most members of this family are transporters of small hydrophobic ligands, although in the case of apoD, neither its physiological function(s) nor its putative ligand(s) have been unequivocally identified. In humans, apoD is expressed in several tissues, including the CNS, and its synthesis is greatly increased during regeneration of rat peripheral nerves. As apoD may have an important function in the nervous system and, particularly, in nerve regeneration, we measured immunoreactive apoD levels in the hippocampus and in CSF of patients with either Alzheimer's disease (AD) or other neuropathologies. In parallel, we determined the concentrations of apolipoprotein E (apoE), another apolipoprotein also implicated in nerve regeneration and in the etiology of AD. Levels of apoD but not apoE were increased in the hippocampus of AD patients compared with controls. ApoD concentrations, as determined by radioimmunoassay, were significantly increased in the CSF of AD patients (4.23 +/- 1.58 microg/ml) and patients with other pathologies (3.29 +/- 1.35 microg/ml) compared with those in the CSF of normal subjects (1.15 +/- 0.71 microg/ml). Although the differences were smaller than for apoD, the mean apoE concentrations in the CSF of both groups of patients were also significantly higher than those of controls. In AD patients, apoD, but not apoE, levels in CSF and hippocampus increased as a function of inheritance of the epsilon4 apoE allele. This study therefore demonstrates that increased apoD levels in the hippocampus and in CSF are a marker of neuropathology, including that associated with AD, and are independent of apoE concentrations.
... Little is known, however, where in the brain such activation of the arachidonic acid cascade takes place during the cerebral inflammation. Although there are several cell groups potentially to be the source of PGs, including neurons (Fujimoto et al., 1992;Van Dam et al., 1993), astrocytes (Katsuura et al., 1989;O'Banion et al., 1996), microglia (Matsuo et al., 1995;Minghetti and Levi, 1995;Fiebich et al., 1996;Bauer et al., 1997;Slepko et al., 1997), and endothelial cells (Moore et al., 1988;Van Dam et al., 1993;de Vries et al., 1995), it has not been clarified yet which of these cell groups plays the major role in the production of PGs during cerebral inflammation in vivo. ...
Activation of the arachidonic acid cascade is an essential step for the development of fever during brain inflammation. We investigated the brain sites where this activation takes place by use of a rat model of brain inflammation. Intracerebroventricular administration of lipopolysaccharide but not of its vehicle evoked fever. The fever was markedly suppressed when the rats had been pretreated with a cyclooxygenase-2-specific inhibitor. In situ hybridization and immunohistochemical studies revealed that cyclooxygenase-2 mRNA and its protein were induced by lipopolysaccharide in blood vessels near the cerebral ventricles and in those in the subarachnoidal space. Double immunohistochemical staining revealed that these cyclooxygenase-2-positive cells were mostly endothelial cells. The time course of fever and that of cyclooxygenase-2 induction in the endothelial cells were in parallel. Cyclooxygenase-2 mRNA in a certain type of telencephalic neurons was also upregulated by the intracerebroventricular administration, but this neuronal response occurred both in vehicle-injected rats and in lipopolysaccharide-injected ones to the same extent. Therefore, the neuronal response was not essential to the development of fever. These results suggest that brain endothelial cells play a crucial role in the development of fever during brain inflammation by activating their arachidonic acid cascade.
... 13 Activated macrophages/microglia have been demonstrated to release in¯ammatory cytokines, 14 quinolinic acid, 15 superoxide anion 16 (which will release iron from ferritin 17 ), nitric oxide and pro-in¯ammatory eicosanoids. 18,19 The active MS lesion is characterized by the presence of in¯ammatory cytokines and the release of toxic chemicals such as free radicals and strong oxidants. Several studies 20,21 have shown that nitric oxide synthase is induced in macrophages/microglia of the active MS lesion. ...
There are still questions regarding whether macrophages found in MS lesions are agents of recovery or of destruction. To address this, we examined in aggregate cultures prepared from dissociated embryonic spinal cord tissue, with or without addition of exogenous macrophages, the effect of menadione-induced oxidative stress. Similar to findings of other laboratories, we observed that in the absence of oxidative stress macrophage enrichment promoted myelinogenesis. In macrophage-poor cultures, menadione at 5 microM had very little effect upon the status of the aggregate cultures; however, increasing this to 10 and 20 microM did result in some damage to axons and myelin. By contrast, in macrophage enriched cultures, menadione at a concentration as little as 5 microM caused the complete destruction of the aggregates. We suggest that in neural tissues that have sufficiently high macrophage numbers, oxidative stress results in a positive inflammatory feedback loop that results in massive tissue destruction. We further suggest that what we see in macrophage-enriched aggregates subjected to oxidative stress may represent what happens in the Marburg-type of MS lesion.
... Activation of microglia is believed to contribute to neurodegenerative processes through the release of proinflammatory and/or cytotoxic factors, including interleukin-1 (IL-1), tumor necrosis factor-␣ (TNF␣), nitric oxide (NO), reactive oxygen intermediates, arachidonic acid metabolites, and quinolinic acid (Chao et al., 1992;Dickson et al., 1993;Lee et al., 1993;Brosnan et al., 1994;Matsuo et al., 1995;Minghetti and Levi, 1995;Espey et al., 1997). Production of these factors by microglia after exposure to lipopolysaccharide (LPS), the human immunodeficiency virus-1 coat protein gp120, or -amyloid has been well documented (Boje and Arora, 1992;Chao et al., 1992;Dawson et al., 1994;Ii et al., 1996;Kong et al., 1996). ...
Inflammation in the brain has been increasingly associated with the development of a number of neurological diseases. The hallmark of neuroinflammation is the activation of microglia, the resident brain immune cells. Injection of bacterial endotoxin lipopolysaccharide (LPS) into the hippocampus, cortex, or substantia nigra of adult rats produced neurodegeneration only in the substantia nigra. Although LPS appeared to impact upon mesencephalic neurons in general, an extensive loss of dopaminergic neurons was observed. Analysis of the abundance of microglia revealed that the substantia nigra had the highest density of microglia. When mixed neuron-glia cultures derived from the rat hippocampus, cortex, or mesencephalon were treated with LPS, mesencephalic cultures became sensitive to LPS at a concentration as low as 10 ng/ml and responded in a dose-dependent manner with the production of inflammatory factors and a loss of dopaminergic and other neurons. In contrast, hippocampal or cortical cultures remained insensitive to LPS treatment at concentrations as high as 10 microg/ml. Consistent with in vivo observations, mesencephalic cultures had fourfold to eightfold more microglia than cultures from other regions. The positive correlation between abundance of microglia and sensitivity to LPS-induced neurotoxicity was further supported by the observation that supplementation with enriched microglia derived from mesencephalon or cortex rendered LPS-insensitive cortical neuron-glia cultures sensitive to LPS-induced neurotoxicity. These data indicate that the region-specific differential susceptibility of neurons to LPS is attributable to differences in the number of microglia present within the system and may reflect levels of inflammation-related factors produced by these cells.
... It is likely that i-cysteinylLTs are synthesized by resident brain cells, although the precise source has not yet been fully clari®ed. Anterior pituitary cells and astroglial cells in culture are able to form cysteinyl-LTs (Hartung & Toyka, 1987;Kiesel et al., 1991;Petroni et al., 1991;Seregi et al., 1990) and more recently, it has been shown that puri®ed microglial cells are mainly involved, rather than astrocytes ( Matsuo et al., 1995). On the other hand, the release of glutamate following pMCAo peaks much earlier (about 2 h post ischaemia induction) ( Matsumoto et al., 1992;Melani et al., 1999) and thus it could be an up-stream event with respect to cysteinyl-LT formation. ...
