Document Information

PMID 17018025  (  )
Title Protective effects of an anti-inflammatory cytokine, interleukin-4, on motoneuron toxicity induced by activated microglia.
Abstract Microglia-mediated cytotoxicity has been implicated in models of neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease, but few studies have documented how neuroprotective signals might mitigate such cytotoxicity. To explore the neuroprotective mechanism of anti-inflammatory cytokines, we applied interleukin-4 (IL-4) to primary microglial cultures activated by lipopolysaccharide as well as to activated microglia cocultured with primary motoneurons. lipopolysaccharide increased nitric oxide and superoxide (O(2) (.-)) and decreased insulin-like growth factor-1 (IGF-1) release from microglial cultures, and induced motoneuron injury in microglia-motoneuron cocultures. However, lipopolysaccharide had minimal effects on isolated motoneuron cultures. IL-4 interaction with microglial IL-4 receptors suppressed and nitric oxide release, and lessened lipopolysaccharide-induced microglia-mediated motoneuron injury. The extent of nitric oxide suppression correlated directly with the extent of motoneuron survival. Although IL-4 enhanced release of free IGF-1 from microglia in the absence of lipopolysaccharide, it did not enhance free IGF-1 release in the presence of lipopolysaccharide. These data suggest that IL-4 may provide a significant immunomodulatory signal which can protect against microglia-mediated neurotoxicity by suppressing the production and release of free radicals. Hospital, Houston, Texas 77030, USA.

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Targets by SciMiner Summary

HUGO ID Symbol Target Name #Occur ActualStr
6014IL4interleukin 4208IL-4 | IL-4-mediated | interleukin 4 | il 4 |
5464IGF1insulin-like growth factor 1 (somatomedin C)49insulin like growth factor 1 | IGF-1 |
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)15NOS | iNOS | nitric oxide synthase |
5962IL10interleukin 1012IL-10 | il 10 |
11892TNFtumor necrosis factor (TNF superfamily, member 2)12tumor necrosis factor alpha | TNF-A |
6015IL4Rinterleukin 4 receptor7IL-4R |
11917TNFRSF1Btumor necrosis factor receptor superfamily, member 1B4p75 |
11766TGFB1transforming growth factor, beta 14TGF-B | transforming growth factor beta |
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))3mSOD1 | SOD | SOD-inhibitable |
6018IL6interleukin 6 (interferon, beta 2)2IL-6 | il 6 |
5438IFNGinterferon, gamma2IFN-G |
5992IL1Binterleukin 1, beta2IL-1 | il 1 |
6025IL8interleukin 82IL-8 | il 8 |
7809NGFRnerve growth factor receptor (TNFR superfamily, member 16)1low affinity nerve growth factor receptor |
6081INSinsulin1insulin |
10627CCL3chemokine (C-C motif) ligand 31MIP-1A |
727ARTNartemin1neurotrophic factor |
1516CATcatalase1catalase |
25079CCDC34coiled-coil domain containing 341L-15 |
10632CCL5chemokine (C-C motif) ligand 51RANTES |
399ALBalbumin1serum albumin |
1678CD4CD4 molecule1CD4 |
14874NOX5NADPH oxidase, EF-hand calcium binding domain 51nadph oxidase |

 

Targets by SciMiner Full list

HUGO ID Symbol Name ActualStr Score FlankingText
6014IL4interleukin 4IL-42.7the neuroprotective mechanism of anti-inflammatory cytokines we applied interleukin-4 (IL-4) IL-4 to primary microglial cultures activated by lipopolysaccharide as well as
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5O 2 _amp_middot _amp_#8722 and decreased insulin-like growth factor-1 (IGF-1) IGF-1 release from microglial cultures and induced motoneuron injury in microglia-motoneuron
6014IL4interleukin 4IL-42.7IL_amp_#8722 4 interaction with microglial IL-4 receptors suppressed and nitric oxide release and lessened lipopolysaccharide-induced microglia-mediated
6014IL4interleukin 4IL-42.7Although IL-4 enhanced release of free IGF-1 from microglia in the absence
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5Although IL-4 enhanced release of free IGF-1 from microglia in the absence of lipopolysaccharide it did not
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5in the absence of lipopolysaccharide it did not enhance free IGF-1 release in the presence of lipopolysaccharide
6014IL4interleukin 4IL-42.7These data suggest that IL-4 may provide a significant immunomodulatory signal which can protect against
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))mSOD11.3is supported by the fact that selective overexpression of the mSOD1 transgene in neurons is not sufficient to cause motoneuron disease
6014IL4interleukin 4IL-42.7We hypothesized that the Th2 lymphocyte-derived anti-inflammatory cytokine interleukin-4 (IL-4) IL-4 may be one of the potential neuroprotective signals
6014IL4interleukin 4IL-42.7IL-4 is an important immunosuppressive mediator in the CNS
6014IL4interleukin 4IL-42.7IL-4 has also been shown to reduce the production of pro-inflammatory
6018IL6interleukin 6 (interferon, beta 2)IL-61.6shown to reduce the production of pro-inflammatory cytokines such as IL-6 IL-8 tumor necrosis factor-alpha (TNF-A), TNF-A MIP-1A and RANTES in
6025IL8interleukin 8IL-81.3to reduce the production of pro-inflammatory cytokines such as IL-6 IL-8 tumor necrosis factor-alpha (TNF-A), TNF-A MIP-1A and RANTES in glial
11892TNFtumor necrosis factor (TNF superfamily, member 2)TNF-A1.7pro-inflammatory cytokines such as IL-6 IL-8 tumor necrosis factor-alpha (TNF-A), TNF-A MIP-1A and RANTES in glial cultures treated with lipopolysaccharide or
10632CCL5chemokine (C-C motif) ligand 5RANTES1.0as IL-6 IL-8 tumor necrosis factor-alpha (TNF-A), TNF-A MIP-1A and RANTES in glial cultures treated with lipopolysaccharide or IL-1 ( Ledeboer
5992IL1Binterleukin 1, betaIL-11.3MIP-1A and RANTES in glial cultures treated with lipopolysaccharide or IL-1 ( Ledeboer et al 2000 Ehrlich et al 1998
10627CCL3chemokine (C-C motif) ligand 3MIP-1A0.3cytokines such as IL-6 IL-8 tumor necrosis factor-alpha (TNF-A), TNF-A MIP-1A and RANTES in glial cultures treated with lipopolysaccharide or IL-1
6014IL4interleukin 4IL-42.7Recently it has been documented that protection of IL-4 was attributed to down-regulation of TNF-A and up-regulation of insulin-like
11892TNFtumor necrosis factor (TNF superfamily, member 2)TNF-A1.7documented that protection of IL-4 was attributed to down-regulation of TNF-A and up-regulation of insulin-like growth factor 1 (IGF-1) IGF-1 from
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5of TNF-A and up-regulation of insulin-like growth factor 1 (IGF-1) IGF-1 from microglia ( Butovsky et al 2005 2006
6014IL4interleukin 4IL-42.7In the present study we examined the effects of IL-4 in primary microglia cultures and in microglia cocultured with primary
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5release more nitric oxide and and lower levels of free IGF-1 an active form of the neurotrophic factor
6014IL4interleukin 4IL-42.7The addition of IL-4 protected against the activated microglia-mediated motoneuron injury by reducing nitric
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5This anti-inflammatory cytokine also enhanced free IGF-1 without lipopolysaccharide stimulation
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5However with lipopolysaccharide there was a minimal increase in free IGF-1 yet the cytokine was still neuroprotective
25079CCDC34coiled-coil domain containing 34L-150.3The cells were resuspended in the L-15 culture medium supplemented with sodium bicarbonate (0.2%), 0.2% glucose (3.6
