Generation and Characterization of Immortalized Human Microglial Cell Lines: Expression of Cytokines and Chemokines

Nagai, Atsushi; Nakagawa, Eiji; Hatori, Kozo; Choi, Hyun B.; McLarnon, James G.; Lee, M.A.; Kim, S.U.

doi:10.1006/nbdi.2001.0437

Cited by 138 publications

(131 citation statements)

References 42 publications

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“…In this regard, the current study clearly demonstrates that following intracortical injection with LPS, there is a significant increase in the levels of IL-13 in the cortex in vivo and that IL-13 is expressed exclusively in activated microglia, but not in astrocytes or in neurons. In marked contrast, expression of IL-13 was not detected even in human pure microglia cultures or in human microglial cell lines in the absence or presence of treatment with LPS (Nagai et al, 2001;Lee et al, 2001). These observations raise the possibility that microglia in culture may not have interactions with other cells and/or the conditions necessary for IL-13 expression when compared to the microglia that exist in vivo.…”

Section: Discussionmentioning

confidence: 89%

Microglia expressing interleukin‐13 undergo cell death and contribute to neuronal survival in vivo

Shin

Lee

Park

et al. 2004

Glia

115

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How to minimize brain inflammation is pathophysiologically important, since inflammation induced by microglial activation can exacerbate brain damage. In the present report, we show that injection of lipopolysaccharide (LPS) into the rat cortex led to increased levels of interleukin-13 (IL-13) and to IL-13 immunoreactivity, followed by the substantial loss of microglia at 3 days post-LPS. IL-13 levels in LPS-injected cortex reached a peak at 12 h post-injection, remained elevated at 24 h, and returned to basal levels at day 4. In parallel, IL-13 immunoreactivity was detected as early as 12 h post-LPS and maintained up to 24 h; it disappeared at 4 days. Surprisingly, IL-13 immunoreactivity was detected exclusively in microglia, but not in neurons or astrocytes. Following treatment with LPS in vitro, IL-13 expression was also induced in microglia in the presence of neurons, but not in the presence of astrocytes or in cultured pure microglia alone. In experiments designed to determine the involvement of IL-13 in microglia cell death, IL-13-neutralizing antibodies significantly increased survival of activated microglia at 3 days post-LPS. Consistent with these results, the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-␣ (TNF-␣) was sustained in activated microglia and neuronal cell death was consequently increased. Taken together, the present study is the first to demonstrate the endogenous expression of IL-13 in LPS-activated microglia in vivo, and to demonstrate that neurons may be required for IL-13 expression in microglia. Our data strongly suggest that IL-13 may control brain inflammation by inducing the death of activated microglia in vivo, resulting in an enhancement of neuronal survival.

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Section: Discussionmentioning

confidence: 89%

Microglia expressing interleukin‐13 undergo cell death and contribute to neuronal survival in vivo

Shin

Lee

Park

et al. 2004

Glia

115

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“…Another possibility is that altered treatment times with PK11195 could lead to differences in efficacy of inhibition. Interestingly, recent work has shown that production of TNF-a accompanies expression of the cytokine in LPS stimulated HMO6 cells, a recently developed microglial human cell line (Nagai et al 2001).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of lipopolysaccharide‐induced cyclooxygenase‐2, tumor necrosis factor‐α and [Ca²⁺]_i responses in human microglia by the peripheral benzodiazepine receptor ligand PK11195

Choi¹,

Khoo²,

Ryu³

et al. 2002

Journal of Neurochemistry

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The anti-inflammatory actions of the mitochondrial peripheral benzodiazepine receptor (PBR) agonist PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide] were investigated in human microglia. Application of the microglial inflammatory stimulus lipopolysaccharide (LPS, at 100 ng/mL for 3 h), induced enhancement of the expressions of the inducible enzyme, cyclooxygenase-2 (COX-2) and the pro-inflammatory cytokine, tumor necrosis factor-a (TNF-a). PK11195 (at 50 lM) significantly inhibited the LPSinduced up-regulation of both inflammatory factors; at a lower concentration of PK11195 (2 lM) expression of TNF-a, but not COX-2, was reduced. Production of both factors, using immunocytochemistry for COX-2 and ELISA for TNF-a, was markedly reduced with 50 lM of PK11195 added to LPS solution. Acute application of LPS induced a transient increase in intracellular Ca 2+ [Ca 2+ ] i exhibiting both a slow development and recovery in kinetic behavior. This increase in [Ca 2+ ] i consisted primarily of a Ca 2+ influx component accompanied by a smaller mobilization from intracellular Ca 2+ stores. In the presence of PK11195, the amplitude of the [Ca 2+ ] i response induced by LPS was reduced by 54%. Another mitochondrial agent cyclosporin A (CsA), which also acts at the permeability transition pore (PTP) of mitochondrial membrane but at a site different from the PBR, was ineffective in reducing either the LPS-induced expression of COX-2 and TNF-a or the endotoxin increase in [Ca 2+ ] i . These results indicate that the mitochondrial effector PK11195 is a specific and effective agent for inhibiting LPS-induced microglial expressions of COX-2 and TNF-a and that modulation of Ca 2+ -mediated signaling pathways could be involved in the anti-inflammatory actions. Keywords: cyclooxygenase-2, human microglia, intracellular calcium, lipopolysaccharide, mitochondrial effector, PK11195, tumor necrosis factor-a. Microglia are resident cells of the CNS that serve a functional role as scavenger cells similar to that of peripheral macrophages. These cells have been implicated as key mediators of inflammation in the CNS, during which microglia proliferate and contribute to the inflammatory process by phagocytosis and secretion of cytokines, eicosanoids and other agents (McGeer and McGeer 1995). Although inflammation of the CNS is intended as a homeostatic means to contain damage to neural tissue, such damage may occur from the inflammatory process itself (McGeer and McGeer 1995;Rogers and Griffin 1998). In this case microglial responses to inflammatory stimuli in the brain may actually exacerbate, rather than inhibit, neuronal damage. Abbreviations used: [Ca 2+ ] i , intracellular calcium concentration; COX-2, cyclooxygenase-2; CsA, cyclosporin A; DAPI, 4,6-diaminodino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; PBR, peripheral benzodiazepine receptor; PK11195...