The aim of this work was to evaluate the role of leukotrienes in brain damage in vivo in a model of focal cerebral ischaemia in the rat, obtained by permanent occlusion of middle cerebral artery.
A significant (P<0.01) elevation of LTC4, LTD4 and LTE4 (cysteinyl-leukotrienes) levels occurred 4 h after ischaemia induction in the ipsilateral cortices of ischaemic compared to sham-operated animals (3998±475 and 897±170 fmol g−1 tissue, respectively, P<0.01).
The NMDA receptor antagonist MK-801 and the adenosine A2A receptor antagonist SCH 58261 were administered in vivo at doses known to reduce infarct size and compared with the leukotriene biosynthesis inhibitor MK-886.
MK-886 (0.3 and 2 mg kg−1 i.v.) and MK-801 (3 mg kg−1 i.p.) decreased cysteinyl-leukotriene levels (−78%, P<0.05; −100%, P<0.01; −92%, P<0.01, respectively) 4 h after permanent occlusion of the middle cerebral artery, whereas SCH 58261 (0.01 mg kg−1 i.v.) had no significant effects.
MK-886 (2 mg kg−1 i.v.) was also able to significantly reduce the cortical infarct size by 30% (P<0.05).
We conclude that cysteinyl-leukotriene formation is associated with NMDA receptor activation, and that it represents a neurotoxic event, the inhibition of which is able to reduce brain infarct area in a focal ischaemic event.
British Journal of Pharmacology (2001) 133, 1323–1329; doi:10.1038/sj.bjp.0704189
The pioneering efforts of a number of key laboratories have led to our current understanding of the pivotal roles for microglia in neuroinflammation and the potential neurotoxic sequalae of such events. This work has generated a rigorous data base that indicates that previous concepts that the brain is an “immunologically privileged” organ are not valid (1). As our knowledge base continues to expand in this area, the potential for defining pharmacological points of intervention in the neuroinflammatory cascade, worthy of clinical evaluation in neurodegenerative disorders, becomes more well grounded. It is the purpose of this chapter to review the basic biochemical characteristics of microglia, to define their potential neurotoxic mediators, and to review some of the in vivo and in vitro pre-clinical models that have been used to study microglial activation. A number of microglial mediators, enzymes, and receptors will be presented throughout this chapter. As an aid to the reader, a summary table of this microglial biochemistry is presented in Appendix 1.
In 1984, when our laboratory first began conducting research on inflammation and Alzheimer’s disease (AD) (1), the brain was still widely considered to be immunologically privileged (reviewed in ref 2). Now, a virtual textbook of inflammatory mediators have been shown to be present in the central nervous system (CNS) or expressed by CNS cells in culture (Table 1). Most of these molecules are known to be increased in expression in AD limbic and neocortex compared to similar samples from nondemented elderly (ND) patients. By understanding the functions of inflammatory mediators in general, and their interactions with AD pathology in particular, a better appreciation of their pathogenic potential may be fostered.
Besides their roles in normal brain function microglia are important effector cells in a variety of pathological conditions, and due to their reactivity to a wide range of stimuli, are believed to foster neuroprotective and repair processes and thus to play a crucial role in host defence. On the other hand, depending on the type or intensity of the noxious stimulus and on the concurrence of other local factors, they may contribute to the establishment or amplification of tissue damage (1, 2). It is therefore important to understand what are the conditions regulating the balance between neuroprotective and neurotoxic activities of these cells.
This article is a revision of the previous edition article by P Morell and A D Toews, Volume 11, pp 201–216, © 1997, Elsevier Ltd.
Cyclooxygenase-2 (COX-2) is an inducible type of enzyme that is involved in prostaglandin biosynthesis. In the present study, we examined whether or not COX-2 is involved in fever that is induced by tumor necrosis factor-alpha (TNF-α) and, if so, where in the brain COX-2 is induced by this factor. Intraperitoneal (i.p.) injection of TNF-α into rats evoked a fever that started 1 h after the TNF injection, peaked 3 h after the injection, and then gradually declined. The fever was suppressed by pretreatment with a COX-2-specific inhibitor. With a time course similar to that of fever, COX-2 mRNA was induced in brain blood vessels. On the other hand, in some of the telencephalic neurons, COX-2 mRNA was constitutively expressed under the normal condition; but its level gradually decreased during the course of fever. Fever was also evoked by an intracerebroventricular (i.c.v.) injection of TNF-α. This febrile response was also suppressed by a COX-2 specific inhibitor and was associated with the induction of COX-2 mRNA in the brain blood vessels. On the other hand, the telencephalic neurons did not show consistent change in COX-2 mRNA level after i.c.v. injection of TNF-α or saline. COX-2-like immunoreactivity was found in some cells of the brain blood vessels 3 h after the TNF-α injection by either i.p. or i.c.v. route. Most of the COX-2-like immunoreactive cells were endothelial cells since COX-2-like immunoreactivity was colocalized with von Willebrand factor, an endothelial cell marker, in the same cells. These results suggest that the brain blood vessels are the major sites where TNF-α enhances PG biosynthesis after peripheral as well as after central injection, and provides further evidence supporting the hypothesis that COX-2 induced in the brain blood vessels is involved in fever.
Acute inflammation following spinal cord injury results in secondary injury and pathological reorganisation of the central nervous system (CNS) architecture. Cyclooxygenases (Prostaglandin Endoperoxide H Synthases, PGH) are key enzymes in the conversion of arachidonic acid into prostanoids which mediate immunomodulation, mitogenesis, apoptosis, blood flow, secondary injury (lipid peroxygenation) and inflammation. Here, we report cyclooxygenase-1 (COX-1) expression following spinal cord injury. In control spinal cords, COX-1 expression was localized by immunohistochemistry to ependymal cells, some neurons, inclusive dorsal and ventral root ganglion cells, few endothelial cells but rarely to brain microglia/macrophages. In injured spinal cords, COX-1+ microglia/macrophages accumulated highly significantly (P
Reported is a case of hemorrhagic shock and encephalopathy syndrome (HSE) with extensive white matter involvement. A three year old, previously healthy boy was presented with an acute onset of fever, loss of consciousness and convulsions. He had disseminated intravascular coagulation, metabolic acidosis, non-ketotic hypoglycemia and hepatorenal dysfunction. The computed tomography (CT) scan of his head on the second day of illness demonstrated symmetric, extensive low-density areas in the cerebral and cerebellar white matter. The child died on the 13th hospital day. A post-mortem histopathological examination of the liver revealed centrilobular necrosis and infiltration of fatty acid droplets. The concentrations of serum 2′,5′-oligoadenylate synthetase and urinary neopterin were markedly elevated, indicating excessively activated cell-mediated immunity. This overproduction of inflammatory cytokines might play an important role in the pathogenesis of the brain lesion as well as in other clinical and laboratory manifestations. The patient had a decreased serum level of 1-antitrypsin, which may have been associated with the development of uncontrolled inflammation and coagulation disorder.
Lipoxin A(4) (LXA(4)), a biologically active eicosanoid with anti-inflammatory and pro-resolution properties, was recently found to have neuroprotective effects in brain ischemia. As 5-lipoxygenase (5-LOX) and leukotrienes are generally considered to aggravate cerebral ischemia/reperfusion (I/R) injury, we investigated their effects on LXA(4)-mediated neuroprotection by studying middle cerebral artery occlusion (MCAO)/reperfusion in rats and oxygen-glucose deprivation (OGD)/recovery in neonatal rat astrocyte primary cultures. LXA(4) effectively reduced infarct volumes and brain edema, and improved neurological scores in the MCAO/reperfusion experiments; this effect was partially blocked by butoxycarbonyl-Phe-Leu-Phe-Leu-Phe (Boc2), a specific antagonist of the LXA(4) receptor (ALXR). Total 5-LOX expression did not change, regardless of treatment, but LXA(4) could inhibit nuclear translocation induced by MCAO or OGD. We also found that LXA(4) inhibits the upregulation of both leukotriene B(4) (LTB(4)) and leukotriene C(4) (LTC(4)) and the phosphorylation of extracellular signal-regulated kinase (ERK) induced by MCAO or OGD. The phosphorylation of the 38-kDa protein kinase (p38) and c-Jun N-terminal kinase (JNK) was not altered throughout the experiment. These results suggest that the neuroprotective effects of LXA(4) are probably achieved by anti-inflammatory mechanisms that are partly mediated by ALXR and through an ERK signal transduction pathway.