6014IL4interleukin 4IL-42.7IL-4 additions were made either 2 h prior to or after
6014IL4interleukin 4IL-42.7added to culture medium 1 h after the addition of IL-4 and lipopolysaccharide
11917TNFRSF1Btumor necrosis factor receptor superfamily, member 1Bp751.2with 2% horse serum and further incubated with mouse anti-rat p75 LNTR monoclonal antibody (p75, p75 1 800 Chemicon overnight at
11917TNFRSF1Btumor necrosis factor receptor superfamily, member 1Bp751.2further incubated with mouse anti-rat p75 LNTR monoclonal antibody (p75, p75 1 800 Chemicon overnight at 4_amp_deg C in blocking solution
11917TNFRSF1Btumor necrosis factor receptor superfamily, member 1Bp751.2As a specific marker for motoneurons p75 (the the low-affinity nerve growth factor receptor immunocytochemistry provides a
11917TNFRSF1Btumor necrosis factor receptor superfamily, member 1Bp751.2Motoneuron survival was assessed by direct counting of large p75 positive cells (cell cell bodies > 30 _amp_#x03BC m displaying
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))SOD-inhibitable1.3assay The release of superoxide ( was determined by the SOD-inhibitable reduction of ferricytochrome c ( Gao et al 2002 Mayer
6014IL4interleukin 4IL-42.7treated with lipopolysaccharide (1 1 ng/mL) ng mL followed by IL-4 (10 10 ng/mL) ng mL 2 h later
6014IL4interleukin 4IL-42.7U/mL), U mL lipopolysaccharide (1 1 ng/mL), ng mL and IL-4 (10 10 ng/mL) ng mL in HBSS were then added
11179SOD1superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))SOD1.6SOD effectively inhibited the reduction of ferricytochrome c which confirmed that
6015IL4Rinterleukin 4 receptorIL-4R1.8The conditions of PCR were as follows for IL-4R the primers 5'-CCT GTT CCC AGC CAG ACT AC-3' and
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5ELISA for free IGF-1 IGF-1 ELISA Duoset kit (R R _amp_ D Systems Minneapolis
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5ELISA for free IGF-1 IGF-1 ELISA Duoset kit (R R _amp_ D Systems Minneapolis MN
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5MN USA was used to determine the concentration of free IGF-1 protein in cell culture supernatant
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5h 100 _amp_#x03BC L of either sample or standard rat IGF-1 (Diagnostic Diagnostic Systems Laboratories Webster TX USA was added to
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5Lipopolysaccharide-activated microglia increased production of nitric oxide and decreased free IGF-1 In our previous study we demonstrated that nitric oxide plays
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5In the present experiments levels of free IGF-1 as well as nitric oxide were measured in microglia activated
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5We also assayed the levels of free IGF-1 in microglia cultures given that only free IGF-1 is active
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5of free IGF-1 in microglia cultures given that only free IGF-1 is active
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5the presence of 1 _amp_#x03BC;g/mL _amp_#x03BC g mL lipopolysaccharide free IGF-1 concentrations were significantly lower than untreated microglia ( Fig 1b
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5In MN Mc cocultures lipopolysaccharide decreased free IGF-1 in a dose-dependent manner ( Fig 2b
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5m MN lipopolysaccharide 1.02 _amp_plusmn 0.10 _amp_#x03BC m and free IGF-1 levels (MN MN only 1.95 _amp_plusmn 0.31 pg/mL, pg mL
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5were all close to background indicating that nitrite nitrate and IGF-1 were produced by microglia
6014IL4interleukin 4IL-42.7IL-4 protected motoneurons from injury induced by activated microglia To determine
6014IL4interleukin 4IL-42.7injury induced by activated microglia To determine the effects of IL-4 on microglia-mediated motoneuron injury IL-4 was added to MN Mc
6014IL4interleukin 4IL-42.7To determine the effects of IL-4 on microglia-mediated motoneuron injury IL-4 was added to MN Mc cocultures 2 h before the
6014IL4interleukin 4IL-42.7In the presence of IL-4 (10 10 ng/mL), ng mL neurite length (92.8% 92.8% _amp_plusmn
6014IL4interleukin 4IL-42.7of nitrite nitrate increased after 24-h treatment with lipopolysaccharide and IL-4 significantly reduced nitric oxide production even in the presence of
6014IL4interleukin 4IL-42.7Addition of IL-4 1-10 ng/mL ng mL significantly prevented motoneuron loss induced by
6014IL4interleukin 4IL-42.7This protective effect of IL-4 was dose-dependent ( Fig 3d
6014IL4interleukin 4IL-42.7With increasing concentrations of IL-4 nitrite nitrate levels in the supernatant decreased ( Fig 3e
6014IL4interleukin 4IL-42.7To test whether IL-4 has any survival effects on motoneurons alone motoneurons were treated
6014IL4interleukin 4IL-42.7any survival effects on motoneurons alone motoneurons were treated with IL-4 (10 10 ng/mL) ng mL for 48 h
6014IL4interleukin 4IL-42.7The survival of motoneurons with IL-4 (99.93 99.93 _amp_plusmn 2.48% was similar to survival of motoneurons
6014IL4interleukin 4IL-42.7was similar to survival of motoneurons in the absence of IL-4 (100% 100% as control
6014IL4interleukin 4IL-42.7Therefore IL-4 had no direct survival effects on motoneurons supporting that the
6014IL4interleukin 4IL-42.7survival effects on motoneurons supporting that the neuroprotective effects of IL-4 in microglia-motoneuron cocultures are mediated indirectly through microglia
6014IL4interleukin 4IL-42.7Nitric oxide was a key factor modulated by IL-4
6014IL4interleukin 4IL-42.7To provide further evidence that the neuroprotective effects of IL-4 may be mediated by reducing nitric oxide production we plotted
6014IL4interleukin 4IL-42.7we plotted the nitrite nitrate content at different concentrations of IL-4
6014IL4interleukin 4IL-42.7treated with lipopolysaccharide the suppression of nitrite nitrate generation by IL-4 was dose-dependent ( Fig 4a
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)iNOS3.7nitric oxide by up-regulation of inducible nitric oxide synthase (iNOS) iNOS ( Zhao et al 2004
6014IL4interleukin 4IL-42.7To further define the mechanisms of IL-4 neuroprotection microglia were treated with lipopolysaccharide in the presence or
6014IL4interleukin 4IL-42.7were treated with lipopolysaccharide in the presence or absence of IL-4
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)iNOS3.7Figure 5 demonstrates that lipopolysaccharide enhanced iNOS protein expression and IL-4 significantly inhibited iNOS expression induced by
6014IL4interleukin 4IL-42.7Figure 5 demonstrates that lipopolysaccharide enhanced iNOS protein expression and IL-4 significantly inhibited iNOS expression induced by lipopolysaccharide
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)iNOS3.7that lipopolysaccharide enhanced iNOS protein expression and IL-4 significantly inhibited iNOS expression induced by lipopolysaccharide
6014IL4interleukin 4IL-42.7Most strikingly the inhibitory effect of IL-4 added 2 h after lipopolysaccharide (lipopolysaccharide lipopolysaccharide IL-4 was even
6014IL4interleukin 4IL-42.7effect of IL-4 added 2 h after lipopolysaccharide (lipopolysaccharide lipopolysaccharide IL-4 was even greater than when IL-4 was added 2 h