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“…The methods used in the isolation and identification of microglia have been described previously (Kim et al, 1986;Satoh and Kim, 1994;Satoh et al, 1995;Nagai et al, 2001). In brief, human embryonic brain tissues (12-18 weeks of gestation) were used for the preparation of microglia.…”

Section: Materials and Methods Preparation And Culture Of Human Micromentioning

confidence: 99%

“…Dissociated cells were plated in T75 flasks in a medium consisting of Dulbecco's modified Eagle's medium (DMEM) with high glucose containing 5% horse serum, 25 g/ml gentamicin, and 2.5 g/ml amphotericin B. Free-floating microglia were harvested from a medium of mixed cell cultures after 7-10 days of growth in culture flasks and plated on aclar coverslips for identification, on poly-Llysine-coated glass coverslips for calcium spectrofluorometry and plated on six-well multiplates for RT-PCR. Immunostaining was performed on isolated cells using CD11b and ricinus communis agglutinin (RCA), specific markers for microglia, and the purity of human microglia was in excess of 98% (Walker et al, 1995;Nagai et al, 2001). The use of embryonic human tissues was approved by the Clinical Screening Committee for Human Subjects of the University of British Columbia.…”

Section: Materials and Methods Preparation And Culture Of Human Micromentioning

confidence: 99%

Differential activation of subtype purinergic receptors modulates Ca²⁺ mobilization and COX‐2 in human microglia

Choi

Hong

Ryu

et al. 2003

Glia

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KEY WORDShuman microglia; adenosine triphosphate; P 2Y and P 2X receptors; COX-2, store-operated channel Ca 2ϩ entry ABSTRACTWe have studied modulation of purinergic receptors (P 2Y and P 2X subtypes) on changes in intracellular Ca 2ϩ [Ca 2ϩ ] i and expression and production of COX-2 in human microglia. Measurements using Ca 2ϩ -sensitive spectrofluorometry showed adenosine triphosphate (ATP) to cause rapid transient increases in [Ca 2ϩ ] i . Application of ATP plus the P 2X antagonist, pyridoxal-phosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (PPADS), or treatment with adenosine diphosphate-␤-S (ADP-␤-S), a selective P 2Y agonist, led to a considerable prolongation in [Ca 2ϩ ] i responses compared with ATP. The prolonged time courses were consistent with sustained activation of store-operated channels (SOC) since SKF96365, an inhibitor of SOC, blocked this component of the response. RT-PCR data showed that microglia expressed no COX-2 either constitutively or following treatment of cells with ATP (100 M for 8 h). However, treatment using ATP plus PPADS or with ADP-␤-S led to marked expression of COX-2. The enhanced COX-2 with ATP plus PPADS treatment was absent in the presence of SKF96365 or using Ca 2ϩ -free solution. Immunocytochemistry, using a specific anti-COX-2 antibody, also revealed a pattern of purinergic modulation whereby lack of P 2X activation enhanced the production of COX-2 protein. These results suggest that modulation of subtypes of purinergic receptors regulates COX-2 in human microglia with a link involving SOCmediated influx of Ca 2ϩ .

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Generation and Characterization of Immortalized Human Microglial Cell Lines: Expression of Cytokines and Chemokines

Cited by 138 publications

References 42 publications

Microglia expressing interleukin‐13 undergo cell death and contribute to neuronal survival in vivo

Microglia expressing interleukin‐13 undergo cell death and contribute to neuronal survival in vivo

Inhibition of lipopolysaccharide‐induced cyclooxygenase‐2, tumor necrosis factor‐α and [Ca²⁺]_i responses in human microglia by the peripheral benzodiazepine receptor ligand PK11195

Differential activation of subtype purinergic receptors modulates Ca²⁺ mobilization and COX‐2 in human microglia

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Generation and Characterization of Immortalized Human Microglial Cell Lines: Expression of Cytokines and Chemokines

Cited by 138 publications

References 42 publications

Microglia expressing interleukin‐13 undergo cell death and contribute to neuronal survival in vivo

Microglia expressing interleukin‐13 undergo cell death and contribute to neuronal survival in vivo

Inhibition of lipopolysaccharide‐induced cyclooxygenase‐2, tumor necrosis factor‐α and [Ca2+]i responses in human microglia by the peripheral benzodiazepine receptor ligand PK11195

Differential activation of subtype purinergic receptors modulates Ca2+ mobilization and COX‐2 in human microglia

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Inhibition of lipopolysaccharide‐induced cyclooxygenase‐2, tumor necrosis factor‐α and [Ca²⁺]_i responses in human microglia by the peripheral benzodiazepine receptor ligand PK11195

Differential activation of subtype purinergic receptors modulates Ca²⁺ mobilization and COX‐2 in human microglia