Inflammation has been increasingly recognized to play an important role in the pathogenesis of Parkinson's disease (PD). Using immunocytochemistry and electron microscopy, we found that intranigral injection of lipopolysaccharide (LPS) caused marked microglial activation and a dose-dependent selective loss of dopaminergic neurons, which was mediated by apoptosis as evidenced by prominent TUNEL labeling. RNase protection assays revealed that mRNA for Bax, Fas and the pro-inflammatory cytokines interleukin (IL)-1α, IL-1β, IL-6 and tumor necrosis factor (TNF)-α were significant increased ipsilaterally in LPS-injected side of SN, while expression of the anti-apoptotic gene Bcl-2 was decreased. Osmotic pump infusion of IL-10, a global inhibitor of cytokine synthesis, protected against LPS-induced cell death of dopaminergic neurons, with a corresponding decrease in the number of activated microglia, suggesting that the reduction in microglia-mediated release of inflammatory mediators may contribute to the anti-inflammatory effect of IL-10. Our results provide evidence that LPS induces apoptotic cell death in SNpc, which is likely through the expression of Fas, Bax, caspase-3, and the pro-inflammatory cytokines.
Current theories state that Alzheimer's disease (AD) is a vascular disorder that initiates its pathology through cerebral microvascular abnormalities. Endothelial dysfunction caused by the injury or death of endothelial cells contributes to progression of AD. Also, functional relationships between neurons, glial cells, and vascular cells within so-called neurovascular unit are dramatically compromised in AD. Several recent studies have highlighted that endothelial cells might be the target for the toxic action of heavily aggregated proteins, glia-derived cytokines, and stimuli inducing oxidative and metabolic stress in AD brains. Here, we describe the properties of the brain endothelium that contribute to its specific functions in the central nervous system, and how endothelial-neuronal-glial cell interactions are compromised in the pathogenesis of AD. We also discuss the ways in which functioning of endothelial cells can be modulated in cerebral microvessels. Understanding of molecular mechanisms of endothelial injury and repair in AD would give us novel diagnostic biomarkers and pharmacological targets.
Concentrations of immunoreactive leukotriene C4 (LTC4) and leukotriene B4 (LTB4) in the cerebrospinal fluid from 18 patients with aseptic meningitis, including 2 patients with encephalitis and 4 patients with febrile seizures, were measured by a sensitive and specific radioimmunoassay; these results were compared with those from control subjects. The concentrations of both LTC4 and LTB4 were elevated significantly in patients with meningitis (LTC4: 115.6 +/- 47.7 pg/ml; LTB4: 1,603.0 +/- 273.5 pg/ml; n = 18) compared to controls (LTC4: 83.2 +/- 21.6 pg/ml; LTB4: 1,219.3 +/- 161.5 pg/ml; n = 12; P < .05 and P < .01, respectively). However, there was no significant increase in LT levels in patients with febrile seizures. These findings suggest that LTs may play an important role in the inflammatory response induced by viral infections of the central nervous system.
In order to elucidate the mechanism(s) of neuronal protection by hypothermia against ischemic damage, we examined the effect of lowering temperature on the microglial activation that is thought to cause the development of ischemia-induced neuronal damages. Cultured microglia from neonatal rats were measured for microglial activation by the following indices: production of superoxide and nitric oxide by the methods of acetyl-cytochrome c reduction and nitrite accumulation in the culture medium, respectively, and cell proliferation evaluated by [3H]thymidine uptake. At 30 degrees C, superoxide production induced by phorbol ester was approximately as low as 30% of the control at 37 degrees C, and nitric oxide production after addition of lipopolysaccharide was decreased to approximately 25% of the control. The time course of nitric oxide production indicates that the induction of nitric oxide synthase seemed to be significantly suppressed by lowering temperature. In addition, the proliferation of microglia was remarkably inhibited at 30 degrees C. The level of proliferation in the hypothermic condition is much lower in microglia (14% of the control) than those in astrocytes cultured from brain cortices (96%) and fibroblasts cultured from brain meninges (53%), suggesting that the microglial activation is highly susceptible to lowering temperature. The present study indicates that hypothermia potently inhibits proliferation, superoxide and nitric oxide production of cultured microglia and that the hypothermic protection against postischemic neuronal damage might be, at least in part, due to the suppression of microglial activation.
Although various methods can be used for detection of arachidonic acid (AA) metabolites, only few of them allow rapid and precise analysis of both cyclooxygenase and lipoxygenase metabolites in a single assay. We have developed a simple and rapid method to evaluate overall profiles of AA metabolites produced by primary cultured glial cells from newborn rats by using reversed-phase, high-performance liquid chromatography (RP-HPLC) and on-line radioisotope detection. © 1997 Elsevier Science B.V. All rights reserved.
Microglial activation and disruption of blood-brain barrier (BBB) are known to occur and contribute to neuronal damages in cerebral ischemic conditions and in some neurodegenerative diseases. To investigate whether a serum factor leaked out from circulation enhances microglial activation, we examined the effect of normal rat serum on superoxide (O2-) production by cultured microglia. Microglia cultured from neonatal rat brains were studied on their O2- production induced by the addition of phorbol myristate acetate by a method of acetyl-cytochrome c reduction. The O2- production was significantly increased by the addition of 0.01% rat serum, and the maximal enhancement was observed at about 0.1% rat serum. After the serum was fractionated using a molecular sieve membrane, we observed the enhancing effect only in a greater molecular weight fraction (>50 kDa). Furthermore, three kinds of bovine serum albumin (BSA) with different purity, and human serum and plasma albumins, also enhanced O2- production to a similar extent to that by rat serum. However, other proteins tested showed no significant effect. The enhancement of O2- production by BSA was observed dose-dependently, and the effect of 50 microg/ml of purified BSA was equivalent to that of 0.1% rat serum, suggesting that albumin itself enhances O2- production by microglia. These results imply that albumin leaked out through impaired BBB may activate microglia and that the potentiation of O2- production by albumin results in the pathogenesis of neuronal damage in cerebral ischemia and some neurodegenerative diseases.
Microglial cells are believed to play an active role in brain inflammatory, immune and degenerative processes. Depending on the magnitude of microglial reaction, on the type of stimulus and on the concurrence of other local factors, microglia can contribute to host defence and repair, or to the establishment and maintenance of brain damage. Many of the effects of microglial cells can be ascribed to the numerous substances that these cells can synthesize and release in response to a variety of stimuli (cytokines, pro-inflammatory substances, neurotransmitters, toxins, etc.). The present article deals with two classes of compounds that activated microglial cells can produce in large amounts: prostanoids (that derive from arachidonic acid through the cyclooxygenase pathway), and nitric oxide (that is synthesized from arginine by nitric oxide synthase). Prostanoids and nitric oxide have a number of common targets, on which they may exert similar or opposite actions, and have a crucial role in the regulation of inflammation, immune responses and cell viability. Their synthesis can massively increase when the inducible isoforms of cyclooxygenase and nitric oxide synthase are expressed. The metabolic pathways of prostanoids and nitric oxide are finely tuned by the respective end-products, by cyclic AMP and by a number of exogenous factors, such as cytokines, glucocorticoids, lipocortin-1 and others. Some of these factors (e.g. transforming growth factor-beta 1, interleukin-10, lipocortin-1) may be secreted by microglial cells themselves, and act in an autocrine-paracrine way. In view of the neuroprotective role attributed to some prostaglandins and to the cytotoxicity of excessive levels of nitric oxide or its derivatives, the balance between prostanoid and nitric oxide levels may be crucial for orienting microglial reactions towards neuroprotection or neurotoxicity.