6014IL4interleukin 4IL-42.7after lipopolysaccharide (lipopolysaccharide lipopolysaccharide IL-4 was even greater than when IL-4 was added 2 h before lipopolysaccharide (IL-4 IL-4 lipopolysaccharide (
6014IL4interleukin 4IL-42.7than when IL-4 was added 2 h before lipopolysaccharide (IL-4 IL-4 lipopolysaccharide ( p _lt_ 0.05
6014IL4interleukin 4IL-42.7IL-4 receptor was up-regulated by lipopolysaccharide and IL-4-mediated neuroprotection was increased
6014IL4interleukin 4IL-4-mediated1.3IL-4 receptor was up-regulated by lipopolysaccharide and IL-4-mediated neuroprotection was increased To help explain why IL-4 was more
6014IL4interleukin 4IL-42.7lipopolysaccharide and IL-4-mediated neuroprotection was increased To help explain why IL-4 was more effective when added after lipopolysaccharide (lipopolysaccharide lipopolysaccharide IL-4
6014IL4interleukin 4IL-42.7IL-4 was more effective when added after lipopolysaccharide (lipopolysaccharide lipopolysaccharide IL-4 expression of IL-4 receptor (IL-4R) IL-4R was assayed by real-time
6014IL4interleukin 4IL-42.7effective when added after lipopolysaccharide (lipopolysaccharide lipopolysaccharide IL-4 expression of IL-4 receptor (IL-4R) IL-4R was assayed by real-time RT-PCR in microglia
6015IL4Rinterleukin 4 receptorIL-4R1.8after lipopolysaccharide (lipopolysaccharide lipopolysaccharide IL-4 expression of IL-4 receptor (IL-4R) IL-4R was assayed by real-time RT-PCR in microglia cultures ( Fig
6015IL4Rinterleukin 4 receptorIL-4R1.8The addition of lipopolysaccharide significantly increased the expression of IL-4R compared with untreated microglia (9-fold 9-fold increase p _lt_ 0.001
6014IL4interleukin 4IL-42.7provides a possible explanation for why adding lipopolysaccharide prior to IL-4 enhanced the protective effects of IL-4
6014IL4interleukin 4IL-42.7adding lipopolysaccharide prior to IL-4 enhanced the protective effects of IL-4
6014IL4interleukin 4IL-42.7In the absence of lipopolysaccharide IL-4 did not significantly change IL-4R mRNA levels compared with untreated
6015IL4Rinterleukin 4 receptorIL-4R1.8In the absence of lipopolysaccharide IL-4 did not significantly change IL-4R mRNA levels compared with untreated control ( p = 0.91
6015IL4Rinterleukin 4 receptorIL-4R1.8Furthermore IL-4R mRNA levels were not statistically changed by IL-4 in lipopolysaccharide-activated
6014IL4interleukin 4IL-42.7Furthermore IL-4R mRNA levels were not statistically changed by IL-4 in lipopolysaccharide-activated microglia (IL-4 IL-4 lipopolysaccharide compared with the group
6014IL4interleukin 4IL-42.7were not statistically changed by IL-4 in lipopolysaccharide-activated microglia (IL-4 IL-4 lipopolysaccharide compared with the group treated with lipopolysaccharide only (
6014IL4interleukin 4IL-42.7Cultures treated with IL-4 2 h after the addition of lipopolysaccharide led to 104.6%
6014IL4interleukin 4IL-42.7higher than the motoneuron survival (83.5% 83.5% _amp_plusmn 4.05 when IL-4 was added 2 h prior to the addition of lipopolysaccharide
6014IL4interleukin 4IL-42.7IL-4 inhibited superoxide production from microglia The effect of the anti-inflammatory
6014IL4interleukin 4IL-42.7IL-4 significantly suppressed the microglial release of in the presence of
6014IL4interleukin 4IL-42.7release of in the presence of lipopolysaccharide (Mc Mc lipopolysaccharide IL-4 105.6% _amp_plusmn 24.5 vs Mc lipopolysaccharide 183.3% _amp_plusmn 11.8 p
6014IL4interleukin 4IL-42.711.8 p = 0.005 and the level of Mc lipopolysaccharide IL-4 was not significantly different from control ( p = 0.79
6014IL4interleukin 4IL-42.7Exogenous nitric oxide and reversed the neuroprotective effects of IL-4 To further prove that neuroprotective effects of IL-4 are through
6014IL4interleukin 4IL-42.7effects of IL-4 To further prove that neuroprotective effects of IL-4 are through reducing nitric oxide and release from microglia exogenous
6014IL4interleukin 4IL-42.7Figure 8 showed that in the presence of lipopolysaccharide and IL-4 addition of nitric oxide donor (NOC-18, NOC-18 25 _amp_#x03BC m
6014IL4interleukin 4IL-42.7exogenous nitric oxide and could reverse the neuroprotective effects of IL-4
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5Free IGF-1 did not increase after addition of IL-4 to lipopolysaccharide-activated microglia
6014IL4interleukin 4IL-42.7Free IGF-1 did not increase after addition of IL-4 to lipopolysaccharide-activated microglia In Fig 1b we noted less free
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5to lipopolysaccharide-activated microglia In Fig 1b we noted less free IGF-1 in the culture media following microglial activation with lipopolysaccharide
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5To determine whether IGF-1 production was influenced by IL-4 the levels of free IGF-1
6014IL4interleukin 4IL-42.7To determine whether IGF-1 production was influenced by IL-4 the levels of free IGF-1 were measured in microglia monocultures
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5IGF-1 production was influenced by IL-4 the levels of free IGF-1 were measured in microglia monocultures or MN Mc cocultures
6014IL4interleukin 4IL-42.7In cultures without lipopolysaccharide IL-4 significantly increased levels of free IGF-1 ( Fig 9a b
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5In cultures without lipopolysaccharide IL-4 significantly increased levels of free IGF-1 ( Fig 9a b
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5The free IGF-1 of motoneuron only cultures were close to background level in
6014IL4interleukin 4IL-42.7close to background level in the presence or absence of IL-4 (data data not shown indicating that microglia were the source
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5indicating that microglia were the source of the increased free IGF-1 after IL-4 treatment
6014IL4interleukin 4IL-42.7microglia were the source of the increased free IGF-1 after IL-4 treatment
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5However the levels of free IGF-1 did not change significantly in lipopolysaccharide-activated microglia or in MN
6014IL4interleukin 4IL-42.7microglia or in MN Mc lipopolysaccharide cocultures regardless of whether IL-4 was added before or after lipopolysaccharide ( Fig 9a b
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5Similar results were seen when IGF-1 mRNA levels were measured with and without IL-4 (data data
6014IL4interleukin 4IL-42.7seen when IGF-1 mRNA levels were measured with and without IL-4 (data data not shown
11892TNFtumor necrosis factor (TNF superfamily, member 2)TNF-A1.7nitric oxide and superoxide ( pro-inflammatory neurotoxic cytokines such as TNF-A and glutamate ( Gonzalez-Scarano and Baltuch 1999 Bal-Price and Brown
6014IL4interleukin 4IL-42.7The present study demonstrates that the anti-inflammatory cytokine IL-4 can protect motoneurons from injury induced by lipopolysaccharide-activated microglia
6014IL4interleukin 4IL-42.7To our knowledge this is the first report that IL-4 has beneficial effects on motoneuron injury mediated by microglia
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)iNOS3.7In our previous study we demonstrated that special iNOS inhibitor L-NIL significantly reduced nitric oxide production and reversed motoneuron
6014IL4interleukin 4IL-42.7nitrite nitrate levels and motoneuron survival in cocultures treated with IL-4 and exogenously added nitric oxide reversed neuroprotection of IL-4
6014IL4interleukin 4IL-42.7with IL-4 and exogenously added nitric oxide reversed neuroprotection of IL-4