In recent years, it has become apparent that astrocytes (at least in vitro) harbor functional receptors to almost all possible neurotransmitters (with the potential noticeable exception of acetylcholine nicotinic receptors). Peptides are no exception, since receptors to all neuropeptides known to be produced in the CNS have been found on cultured astrocytes, and the presence of many of these has been confirmed on astrocytes in vivo. A variety of methodologies have been used to detect peptide receptors on astrocytes, as summarized in the current review. Special emphasis is also put on the possible roles that peptides may play in the regulation of astrocyte functions. These include proliferation, morphology, release of eicosanoids and arachidonic acid, induction of calcium transients and calcium waves, and control of internal pH, glucose uptake, glycogen metabolism, and gap junctional conductance. Recent data concerning the effects of natriuretic peptides on astrocytes are reviewed, and why these peptides may constitute priviledged tools to test the effects of peptides on astrocyte-neuron interactions is also discussed.
In neuropathological conditions such as Alzheimer's disease, Parkinson's disease, AIDS dementia complex and multiple selerosis, activation of microglial cells and astroglial cells is evident. Under these neuropathological conditions cellular damage in the brain is considered to arise indirectly from cytotoxic substances produced by activated glial cells. One of these toxins is NO which has been demonstrated to be produced during several neuropathological conditions. High NO levels are produced by glial cells and exert neurotoxic effects. Astroglial cells and microglial cells communicate in various ways to reduce NO production by microglial cells which is essential to maintain homeostasis in the brain. The production of TGFβ by glial cells and its activation by astrocyte-derived tPA represents one mechanism by which astroglia limit NO production in the brain.
In anaesthetized rats, the intraspinal release of immunoreactive prostaglandin E2 was measured using antibody microprobes. We addressed the question of whether the release of immunoreactive prostaglandin E2 is altered during development of acute inflammation in the knee evoked by intra-articular injections of kaolin and carrageenan. We also examined cyclo-oxygenase-1 and cyclo-oxygenase-2 protein levels in the spinal cord during the development of inflammation using the same model of arthritis. Densitometric analysis of microprobes showed that basal release of immunoreactive prostaglandin E2 in the period 175-310 min after kaolin was slightly higher than in the absence of inflammation. A pronounced enhancement of basal release of immunoreactive prostaglandin E2 was observed 430-530 min after kaolin. Enhanced levels of immunoreactive prostaglandin E2 were observed throughout the dorsal and ventral horns. Release of immunoreactive prostaglandin E2 was not altered further by the application of innocuous and noxious pressure onto the inflamed knee. Western blot analysis revealed that cyclo-oxygenase-2 but not cyclo-oxygenase-1 protein levels were elevated in the spinal cords of animals with inflammation compared to normal animals. This effect was evident as early as 3 h after the induction of arthritis. The maximum elevation of cyclo-oxygenase-2 protein levels (six-fold) was observed 12 h after the induction of arthritis. The results show that there is a tonic release of immunoreactive prostaglandin E2 from the spinal cord following the induction of arthritis, which is accompanied by enhanced expression of cyclo-oxygenase-2 protein in the spinal cord. We suggest that intraspinal prostaglandins may play a role in inflammation-evoked central sensitization of spinal cord neurons.
Increases in intracellular Ca(2+) concentration in human microglial cells in response to platelet-activating factor (PAF) were studied using Ca(2+)-sensitive fluorescence microscopy. In normal physiological solution (PSS), PAF-induced transient increases in [Ca2+](i) which recovered to baseline values within 200 s. Application of PAF in zero-Ca(2+) solution caused the peak response to be decreased to a value near 20% of that recorded in PSS suggesting a primary contribution of Ca(2+) influx for the [Ca2+](i) increase in PSS. To investigate PAF-induced Ca(2+) influx, the contents of intracellular stores were modulated using the SERCA blocker cyclopiazonic acid (CPA). The Ca(2+) signal induced by CPA (10 microM) in zero-Ca(2+) solution showed a peak response about 20% of the amplitude in the presence of external Ca(2+), suggesting the latter response included significant contributions from store-operated Ca(2+) entry. The influx of divalent cations with PAF or CPA was directly measured using Mn(2+) quenching of the fluorescence signal. Although both PAF and CPA induced a similar degree of Mn(2+) influx over time, the PAF effect was very rapid, whereas the CPA action was delayed and only evident about 200 s after application. Overall, the results show that the primary source of the PAF-induced increase of [Ca2+](i) in human microglia was the influx of Ca(2+) from the extracellular space and intracellular Ca(2+)-release contributed only a small part of the total Ca(2+) signal. Nevertheless, Ca(2+)-release induced by PAF (or CPA) serves as an important factor in controlling Ca(2+) entry presumably mediated by activation of store-operated-Ca(2+) channels.
Prostaglandin (PG) D synthase catalyzes the isomerization of PGH2, a common precursor of various prostanoids, to produce PGD2 in the presence of sulfhydryl compounds. PGD2 induces sleep, regulates nociception, inhibits platelet aggregation, acts as an allergic mediator, and is further converted to 9 alpha, 11 beta-PGF2 or the J series of prostanoids, such as PGJ2, delta 12-PGJ2, and 15-deoxy-delta 12,14-PGJ2. We have purified two distinct types of PGD synthase; one is the lipocalin-type enzyme and the other is the hematopoietic enzyme. We isolated the cDNA and the gene for each enzyme and determined the tissue distribution profile and the cellular localization in several animal species. Lipocalin-type PGD synthase is localized in the central nervous system and male genital organs of various mammals and the human heart and is secreted into cerebrospinal fluid, seminal plasma, and plasma, respectively. The human enzyme was identified as beta-trace, which is a major protein in human cerebrospinal fluid. This enzyme is considered to be a dual-function protein; it acts as a PGD2-producing enzyme and also as a lipophilic ligand-binding protein, because the enzyme binds retinoids, thyroids, and bile pigments, with high affinities. Hematopoietic PGD synthase is widely distributed in the peripheral tissues and localized in the antigen-presenting cells, mast cells, and megakaryocytes. The hematopoietic enzyme is the first recognized vertebrate homolog of the sigma class of glutathione S-transferase. X-ray crystallographic analyses and generation of gene-knockout and transgenic mice for each enzyme have been performed.