6014IL4interleukin 4IL-42.7suggest that nitric oxide is a key factor modulated by IL-4 in our cell culture system
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)iNOS3.7Nitric oxide is synthesized by iNOS in microglia iNOS protein expression was down-regulated by IL-4 in
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)iNOS3.7Nitric oxide is synthesized by iNOS in microglia iNOS protein expression was down-regulated by IL-4 in activated microglia when
6014IL4interleukin 4IL-42.7by iNOS in microglia iNOS protein expression was down-regulated by IL-4 in activated microglia when IL-4 was given either before or
6014IL4interleukin 4IL-42.7protein expression was down-regulated by IL-4 in activated microglia when IL-4 was given either before or after the triggering signal lipopolysaccharide
6014IL4interleukin 4IL-42.7More strikingly we found that IL-4 increased motoneuron survival to a greater extent when added to
6014IL4interleukin 4IL-42.7the cause of this increase we examined the levels of IL-4 mRNA
6015IL4Rinterleukin 4 receptorIL-4R1.8We detected increased IL-4R mRNA levels after microglia were activated with lipopolysaccharide
6015IL4Rinterleukin 4 receptorIL-4R1.8Thus the greater expression of IL-4R with the subsequent greater efficacy of the available IL-4 could
6014IL4interleukin 4IL-42.7of IL-4R with the subsequent greater efficacy of the available IL-4 could provide one potential explanation of the increased suppressive effects
6014IL4interleukin 4IL-42.7provide one potential explanation of the increased suppressive effects of IL-4 on iNOS expression in activated microglia
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)iNOS3.7potential explanation of the increased suppressive effects of IL-4 on iNOS expression in activated microglia
6014IL4interleukin 4IL-42.7Two studies have reported the inhibitory effects of IL-4 on iNOS in lipopolysaccharide-stimulated astrocytes and mixed glial cultures (
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)iNOS3.7Two studies have reported the inhibitory effects of IL-4 on iNOS in lipopolysaccharide-stimulated astrocytes and mixed glial cultures ( Brodie et
6014IL4interleukin 4IL-42.7although in their studies the cells were all treated with IL-4 first and then stimulated cell with other triggering signals
6014IL4interleukin 4IL-42.7Our present findings that IL-4 also inhibits production in lipopolysaccharide-activated microglia and that neuroprotection of
6014IL4interleukin 4IL-42.7also inhibits production in lipopolysaccharide-activated microglia and that neuroprotection of IL-4 was reversed by exogenous illustrate another potential neuroprotective aspect of
6014IL4interleukin 4IL-42.7(1995 1995 reported that IL-4 suppressed production in human microglia activated by TNF-A or interferon
11892TNFtumor necrosis factor (TNF superfamily, member 2)TNF-A1.7reported that IL-4 suppressed production in human microglia activated by TNF-A or interferon (IFN)-G IFN -G
5438IFNGinterferon, gammaIFN0.3production in human microglia activated by TNF-A or interferon (IFN)-G IFN -G
6014IL4interleukin 4IL-42.7It has been shown that IL-4 reduces formation in macrophages via down-regulation of gp9l-phox at the
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)iNOS3.7However simultaneous activation of iNOS (to to produce nitric oxide and NADPH oxidase (to to
6014IL4interleukin 4IL-42.7The ability of IL-4 to suppress the production of both nitric oxide and suggests
6014IL4interleukin 4IL-42.7suppress the production of both nitric oxide and suggests that IL-4 can decrease the formation of peroxynitrite
11892TNFtumor necrosis factor (TNF superfamily, member 2)TNF-A1.7(2005 2005 demonstrated that AB-stimulated microglia released TNF-A and glutamate which synergistically increased NOS expression and activity in
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)NOS2.7that AB-stimulated microglia released TNF-A and glutamate which synergistically increased NOS expression and activity in neurons by western blot and immunocytochemistry
11892TNFtumor necrosis factor (TNF superfamily, member 2)TNF-A1.72004 we also showed that lipopolysaccharide induced microglia to release TNF-A and higher levels of glutamate (30-40 30-40 _amp_#x03BC m present
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)iNOS3.7Therefore in addition to microglia iNOS expression neuronal NOS may be another source of nitric oxide
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)NOS2.7Therefore in addition to microglia iNOS expression neuronal NOS may be another source of nitric oxide contributing to motoneuron
11892TNFtumor necrosis factor (TNF superfamily, member 2)TNF-A1.7It has also been shown that TNF-A had neurotoxic effects through up-regulating iNOS nitric oxide and production
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)iNOS3.7also been shown that TNF-A had neurotoxic effects through up-regulating iNOS nitric oxide and production from microglia ( Kuno et al
6014IL4interleukin 4IL-42.7It has also been documented that neuroprotection of IL-4 was attributed to down-regulation of TNF-A ( Butovsky et al
11892TNFtumor necrosis factor (TNF superfamily, member 2)TNF-A1.7documented that neuroprotection of IL-4 was attributed to down-regulation of TNF-A ( Butovsky et al 2005
11892TNFtumor necrosis factor (TNF superfamily, member 2)TNF-A1.7In accordance with this we did find lipopolysaccharide dramatically enhanced TNF-A produced by microglia ( Zhao et al 2004 and IL-4
6014IL4interleukin 4IL-42.7TNF-A produced by microglia ( Zhao et al 2004 and IL-4 significantly decreased TNF-A levels in MN Mc lipopolysaccharide cocultures (data
11892TNFtumor necrosis factor (TNF superfamily, member 2)TNF-A1.7microglia ( Zhao et al 2004 and IL-4 significantly decreased TNF-A levels in MN Mc lipopolysaccharide cocultures (data data not shown
11892TNFtumor necrosis factor (TNF superfamily, member 2)TNF-A1.7Therefore down-regulation of TNF-A is one of mechanisms by which IL-4 inhibited nitric oxide
6014IL4interleukin 4IL-42.7Therefore down-regulation of TNF-A is one of mechanisms by which IL-4 inhibited nitric oxide and production and protected motoneurons from microglia-mediated
6014IL4interleukin 4IL-42.7Another finding in the current study is that IL-4 increased free IGF-1 the active form of this neuroprotective factor
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5finding in the current study is that IL-4 increased free IGF-1 the active form of this neuroprotective factor in untreated microglia
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5factor in untreated microglia cultures and that lipopolysaccharide decreased free IGF-1
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5At first we hypothesized that the induction of free IGF-1 in microglia might be another aspect of IL-4 neuroprotection however
6014IL4interleukin 4IL-42.7of free IGF-1 in microglia might be another aspect of IL-4 neuroprotection however our subsequent results suggested it might not be
6014IL4interleukin 4IL-42.7IL-4 increased motoneuron survival in both lipopolysaccharide-treated MN Mc cocultures however
6014IL4interleukin 4IL-42.7increased motoneuron survival in both lipopolysaccharide-treated MN Mc cocultures however IL-4 did not increase free IGF-1 levels in the same cocultures
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5lipopolysaccharide-treated MN Mc cocultures however IL-4 did not increase free IGF-1 levels in the same cocultures