Stlmmsr~ Human immunodeficiency virus (HIV) infection of brain macrophages and astroglial proliferation are central features of HIV-induced central nervous system (CNS) disorders. These observations suggest that glial cellular interactions participate in disease. In an experimental system to examine this process, we found that cocultures of HIV-infected monocytes and astroglia release high levels of cytokines and arachidonate metabolites leading to neuronotoxicity. HIV-l^D^-infected monocytes cocultured with human glia (astrocytoma, neuroglia, and primary human astrocytes) synthesized tumor necrosis factor (TNF-o 0 and interleukin 1B (IblB) as assayed by coupled reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and biological activity. The cytokine induction was selective, cell specific, and associated with induction of arachidonic acid metabolites. TNF-B, Iblc~, IL-6, interferon c~ (IFN-c~), and IFN-'y were not produced. Leukotriene B4, leukotriene D4, lipoxin A4, and platelet-activating factor were detected in large amounts after high-performance liquid chromatography separation and correlated with cytokine activity. Specific inhibitors of the arachidonic cascade markedly diminished the cytokine response suggesting regulatory relationships between these factors. Cocultures of HIV-infected monocytes and neuroblastoma or endothelial cells, or HIV-infected monocyte fluids, sucrose gradient-concentrated viral particles, and paraformaldehyde-fixed or freeze-thawed HIV-infected monocytes placed onto astroglia failed to induce cytokines and neuronotoxins. This demonstrated that viable monocyte-astroglia interactions were required for the cell reactions. The addition of actinomycin D or cycloheximide to the HIV-infected monocytes before coculture reduced, >2.5-fold, the levels of TNF-o~. These results, taken together, suggest that the neuronotoxicity associated with HIV central nervous system disorders is mediated, in part, through cytokines and arachidonic acid metabolites, produced during cell-to-cell interactions between HIV-infected brain macrophages and astrocytes.
Primary rat astrocyte cultures were used to isolate a macrophage population that does not adhere to the confluent glial cells. The cells multiplied vigorously in coculture with astrocytes during the 14 d culture period, provided that functionally active lipopolysaccharide (LPS) was either absent or present in very low concentrations. Based on morphological, immunocytochemical, and pharmacological data, it was concluded that the isolated cells were microglia, the resident macrophages of the brain. The findings characterized them as a distinct cell population that shares features both of peritoneal macrophages and of astroglial cells. Like peritoneal macrophages, the isolated cells were able to phagocytize as shown by their ingestion of latex beads and uptake of L-leucyl methylester. Furthermore, they were immunocytochemically stainable by a specific monoclonal antibody (ED 1) against a macrophage-specific antigen (Dijkstra et al., 1985). They also synthesized prostaglandin E2 (PGE2) and secreted interleukin 1 (IL-1) upon stimulation with LPS. Upon stimulation with the ionophore A23187, PGD2, the predominant prostaglandin of the brain, was the major PG metabolite released by these cells. In contrast to peritoneal macrophages, microglial cells were able to multiply. Proliferation of microglial cells in coculture with astrocytes was suppressed when 2 ng LPS/ml or higher concentrations were added to astroglial culture media. These astrocyte cultures, which contained approximately 1% microglia, were used to investigate the influence of LPS on prostaglandin and IL-1 secretion in order to compare astroglial and microglial features. Increasing LPS concentrations induced increased PGE2 secretion, whereas PGD2 secretion was essentially unaffected by LPS. The critical influence of LPS contaminations in most of the commercially available animal sera used for astrocyte cultures on cellular composition in general and on metabolism of hormones and growth factors in particular is discussed.
Rat ameboid microglia are able to lyse rat oligodendrocytes in vitro. The lysis is inhibited by transforming growth factor-beta, antagonists of nitric oxide (NO) production, as well as antibodies to TNF-alpha, intercellular adhesion molecule-1 (ICAM-1), and leukocyte functional Ag-1. Ameboid microglial cells spontaneously produce detectable levels of the NO metabolite nitrite (NO2-). Stimuli such as PMA, LPS, and/or IFN-gamma induce micromolar concentrations of NO2- within 24 h. TNF-alpha increases IFN gamma but not LPS-induced NO2- production. Incubation with target oligodendrocytes also increases NO2- production in a contact-dependent manner. NO2- production is inhibited by NO synthase antagonists, transforming growth factor-beta, and anti TNF-alpha. Neither antileukocyte functional Ag-1 nor anti-ICAM-1 inhibit NO2- production by microglia in the presence or absence of oligodendrocytes. Indeed, anti-ICAM-1 treatment increases NO2- production. There is a correlation between ameboid microglial cell killing of oligodendrocytes and NO2- production suggesting NO may be a mechanism of death of the oligodendrocyte and possibly play a role in lesion formation in multiple sclerosis.
Interactions between rat microglia and oligodendrocytes were examined in vitro in order to characterize the stages of adherence, phagocytosis and cytotoxicity against oligodendrocytes by microglia. Under resting conditions, microglia showed minimal contact with oligodendrocytes and exhibited surface staining of myelin basic protein and myelin debris only; they were not cytotoxic for oligodendrocytes and did not produce tumour necrosis factor (TNF). On activation with either Γ-interferon or lipopolysaccharide and interferon, these cells increased surface binding for myelin basic protein, showed greater contact with living oligodendrocytes and produced TNF in both secreted and cell surface bound forms. Secreted TNF was capable of killing oligodendrocytes but the cell surface bound form did this more efficiently. In the presence of complement, activated microglia showed significant phagocytosis of myelin basic protein which was not obvious in the unactivated cells. These results suggest that activated microglia in the presence of complement are sufficient to kill and phagocytose the oligodendrocyte-myelin complex in vitro
Prostaglandin (PG) and thromboxane (TX) biosynthesis in primary neuronal and astroglial cell cultures was studied. Cultures obtained from fetal (15-16 days old) and neonatal rat brain hemispheres were characterized by chemical and immunocytochemical staining techniques as predominantly neurons or mature and immature astrocytes, respectively. Six-day old neuronal cell cultures grown in the presence of cytosine arabinoside (2 ?M) from the day 3 onwards were contaminated up to 10% with glioblasts. In astroglial cultures up to 3% of the cells were postively stained with a marker for oligodendroglial cells. Fibroblast contamination was below 1% in both cultures. Prostanoid formation (measured by specific radioimmunoassays) in 6-day old neuronal cell cultures was low (sum of the amount of PGs and TX formed: 1.16 +/- 0.17 (ng/mg protein/15 min) as compared to 14-day old cultured astroglial cells: 21.27 +/- 2.53 (ng/mg protein/15 min). Also the pattern of prostanoids formed was different in neuronal (PGD(2) ? PGF(2?) > TXB(2) ? PGE(2)) and astroglial cells (PGD(2) > TXB(2) ? PGF(2?) ? PGE(2) ? 6-ketoPGF(1?)). Preincubation with arachidonic acid (1 ?g/ml) did not affect prostanoid formation in both cultures, whereas it was stimulated 4-6-fold by addition of the calcium ionophore A23187 (1 ?M). These results, although found on cultured neuronal and glial cells of different stages of development, support the view that astroglial cells might play a crucial role in brain prostanoid synthesis.
In this study we evaluated the release of some mediators of inflammatory reactions such as histamine (H), leukotriene B4 (LTB4), leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) in the cerebrospinal fluid (CSF) of 15 patients with acquired immunodeficiency syndrome (AIDS), eight with opportunistic infections of the central nervous system (CNS) and seven without HIV-related neurological pathology, and of 25 HIV-negative control subjects with other neurological diseases. The cerebrospinal LTB4 level was increased in all the AIDS patients (mean 348 pg/ml); the control group revealed normal levels of LTB4 in the CSF (mean 63.2 pg/ml). The PGD2 level in the HIV-positive (mean 264 pg/ml) patients was higher than of the control subjects (mean 50 pg/ml), while low LTC4 levels were found both in the HIV-positive and control groups. We did not find any significant concentration of H in the CSF of either the HIV-positive or the control subjects. These findings may be due to the presence of chronic HIV infection or to the opportunistic infections of the CNS that so often occur in the latest stages of the disease.
Interactions between rat microglia and oligodendrocytes were examined in vitro in order to characterize the stages of adherence, phagocytosis and cytotoxicity against oligodendrocytes by microglia. Under resting conditions, microglia showed minimal contact with oligodendrocytes and exhibited surface staining of myelin basic protein and myelin debris only; they were not cytotoxic for oligodendrocytes and did not produce tumour necrosis factor (TNF). On activation with either gamma-interferon or lipopolysaccharide and interferon, these cells increased surface binding for myelin basic protein, showed greater contact with living oligodendrocytes and produced TNF in both secreted and cell surface bound forms. Secreted TNF was capable of killing oligodendrocytes but the cell surface bound form did this more efficiently. In the presence of complement, activated microglia showed significant phagocytosis of myelin basic protein which was not obvious in the unactivated cells. These results suggest that activated microglia in the presence of complement are sufficient to kill and phagocytose the oligodendrocyte-myelin complex in vitro.
There is now evidence approaching, if not having already surpassed, overwhelming in support of microglial cells as macrophages. Consistent with this cellular identity, they appear to arise from monocytes in developing brain where amoeboid microglia function in removing cell death-associated debris and in regulating gliogenesis. In normal adult tissue, ramified microglial cells with down-regulated macrophage functional properties may serve a constitutive role in cleansing the extracellular fluid. Under all conditions of brain injury, microglia appear to activate and convert into active macrophages. Activated and reactive microglia participate in inflammation, removal of cellular debris and wound-healing, the latter through regulation of gliosis in scar formation and a potential contribution to neural regeneration and neovascularization. In the activated state, microglia also express MHC's and, thus, may function in antigen presentation and lymphocyte activation for CNS immune responses. As uniquely adapted tissue resident macrophages within the CNS, microglia serve a variety of functional roles over the lifespan of this tissue. These cells may therefore be involved in or contribute to some disease states; such has been indicated in multiple sclerosis and AIDS dementia complex.
The aim of our study has been to investigate the metabolism of endogenous arachidonic acid or that of radiolabeled arachidonate in astroglial cells, stimulated with platelet activating factor (PAF) and with the calcium-ionphore A23187. Primary cultures of astroglial cells were obtained from brain cortex of one-day-old rats and were characterized by immunofluorescent staining vs glial fibrillary acidic protein. In labeled cells, diacylglycerol was formed after stimulation with platelet activating factor, whereas mainly the release of labeled arachidonic acid from phospholipids was observed after stimulation with calcium-ionophore. Both PAF and the calcium-ionophore A23187 actively stimulated the formation of the cycloxygenase products PGD2, TXB2 and 6-keto-PGF1 alpha, measured by radio- or enzyme-immunoassay. Differences were observed, instead, in the formation of the lipoxygenase metabolites, the hydroxyeicosateraenoic acids, which were measured by high pressure liquid chromatography (HPLC) with on line radiodetection for the labeled products, and Leukotriene C4, measured by radioimmunoassay. The formation of hydroxyacids by stimulated cells was confirmed by gas chromatography-mass spectrometry (GC-MS). In labeled cells, both agonists induced the formation of 12- and 15-hydroxyeicosatetraenoic acids, whereas stimulation of unlabeled cells with calcium ionophore resulted in formation of 12-hydroxyeicosatetraenoic acid and Leukotriene C4. Our results suggest that in astroglial cells, PAF, a compound which is produced in several tissues including brain, mobilizes a selected arachidonic acid pool, possibly associated with diacylglycerol production, from phospholipids, thus activating the conversion of the released fatty acid via the cyclo and the 12-lipoxygenase pathways.
Peptidoleukotrienes (SP-LTs) (both total product and individual LTC4 and LTE4 and LTB4 were measured by radioimmunoassay in cerebrospinal fluid (CSF) collected from the third ventricle of conscious cats. Total SP-LT was expressed as LTE4 after treating samples with crude gamma-glutamyltranspeptidase. Prostaglandin (PG) E2 and thromboxane (TX) B2, the stable metabolite of TXA2, were also assayed in part of the experiments. Under basal conditions, SP-LT and LTC4 were consistently measurable (respectively, 327 +/- 14 and 244 +/- 41 pg/ml), while native LTE4 was below the threshold of the assay (60-280 pg/ml) in most cases. LTB4 was barely detectable (30 +/- 2 pg/ml) or not detectable at all. PGE2 was normally less abundant than TXB2 (31 +/- 4 vs 281 +/- 47 pg/ml). Intracerebroventricular (i.c.v.) administration of arachidonic acid (40 microgram) caused a 4-fold increase in SP-LT levels which was relatively small and transient compared to PGE2 (76-fold) and TXB2 (23-fold), while there was no change in either native LTE4 or LTB4. A similar response was obtained with platelet-activating factor (PAF, 1 microgram i.c.v.), though SP-LT elevation (4-fold) was more persistent. A further rise in SP-LT (9-fold) was noted when PAF administration was preceded by indomethacin (500 microgram i.c.v.), whereas PAF effect was reversed by pretreatment with either the PAF antagonist, BN52021 (1 microgram i.c.v.), or the 5-lipoxygenase inhibitors, U-60,257 (75 micrograms i.c.v.) and L-651,392 (10 mg/kg p.o.). PAF was also effective in causing a 3-fold rise in LTC4. Unlike PAF, pyrogens (endotoxin i.c.v. or i.v.; interleukin-1 i.v.) at doses above threshold for fever had no effect on LT levels in CSF, both in the absence and presence of indomethacin pretreatment. We conclude that SP-LTs are a normal constituent of CSF, LTC4, being the major species. The response to PAF accords with a pathogenetic role of the compounds in inflammatory processes and the reactive changes to injury. No evidence was obtained for the involvement of SP-LTs in the central mechanism of fever.
The formation and composition of cysteinyl-leukotrienes (LT) in primary astroglial cell cultures prepared from newborn rat brain has been studied. Small amounts of cysteinyl-LT determined in terms of LTC4-like material in the supernatants of the cultures, became detectable after stimulation of the cells with 10(-5) M ionophore A23187. Cysteinyl-LT formation increased with time, reaching about 600 pg (mg protein)-1 after 60 min incubation. In contrast, considerable thromboxane (TX) B2 synthesis was found at 5 min following A23187-stimulation (about 30 ng TXB2 (mg protein)-1). The synthesis of cysteinyl-LT was abolished by 5 x 10(-5) M nordihydroguaiaretic acid (NDGA). Irrespective of the duration of incubation, blockage of prostanoid synthesis by 10(-6) M indomethacin did not result in increased cysteinyl-LT production. Reversed phase HPLC combined with radioimmunological detection showed that, after 60 min incubation in the presence of A23187, LTC4 and LTD4 accounted for practically all the LTC4-like immunoreactive material in the supernatants of cell cultures. No significant amounts of LTE4 could be detected. The results show that astrocytes may contribute to brain LTC4 and LTD4 synthesis. However, the cellular site of cerebral LTE4 formation seems to be other than the astroglia.