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5In addition we saw no increase in IGF-1 mRNA in these lipopolysaccharide-activated cultures although it has been reported
6014IL4interleukin 4IL-42.7in these lipopolysaccharide-activated cultures although it has been reported that IL-4 up-regulated IGF-1 mRNA in both untreated and lipopolysaccharide-activated microglia (
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5lipopolysaccharide-activated cultures although it has been reported that IL-4 up-regulated IGF-1 mRNA in both untreated and lipopolysaccharide-activated microglia ( Butovsky et
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5Additionally even though free IGF-1 was up-regulated by IL-4 in untreated microglia cocultured with motoneurons
6014IL4interleukin 4IL-42.7Additionally even though free IGF-1 was up-regulated by IL-4 in untreated microglia cocultured with motoneurons IL-4 did not increase
6014IL4interleukin 4IL-42.7was up-regulated by IL-4 in untreated microglia cocultured with motoneurons IL-4 did not increase motoneuron survival under the same conditions
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5Finally we also found a poor correlation between free IGF-1 levels and motoneuron survival in MN Mc cocultures
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5Therefore these results suggest that IGF-1 may not be the primary mechanism of IL-4 neuroprotection
6014IL4interleukin 4IL-42.7suggest that IGF-1 may not be the primary mechanism of IL-4 neuroprotection
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5Firstly IGF-1 is clearly a generally neuroprotective trophic factor that has been
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5that there may have been an immediate local increase in IGF-1 that was quickly sequestered by binding proteins it is likely
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5sequestered by binding proteins it is likely that the free IGF-1 levels induced by IL-4 either before or after lipopolysaccharide in
6014IL4interleukin 4IL-42.7it is likely that the free IGF-1 levels induced by IL-4 either before or after lipopolysaccharide in our system were not
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5Secondly because free IGF-1 levels are determined by total IGF-1 and the levels of
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5Secondly because free IGF-1 levels are determined by total IGF-1 and the levels of the five known binding proteins (
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5et al 2005 there may have been increases in both IGF-1 and one or more of the binding proteins that resulted
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5binding proteins that resulted in no net change in free IGF-1
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5Finally it is possible that neurotrophic factors other than IGF-1 may enhance the neuroprotective actions of this anti-inflammatory cytokine
6014IL4interleukin 4IL-42.7Several studies have demonstrated the beneficial effects of IL-4 in the CNS
6014IL4interleukin 4IL-42.7(1993 1993 reported that IL-4 blocked microglia-mediated cerebral neuron injury
6014IL4interleukin 4IL-42.7It has also been reported that IL-4 increased neuronal survival in hippocampal mixed cultures ( Araujo and
6014IL4interleukin 4IL-42.7IL-4 delivered by transfected cells inhibited the progression and the severity
6014IL4interleukin 4IL-42.7Furthermore gene transfer of IL-4 to the retina enhanced the survival of axotomized retinal ganglion
6014IL4interleukin 4IL-42.7While these studies demonstrate that IL-4 can be neuroprotective few studies have examined the mechanisms of
6014IL4interleukin 4IL-42.7Our present study provides evidence that IL-4 protects motoneurons from injury partly by decreasing free radicals released
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5free radicals released from microglia and may not involve free IGF-1
6014IL4interleukin 4IL-42.7Whereas one paper reported that IL-4 did not significantly suppress nitric oxide production from microglia when
5438IFNGinterferon, gammaIFN-G0.3from microglia when given 24 h after the triggering signals IFN-G and lipopolysaccharide ( Chao et al 1993 we found that
6014IL4interleukin 4IL-42.7and lipopolysaccharide ( Chao et al 1993 we found that IL-4 was more neuroprotective when added 2 h after lipopolysaccharide than
6014IL4interleukin 4IL-42.7Besides IL-4 IL-10 and transforming growth factor-beta (TGF-B) TGF-B are two other
5962IL10interleukin 10IL-101.3Besides IL-4 IL-10 and transforming growth factor-beta (TGF-B) TGF-B are two other major
11766TGFB1transforming growth factor, beta 1TGF-B2.2Besides IL-4 IL-10 and transforming growth factor-beta (TGF-B) TGF-B are two other major anti-inflammatory cytokines
5962IL10interleukin 10IL-101.3We had detected the effects of IL-10 and TGF-B in our MN Mc coculture system as well
11766TGFB1transforming growth factor, beta 1TGF-B2.2We had detected the effects of IL-10 and TGF-B in our MN Mc coculture system as well as IL-4
6014IL4interleukin 4IL-42.7TGF-B in our MN Mc coculture system as well as IL-4
11766TGFB1transforming growth factor, beta 1TGF-B2.2We did not observe a neuroprotective effect of TGF-B in the cultures (data data not shown
6014IL4interleukin 4IL-42.7We found that IL-4 had stronger protective effects on motoneurons than IL-10
5962IL10interleukin 10IL-101.3found that IL-4 had stronger protective effects on motoneurons than IL-10
6014IL4interleukin 4IL-42.7Like IL-4 IL-10 inhibited iNOS expression and nitric oxide production from microglia
5962IL10interleukin 10IL-101.3Like IL-4 IL-10 inhibited iNOS expression and nitric oxide production from microglia (data
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)iNOS3.7Like IL-4 IL-10 inhibited iNOS expression and nitric oxide production from microglia (data data not
5962IL10interleukin 10IL-101.3However IL-10 increased microglial release and also decreased rather than increased IGF-1
5464IGF1insulin-like growth factor 1 (somatomedin C)IGF-13.5IL-10 increased microglial release and also decreased rather than increased IGF-1 release from microglia (data data not shown
5962IL10interleukin 10IL-101.3Further studies are clearly necessary to define the mechanism of IL-10 effects
6014IL4interleukin 4IL-42.7One potential source of the neuroprotective signal IL-4 is Th2 lymphocytes
1678CD4CD4 moleculeCD40.3Furthermore CD4 T cells have been documented to enhance facial motoneuron survival
6014IL4interleukin 4IL-42.7Our present data suggest that IL-4 may be one of the significant signals in T-cell-mediated neuroprotection
6014IL4interleukin 4IL-42.7increase the presence of Th2 lymphocytes and the release of IL-4 would be a promising direction for treatment of neurodegenerative disorders
6014IL4interleukin 4IL-42.7In vivo studies will clearly be required to determine whether IL-4 achieves neuroprotection by mitigating the neurotoxic effects of microglia
5464IGF1insulin-like growth factor 1 (somatomedin C)insulin like growth factor 11.0tivated by lipopolysaccharide as well as to activated microglia cocultured with primary motoneurons. lipopolysaccharide increased nitric oxide and superoxide o 2 _amp_middot;_amp_#8722; and decreased insulin like growth factor 1 igf 1 release from microglial cultures and induced motoneuron injury in microglia motoneuron cocultures.