The specific mechanism underlying the genesis of vasogenic brain edema is still debated: the role of arachidonic acid is considered extremely important, as it is a possible activator of self-maintaining reactions enhancing the release of vasoactive and cytotoxic compounds. The relationship between arachidonic acid metabolism and brain edema has been studied primarily in brain tissue samples or in the extracellular fluid, whereas the residual capacity of perilesional tissue to synthesize and release eicosanoids has not been investigated. In the present study, perilesional samples of brain tissue were available from 4 patients operated on for brain metastasis, from 8 patients who had malignant neuroepithelial tumors, from 4 with meningiomas, and from 5 with subarachnoid hemorrhage. A brain edema index was calculated from the preoperative computed tomographic scan. The "ex vivo" method allowed determination of the residual capacity of endogenous arachidonic acid metabolism. The edema index is significantly higher in patients with brain metastasis (6.5 +/- 0.8) and neuroepithelial tumors (3.6 +/- 0.2) than in those with meningiomas (1.5 +/- 0.06), subarachnoid hemorrhage (1.7 +/- 0.18), and in controls. In patients with metastatic and neuroepithelial tumors there is a significant correlation between peritumoral brain edema and the capacity to synthesize leukotriene C4 (P less than 0.05); the capacity to synthesize leukotriene C4 is also significantly elevated after subarachnoid hemorrhage (13.91 +/- 2.6 ng/ml of incubation medium) when compared with control cases (5.56 +/- 0.91). The capacity to synthesize prostacyclin is significantly higher in patients with brain metastasis than in those with neuroepithelial tumors and meningiomas (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Since a review on this topic in this Journal appeared (Wolfe, 1982), the CNS has proved to be a major focus in eicosanoid research. Although our knowledge is limited at the moment, the research in this field is rapidly growing. In this short review, we summarize recent progress of research (1982-1989) in this field with special attention directed to eicosanoid metabolism, functions of eicosanoids in the neuroendocrine system and synaptic transmission, current information on eicosanoid receptors, and the link between eicosanoids and cerebral circulation. Knowledge of the eicosanoids has paved the way to a better understanding of intercellular signal transduction systems, including neuronal functions.
Transient ischemia to the lumbar and sacral sections of the spinal cord of rabbit leads to a deterioration in neurological function that usually worsens 24 to 48 hours after injury. This decline in movement of the hindlimbs develops simultaneously with the appearance of mononuclear phagocytes in the gray matter of injured neural tissue. Chloroquine and colchicine inhibit phagocytic and secretory functions in mononuclear phagocytes. When given up to 6 hours after an induced ischemic lesion in rabbit, this drug combination decreased the number of mononuclear phagocytes found within the gray matter of damaged spinal cord, improved the recovery of function of the hindlimbs and bladder, preserved spinal somatosensory evoked potentials, and promoted the survival of motor neurons. In contrast, the glucocorticoid dexamethasone, a weak inhibitor of mononuclear phagocytes in vivo, did not reduce the number of inflammatory cells in the spinal cord and did not improve motor and bladder functions. The suppression of mononuclear phagocytes soon after ischemic injury may offer a new approach in the treatment of vascular disease in the central nervous system.
Arachidonic acid metabolism in primary cultures of astroglial cells prepared from cerebra of rat embryos was examined. Arachidonic acid was mainly metabolized through the lipoxygenase pathway and the major metabolites formed were 12-hydroxyeicosatetraenoic acid (12-HETE), 11-HETE and 15-HETE. By contrast, in C6 cells, which are considered to be of astroglial origin, arachidonic acid was mainly metabolized through the cyclooxygenase pathway and the major metabolites formed were prostaglandin (PG)E2, PGF2 alpha and thromboxane B2.
Astrocytes are important elements of cellular immune responses in the central nervous system. Eicosanoids exhibit immunomodulatory properties. We studied whether astroglial cells have the capacity to convert arachidonic acid via the lipoxygenase pathway to leukotriene B4 (LTB4). Calcium ionophore A23187 evoked a dose-related release of LTB4 into supernatants of primary culture rat astrocytes. This stimulatory effect was abrogated by the addition of the lipoxygenase inhibitors BW755c and nordihydroguaretic acid. Astrocyte production of the phlogistic and immunoregulatory mediator LTB4 may be one mechanism by which these cells influence immunity and inflammation in the central nervous system.
Vasoactive arachidonic acid metabolites are postulated to play a role in the pathogenesis of cerebral ischemia. In order to characterize the local generation of cyclooxygenase and lipoxygenase metabolites of arachidonic acid in transient ischemia with reperfusion, Mongolian gerbils were studied for regional cerebral blood flow (CBF), using the hydrogen clearance technique, and for cerebral levels of the thromboxane metabolite TXB 2 , and prostaglandins 6-keto-PGF 1α and PGE 2 , as well as the leukotriene LTB 4 . The gerbils were anesthetized with pentobarbital, and half of the animals were pretreated with the cyclooxygenase inhibitor indomethacin. All received 10 or 20 minutes of dense forebrain ischemia followed by reperfusion of 10 minutes, 50 minutes, or 100 minutes. A separate control group received no ischemic lesion.
Regional CBF decreased significantly from 23.7 ± 2.6 to 4.3 ± 1.7 cc/100 gm/min during ischemia (p < 0.01). Reperfusion resulted in initially normal flows (22.5 ± 5.1 cc/100 gm/min) followed by a progressive hypoperfusion (11.3 ± 2.7 cc/100 gm/min). All metabolites showed parallel significant (p < 0.05) increases after transient ischemia and reperfusion compared to baseline levels (values (in pg/mg protein) were: TXB 2 45.5 ± 7.1 vs 23.3 ± 3.6; 6-keto-PGF 1α 262.8 ± 47.9 vs 175.8 ± 26.8; PGE 2 256.5 ± 35.6 vs 112.5 ± 11.2; and LTB 4 37.8 ± 4.6 vs 24.6 ± 6). These levels were all significantly decreased (p < 0.05) by pretreatment with indomethacin except for the leukotriene LTB 4 , which was increased. Transient cerebral ischemia results in a reperfusion abnormality and the local generation of cyclooxygenase products, which are reduced by pretreatment with indomethacin; however, cyclooxygenase inhibition may result in increased substrate availability for the lipoxygenase system. Studies of such an interaction may lead to new understandings of the pharmacological modification of detrimental vascular changes after transient cerebral ischemia.
Our understanding of the biochemistry and biologic actions of AA metabolites has been greatly expanded in recent years. The discoveries of TXA2, PGI2, and LTs have fostered new concepts of the pathophysiology of cerebral ischemia. New approaches to treatment of ischemia include seeking an optimal dose of aspirin, developing drugs that selectively inhibit or antagonize TXA2 or LTs, and administering PGI2 or its analogues. Altering the dietary content of essential fatty acids for prophylaxis is also being studied. Though the results of this thrust are still preliminary, the exploration of these therapeutic strategies in cerebrovascular disorders based on further understanding of the pathophysiologic roles of TXA2, PGI2, LTs and probably other AA metabolites is anticipated with some optimism.
Astrocytes metabolize arachidonic acid via the cyclooxygenase pathway to prostanoids. We examined whether primary culture astrocytes from neonatal rat brain can be induced to generate and release the lipoxygenase derivative leukotriene C4 (LTC4). While there was only minute constitutive production of immunoreactive LTC4 this metabolite was liberated by astroglial cells in response to calcium ionophore A23187. The phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) failed to precipitate leukotriene release. However, when threshold doses of A23187 were added to astrocyte cultures challenged with TPA, LTC4 was recovered from their supernatants. It is suggested that leukotriene generation by astrocytes bears relevance to immunoinflammatory responses in the central nervous system and may be involved in brain edema formation.
Prostanoid forming capacity in vitro and convulsion-induced prostanoid formation in vivo were studied in the developing rat brain. For comparison, prostanoid synthesis in homogenates of primary astrocyte cultures of different ages was also examined. There was no significant prostanoid production in homogenates from primary astrocyte cultures prepared one week after cultivation. Two-week-old astrocyte cultures possessed a prostanoid synthesizing system of high specific activity. The relative proportions of the products were similar to those obtained in brain homogenates of adult rats, prostaglandin D2 (PGD2) being the major product. Prostanoid forming capacity of brain homogenates was low at birth, increased during development and nearly reached adult values by day 21. Generalized convulsions could be evoked by pentylenetetrazol (PTZ) irrespective of age, but convulsion-induced prostanoid formation characteristic of adult rodents did not take place before the third week of postnatal life. The close similarities between the characteristic features of prostanoid synthesis in both brain and astroglial homogenates, together with the coincidence during brain development of the expression of cerebral prostanoid synthesis with the appearance of mature astrocytes suggest that astrocytes are an important source of brain prostanoids.