6014IL4interleukin 4interleukin 41.0to explore the neuroprotective mechanism of anti inflammatory cytokines we applied interleukin 4 il 4 to primary microglial cultures activated by lipopolysaccharide as well as to activated microglia cocultured with primary motoneurons. lipopolysaccharide increased nitric oxide and superoxide o 2
6014IL4interleukin 4il 41.0to explore the neuroprotective mechanism of anti inflammatory cytokines we applied interleukin 4 il 4 to primary microglial cultures activated by lipopolysaccharide as well as to activated microglia cocultured with primary motoneurons. lipopolysaccharide increased nitric oxide and superoxide o 2 _amp
6014IL4interleukin 4il 41.0il_amp_#8722;4 interaction with microglial il 4 receptors suppressed and nitric oxide release and lessened lipopolysaccharide induced microglia mediated motoneuron injury.
6014IL4interleukin 4il 41.0although il 4 enhanced release of free igf 1 from microglia in the absence of lipopolysaccharide it did not enhance free igf 1 release in the presence of lipopolysaccharide.
6014IL4interleukin 4il 41.0these data suggest that il 4 may provide a significant immunomodulatory signal which can protect against microglia mediated neurotoxicity by suppressing the production and release of free radicals.
6014IL4interleukin 4interleukin 41.0we hypothesized that the th2 lymphocyte derived anti inflammatory cytokine interleukin 4 il 4 may be one of the potential neuroprotective signals.
6014IL4interleukin 4il 41.0we hypothesized that the th2 lymphocyte derived anti inflammatory cytokine interleukin 4 il 4 may be one of the potential neuroprotective signals.
6014IL4interleukin 4il 41.0il 4 is an important immunosuppressive mediator in the cns.
5992IL1Binterleukin 1, betail 11.04 has also been shown to reduce the production of pro inflammatory cytokines such as il 6 il 8 tumor necrosis factor alpha tnf a mip 1a and rantes in glial cultures treated with lipopolysaccharide or il 1 ledeboer et al 2000 ; ehrlich et al 1998 .
11892TNFtumor necrosis factor (TNF superfamily, member 2)tumor necrosis factor alpha1.0il 4 has also been shown to reduce the production of pro inflammatory cytokines such as il 6 il 8 tumor necrosis factor alpha tnf a mip 1a and rantes in glial cultures treated with lipopolysaccharide or il 1 ledeboer et al 2000 ; ehrlich et al 1998 .
6014IL4interleukin 4il 41.0il 4 has also been shown to reduce the production of pro inflammatory cytokines such as il 6 il 8 tumor necrosis factor alpha tnf a mip 1a and rantes in glial cultures treated with lipopolysaccharide or i
6025IL8interleukin 8il 81.0il 4 has also been shown to reduce the production of pro inflammatory cytokines such as il 6 il 8 tumor necrosis factor alpha tnf a mip 1a and rantes in glial cultures treated with lipopolysaccharide or il 1 ledeboer et al 2000 ; ehrlich et al 1998 .
6018IL6interleukin 6 (interferon, beta 2)il 61.0il 4 has also been shown to reduce the production of pro inflammatory cytokines such as il 6 il 8 tumor necrosis factor alpha tnf a mip 1a and rantes in glial cultures treated with lipopolysaccharide or il 1 ledeboer et al 2000 ; ehrlich et al 1998 .
5464IGF1insulin-like growth factor 1 (somatomedin C)insulin like growth factor 11.0recently it has been documented that protection of il 4 was attributed to down regulation of tnf a and up regulation of insulin like growth factor 1 igf 1 from microglia butovsky et al 2005 2006 .
6014IL4interleukin 4il 41.0recently it has been documented that protection of il 4 was attributed to down regulation of tnf a and up regulation of insulin like growth factor 1 igf 1 from microglia butovsky et al 2005 2006 .
6014IL4interleukin 4il 41.0in the present study we examined the effects of il 4 in primary microglia cultures and in microglia cocultured with primary motoneurons.
727ARTNarteminneurotrophic factor1.0following activation with lipopolysaccharide microglia were noted to release more nitric oxide and and lower levels of free igf 1 an active form of the neurotrophic factor.
6014IL4interleukin 4il 41.0the addition of il 4 protected against the activated microglia mediated motoneuron injury by reducing nitric oxide production and suppressing microglial production.
399ALBalbuminserum albumin1.0after centrifugation at 800 g for 15 min the sharp band on top of the histodenz cushion was collected and centrifuged through a 4% bovine serum albumin bsa cushion at 400 g for 10 min.
6081INSinsulininsulin1.0the cells were resuspended in the l 15 culture medium supplemented with sodium bicarbonate 0.2% glucose 3.6 mg/ml progesterone 20 n m insulin 5 _amp_#x03bc;g/ml putrescine 0.1 m m conalbumin 0.1 mg/ml sodium selenite 30 n m penicillin 100 iu/ml streptomycin 100 _amp_#x03bc;g/ml and horse serum 2% .
6014IL4interleukin 4il 41.0il 4 additions were made either 2 h prior to or after the addition of lipopolysaccharide.
6014IL4interleukin 4il 41.0noc 18 nitric oxide donor and pyrogallol donor were added to culture medium 1 h after the addition of il 4 and lipopolysaccharide.
7809NGFRnerve growth factor receptor (TNFR superfamily, member 16)low affinity nerve growth factor receptor1.0as a specific marker for motoneurons p75 the low affinity nerve growth factor receptor immunocytochemistry provides a clear labeling of the motoneurons over microglia layer and simplifies identification and counting under brightfield microscope.
6014IL4interleukin 4il 41.0briefly microglia cultures 4 _amp_#x00d7; 10 4 cells/well grown in 96 well plates were treated with lipopolysaccharide 1 ng/ml followed by il 4 10 ng/ml 2 h later.
1516CATcatalasecatalase1.0to each well ferricytochrome c 80 _amp_#x03bc; m catalase 10 u/ml lipopolysaccharide 1 ng/ml and il 4 10 ng/ml in hbss were then added.
6014IL4interleukin 4il 41.0to each well ferricytochrome c 80 _amp_#x03bc; m catalase 10 u/ml lipopolysaccharide 1 ng/ml and il 4 10 ng/ml in hbss were then added.
6014IL4interleukin 4il 41.0il 4 protected motoneurons from injury induced by activated microglia to determine the effects of il 4 on microglia mediated motoneuron injury il 4 was added to mn + mc cocultures 2 h before the addition of lipopolysaccharide.
6014IL4interleukin 4il 41.0in the presence of il 4 10 ng/ml neurite length 92.8% _amp_plusmn; 7.82 increased significantly compared with mn + mc + lipopolysaccharide fig 3a b .
6014IL4interleukin 4il 41.0in coculture supernatants levels of nitrite + nitrate increased after 24 h treatment with lipopolysaccharide and il 4 significantly reduced nitric oxide production even in the presence of lipopolysaccharide fig 3c .
6014IL4interleukin 4il 41.0addition of il 4 1 10 ng/ml significantly prevented motoneuron loss induced by lipopolysaccharide activated microglia.
6014IL4interleukin 4il 41.0this protective effect of il 4 was dose dependent fig 3d .
6014IL4interleukin 4il 41.0with increasing concentrations of il 4 nitrite + nitrate levels in the supernatant decreased fig 3e and motoneuron survival increased fig 3d .
6014IL4interleukin 4il 41.0to test whether il 4 has any survival effects on motoneurons alone motoneurons were treated with il 4 10 ng/ml for 48 h.
6014IL4interleukin 4il 41.0the survival of motoneurons with il 4 99.93 _amp_plusmn; 2.48% was similar to survival of motoneurons in the absence of il 4 100% as control .
6014IL4interleukin 4il 41.0therefore il 4 had no direct survival effects on motoneurons supporting that the neuroprotective effects of il 4 in microglia motoneuron cocultures are mediated indirectly through microglia.
6014IL4interleukin 4il 41.0nitric oxide was a key factor modulated by il 4
6014IL4interleukin 4il 41.0to provide further evidence that the neuroprotective effects of il 4 may be mediated by reducing nitric oxide production we plotted the nitrite + nitrate content at different concentrations of il 4.
6014IL4interleukin 4il 41.0in mn + mc cocultures treated with lipopolysaccharide the suppression of nitrite + nitrate generation by il 4 was dose dependent fig 4a .
7873NOS2Anitric oxide synthase 2A (inducible, hepatocytes)nitric oxide synthase1.0we have previously demonstrated that activated microglia produce nitric oxide by up regulation of inducible nitric oxide synthase inos zhao et al 2004 .
6014IL4interleukin 4il 41.0to further define the mechanisms of il 4 neuroprotection microglia were treated with lipopolysaccharide in the presence or absence of il 4.
6014IL4interleukin 4il 41.0figure 5 demonstrates that lipopolysaccharide enhanced inos protein expression and il 4 significantly inhibited inos expression induced by lipopolysaccharide.
6014IL4interleukin 4il 41.0most strikingly the inhibitory effect of il 4 added 2 h after lipopolysaccharide lipopolysaccharide + il 4 was even greater than when il 4 was added 2 h before lipopolysaccharide il 4 + lipopolysaccharide p _lt_ 0.05 .
6014IL4interleukin 4il 41.0il 4 receptor was up regulated by lipopolysaccharide and il 4 mediated neuroprotection was increased to help explain why il 4 was more effective when added after lipopolysaccharide lipopolysaccharide + il 4 expression of il 4 receptor il 4r was assayed by real time rt pcr in microglia cultures fig 6a .
6014IL4interleukin 4il 41.0 was more effective when added after lipopolysaccharide lipopolysaccharide + il 4 expression of il 4 receptor il 4r was assayed by real time rt pcr in microglia cultures fig 6a .
6014IL4interleukin 4il 41.0charide significantly increased the expression of il 4r compared with untreated microglia 9 fold increase p _lt_ 0.001 which provides a possible explanation for why adding lipopolysaccharide prior to il 4 enhanced the protective effects of il 4.
6014IL4interleukin 4il 41.0 enhanced the protective effects of il 4.
6014IL4interleukin 4il 41.0in the absence of lipopolysaccharide il 4 did not significantly change il 4r mrna levels compared with untreated control p = 0.91 .
6014IL4interleukin 4il 41.0furthermore il 4r mrna levels were not statistically changed by il 4 in lipopolysaccharide activated microglia il 4 + lipopolysaccharide compared with the group treated with lipopolysaccharide only p = 0.13 .
6014IL4interleukin 4il 41.0cultures treated with il 4 2 h after the addition of lipopolysaccharide led to 104.6% _amp_plusmn; 7.62 motoneuron survival which was significantly higher than the motoneuron survival 83.5% _amp_plusmn; 4.05 when il 4 was adde
6014IL4interleukin 4il 41.0 2 h after the addition of lipopolysaccharide led to 104.6% _amp_plusmn; 7.62 motoneuron survival which was significantly higher than the motoneuron survival 83.5% _amp_plusmn; 4.05 when il 4 was added 2 h prior to the addition of lipopolysaccharide fig 6b .
6014IL4interleukin 4il 41.0il 4 inhibited superoxide production from microglia the effect of the anti inflammatory cytokine on microglial production is shown in fig. 7 .
6014IL4interleukin 4il 41.0il 4 significantly suppressed the microglial release of in the presence of lipopolysaccharide mc + lipopolysaccharide + il 4: 105.6% _amp_plusmn; 24.5 vs mc + lipopolysaccharide 183.3% _amp_plusmn; 11.8 p = 0.005 and the level of mc + lipopolysaccharide + il 4 was not significantly different from control p = 0.79 .
6014IL4interleukin 4il 41.0: 105.6% _amp_plusmn; 24.5 vs mc + lipopolysaccharide 183.3% _amp_plusmn; 11.8 p = 0.005 and the level of mc + lipopolysaccharide + il 4 was not significantly different from control p = 0.79 .
6014IL4interleukin 4il 41.0exogenous nitric oxide and reversed the neuroprotective effects of il 4 to further prove that neuroprotective effects of il 4 are through reducing nitric oxide and release from microglia exogenous nitric oxide and were added to the motoneuron and microglia cocultures.
6014IL4interleukin 4il 41.0figure 8 showed that in the presence of lipopolysaccharide and il 4 addition of nitric oxide donor noc 18 25 _amp_#x03bc; m and donor pyrogallol pyr 30 _amp_#x03bc; m significantly decreased motoneuron survival to levels similar to those of mn + mc + lipopolysacchari
6014IL4interleukin 4il 41.0p_#x03bc; m significantly decreased motoneuron survival to levels similar to those of mn + mc + lipopolysaccharide showing that exogenous nitric oxide and could reverse the neuroprotective effects of il 4.
6014IL4interleukin 4il 41.0free igf 1 did not increase after addition of il 4 to lipopolysaccharide activated microglia in fig. 1b we noted less free igf 1 in the culture media following microglial activation with lipopolysaccharide.
6014IL4interleukin 4il 41.0to determine whether igf 1 production was influenced by il 4 the levels of free igf 1 were measured in microglia monocultures or mn + mc cocultures.