Leukotrienes are pharmacologically active compounds that promote vascular permeability. In this study we sought to determine whether tissue leukotriene-like immunoreactivity was increased in intracranial tumors associated with peritumoral edema. In 20 patients undergoing craniotomy tissue specimens were immediately frozen after removal and tissue leukotriene C4 levels were determined by radioimmunoassay. An index of peritumoral edema was estimated from preoperative contrast-enhanced computed tomographic scans. There was a significant correlation between brain edema and tissue leukotriene levels (p less than 0.003). Metastatic tumors (n = 8) had the highest leukotriene C4 level at 13.8 +/- 8.5 pg/mg tissue (mean +/- SE) and the highest index of edema 5.7 +/- 1.8. The mean leukotriene C4 level in the gliomas (n = 5) was 6.2 +/- 2.3 pg/mg tissue and the edema index was 2.1 +/- 0.6. There was no edema and no neoplasm in the temporal lobes removed for seizure (n = 2), and their level of leukotriene C4 was 0.4 +/- 0.1 pg/mg tissue. The formation of leukotriene C4 is stimulated by intracranial tumors. Leukotrienes increase blood-brain barrier permeability and may be important in the formation of vasogenic edema surrounding tumors.
The presence (1) and synthesis of prostanoids under (2–10) and (11–15) conditions has been well established in the CNS of several species including man (16). These results, however, do not permit to draw conclusions on the neuronal or extraneuronal origin of prostanoids, except for prostaglandin (PG) I2, shown to be formed principally in blood vessels (6, 16, 17). Informations on their site of origin may be helpful to better understand the role of PGs and thromboxane (TX) in the CNS. As a model to examine the involvement of neuronal and/or glial elements in brain PG and TX biosynthesis we have chosen primary brain cultures. In this communication, we report on our first results concerning prostanoid formation in homogenates of primary neuronal and astroglial cell cultures.
6-Sulfidopeptide-containing leukotriene-like immunoreactivity was synthesized in gerbil forebrains after bilateral common carotid occlusion and reperfusion. At 5, 10, or 15 minutes of ischemia, concentrations increased significantly and became more marked on reperfusion. Immunoreactivity was highest in forebrain gray matter and was below the detection limit of the assay in brain regions remote from the zone of ischemia. In vitro experiments with vascular cells and organ cultures of cerebral arteries indicate that the cerebral blood vessel wall is not a major source of biosynthetic activity in the brain. These experiments demonstrate leukotriene biosynthesis by the brain. Because synthesis occurs during ischemia and reperfusion and because leukotrienes are potent vasoconstrictors and promoters of tissue edema, they may play a role in the pathophysiology of cerebral ischemia.
The dementia associated with human immunodeficiency virus (HIV) is poorly understood. Dementia is accompanied by infection and activation of macrophage lineage cells in the brain and production of toxic products by these cells has been postulated to play a role in the pathogenesis of dementia. Eicosanoids are potential products of activated macrophages that can mediate cell injury. We measured the levels of prostaglandin E2 in the cerebrospinal fluid of HIV-positive individuals with dementia and/or myelopathy and compared these levels with those of HIV-negative patients with other neurological diseases and HIV-positive patients without dementia. Cerebrospinal fluid prostaglandin E2 levels were increased in dementia. This increase was associated with severity of dementia and correlated with cerebrospinal fluid levels of neopterin and beta 2-microglobulin. Prostaglandins F2 alpha and thromboxane B2, additional products of the cyclooxygenase pathway of arachidonic acid metabolism, were also elevated in dementia, but leukotriene C4, a product of the lipoxygenase pathway was not. Since synthesis of prostaglandins is regulated in part by the levels of inducible forms of cyclooxygenase, we measured the levels of cyclooxygenase-1 and 2 mRNAs in the brains of HIV-positive individuals with and without dementia by reverse transcriptase polymerase chain reaction. Levels of intact cyclooxygenase-1 mRNA were higher in the brains of demented individuals, but this did not reach statistical significance. These data demonstrate that prostaglandins are increased in the central nervous system in HIV-associated dementia and may play a role in the development of neurological dysfunction.
Microglia may play an important role in host defense of the central nervous system against Toxoplasma gondii, and cytokines produced by these glial cells may participate in their antitoxoplasma activity. In our study, the antitoxoplasma activity of human fetal microglia was investigated. The RH strain of T. gondii multiplied readily in these glial cells. IFN-gamma/LPS-treated microglia limited (p < 0.01) T. gondii growth by reducing entry of this parasite rather than intracellular multiplication. More than 90% of the antitoxoplasma activity of activated microglia was blocked (p < 0.01) by neutralizing antibodies to TNF-alpha or IL-6 (but not to IL-1 or TGF-beta), suggesting that these proinflammatory cytokines play a role in the inhibitory process. Consistent with this hypothesis, treatment of microglia with TNF-alpha or IL-6 (in the presence or absence of IFN-gamma) inhibited (p < 0.01), in a dose-dependent manner, T. gondii growth. Inasmuch as NGMA did not affect cytokine-mediated antitoxoplasma activity of microglia, nitric oxide appears not to be involved in this host defense function of human fetal microglia. Results of our study suggest that the host defense activity of human microglia against T. gondii is dependent primarily on the activating properties of IFN-gamma, TNF-alpha, and IL-6.
Macrophages have long been known to be involved in cytotoxic actions in many tissues in the body following infection. Knowledge of the post-injury actions of blood-borne macrophages in the brain, and their resident counterparts, the microglia, have been limited to the "mopping-up" of cellular debris. However, other functions are now coming to light and there is evidence that they contribute to both growth promotion and cytotoxicity following injury in the brain. This review raises the possibility that macrophages may contribute to delayed neuronal death following ischemia. Growth factors including certain cytokines produced by these cells protect against ischemia-induced neuronal death. In contrast, cytokines can also induce macrophages to synthesize nitric oxide synthase and indoleamine-2,3-dioxygenase which results in the production of the cytotoxins nitric oxide and quinolinic acid. It is hypothesized that viable cells produce or concentrate growth factors which prevent the induction of these enzymes, whereas damaged cells cannot.
The cause of cerebral atrophy in patients with acquired immunodeficiency syndrome (AIDS) is obscure because human immunodeficiency virus type 1 (HIV-1)-related histopathological changes hardly correlate with cerebral atrophy. In this study, brain ventricular expansion was compared to the frontal lobe density of mononuclear and astroglial cells at autopsy. Twenty-eight male patients with AIDS displaying varying degrees of atrophy were compared to 17 age-matched male control subjects without AIDS or atrophy. An index of ventricular expansion was measured in uniformly sliced, formalin-fixed brain specimens, and immunochemically marked cells in coronal sections of the left superior frontal gyrus (Brodmann area 8) were quantified by field counting and planimetry. In the cortex, diffuse ferritin-stained microglia and glial fibrillary acidic protein-positive astrocytes were about twice as numerous in the patients with AIDS. Sixty-five percent (18/28) of the patients with AIDS had a microglial cell density greater than 2 standard deviations above the control mean. Microglial cell density was correlated positively with the severity of ventricular expansion (r = 0.71, p < 0.0001), while hypertrophied astroglial cells were very weakly related. In white matter, Ham-56-positive macrophages and glial fibrillary acidic protein-positive astrocytes were not meaningfully correlated with the index of ventricular expansion. Brain ventricular expansion and diffuse cortical microgliosis are highly prevalent anomalies in patients with AIDS, and their interrelationship may be more important than previously recognized.
Cell and tissue cultures
- Herz
Interactions between oligodendrocytes and microglia
- Zajicek