6014IL4interleukin 4il 41.0in cultures without lipopolysaccharide il 4 significantly increased levels of free igf 1 fig 9a b .
6014IL4interleukin 4il 41.0the free igf 1 of motoneuron only cultures were close to background level in the presence or absence of il 4 data not shown indicating that microglia were the source of the increased free igf 1 after il 4 treatment.
6014IL4interleukin 4il 41.0however the levels of free igf 1 did not change significantly in lipopolysaccharide activated microglia or in mn + mc + lipopolysaccharide cocultures regardless of whether il 4 was added before or after lipopolysaccharide fig 9a b .
6014IL4interleukin 4il 41.0similar results were seen when igf 1 mrna levels were measured with and without il 4 data not shown .
6014IL4interleukin 4il 41.0the present study demonstrates that the anti inflammatory cytokine il 4 can protect motoneurons from injury induced by lipopolysaccharide activated microglia.
6014IL4interleukin 4il 41.0to our knowledge this is the first report that il 4 has beneficial effects on motoneuron injury mediated by microglia.
6014IL4interleukin 4il 41.0our present results reveal a strong negative correlation between nitric oxide levels measured by nitrite + nitrate levels and motoneuron survival in cocultures treated with il 4 and exogenously added nitric oxide reversed neuroprotection of il 4.
6014IL4interleukin 4il 41.0 and exogenously added nitric oxide reversed neuroprotection of il 4.
6014IL4interleukin 4il 41.0these suggest that nitric oxide is a key factor modulated by il 4 in our cell culture system.
6014IL4interleukin 4il 41.0nitric oxide is synthesized by inos in microglia. inos protein expression was down regulated by il 4 in activated microglia when il 4 was given either before or after the triggering signal lipopolysaccharide.
6014IL4interleukin 4il 41.0more strikingly we found that il 4 increased motoneuron survival to a greater extent when added to mn + mc cocultures after lipopolysaccharide than when added prior to lipopolysaccharide.
6014IL4interleukin 4il 41.0in an effort to investigate the cause of this increase we examined the levels of il 4 mrna.
6014IL4interleukin 4il 41.0thus the greater expression of il 4r with the subsequent greater efficacy of the available il 4 could provide one potential explanation of the increased suppressive effects of il 4 on inos expression in activated microglia.
6014IL4interleukin 4il 41.0two studies have reported the inhibitory effects of il 4 on inos in lipopolysaccharide stimulated astrocytes and mixed glial cultures brodie et al 1998 ; kitamura et al 2000 although in their studies the cells were all treated with il 4 first and then stim
6014IL4interleukin 4il 41.0 on inos in lipopolysaccharide stimulated astrocytes and mixed glial cultures brodie et al 1998 ; kitamura et al 2000 although in their studies the cells were all treated with il 4 first and then stimulated cell with other triggering signals.
6014IL4interleukin 4il 41.0our present findings that il 4 also inhibits production in lipopolysaccharide activated microglia and that neuroprotection of il 4 was reversed by exogenous illustrate another potential neuroprotective aspect of this particular anti inflammatory cytokine.
6014IL4interleukin 4il 41.0 1995 reported that il 4 suppressed production in human microglia activated by tnf a or interferon ifn g.
6014IL4interleukin 4il 41.0it has been shown that il 4 reduces formation in macrophages via down regulation of gp9l phox at the mrna level zhou et al 1995 .
14874NOX5NADPH oxidase, EF-hand calcium binding domain 5nadph oxidase1.0however simultaneous activation of inos to produce nitric oxide and nadph oxidase to produce resulted in massive microglia mediated neuronal death mander and brown 2005 .
6014IL4interleukin 4il 41.0the ability of il 4 to suppress the production of both nitric oxide and suggests that il 4 can decrease the formation of peroxynitrite.
6014IL4interleukin 4il 41.0it has also been documented that neuroprotection of il 4 was attributed to down regulation of tnf a butovsky et al 2005 .
6014IL4interleukin 4il 41.0in accordance with this we did find lipopolysaccharide dramatically enhanced tnf a produced by microglia zhao et al 2004 and il 4 significantly decreased tnf a levels in mn + mc + lipopolysaccharide cocultures data not shown .
6014IL4interleukin 4il 41.0therefore down regulation of tnf a is one of mechanisms by which il 4 inhibited nitric oxide and production and protected motoneurons from microglia mediated toxicity.
6014IL4interleukin 4il 41.0another finding in the current study is that il 4 increased free igf 1 the active form of this neuroprotective factor in untreated microglia cultures and that lipopolysaccharide decreased free igf 1.
6014IL4interleukin 4il 41.0at first we hypothesized that the induction of free igf 1 in microglia might be another aspect of il 4 neuroprotection; however our subsequent results suggested it might not be.
6014IL4interleukin 4il 41.0il 4 increased motoneuron survival in both lipopolysaccharide treated mn + mc cocultures; however il 4 did not increase free igf 1 levels in the same cocultures.
6014IL4interleukin 4il 41.0in addition we saw no increase in igf 1 mrna in these lipopolysaccharide activated cultures although it has been reported that il 4 up regulated igf 1 mrna in both untreated and lipopolysaccharide activated microglia butovsky et al 2005 .
6014IL4interleukin 4il 41.0additionally even though free igf 1 was up regulated by il 4 in untreated microglia cocultured with motoneurons il 4 did not increase motoneuron survival under the same conditions.
6014IL4interleukin 4il 41.0therefore these results suggest that igf 1 may not be the primary mechanism of il 4 neuroprotection.
6014IL4interleukin 4il 41.0gh we cannot discount the possibility that there may have been an immediate local increase in igf 1 that was quickly sequestered by binding proteins it is likely that the free igf 1 levels induced by il 4 either before or after lipopolysaccharide in our system were not sufficient to elicit the neuroprotective effects.
6014IL4interleukin 4il 41.0several studies have demonstrated the beneficial effects of il 4 in the cns.
6014IL4interleukin 4il 41.0 1993 reported that il 4 blocked microglia mediated cerebral neuron injury.
6014IL4interleukin 4il 41.0it has also been reported that il 4 increased neuronal survival in hippocampal mixed cultures araujo and cotman 1993 .
6014IL4interleukin 4il 41.0il 4 delivered by transfected cells inhibited the progression and the severity of experimental autoimmune encephalomyelitis shaw et al 1997 ; furlan et al 2001 .
6014IL4interleukin 4il 41.0furthermore gene transfer of il 4 to the retina enhanced the survival of axotomized retinal ganglion cells koeberle et al 2004 .
6014IL4interleukin 4il 41.0while these studies demonstrate that il 4 can be neuroprotective few studies have examined the mechanisms of neuroprotection.
6014IL4interleukin 4il 41.0our present study provides evidence that il 4 protects motoneurons from injury partly by decreasing free radicals released from microglia and may not involve free igf 1.
6014IL4interleukin 4il 41.0whereas one paper reported that il 4 did not significantly suppress nitric oxide production from microglia when given 24 h after the triggering signals ifn g and lipopolysaccharide chao et al 1993 we found that il 4 was more neuroprotec
6014IL4interleukin 4il 41.0 did not significantly suppress nitric oxide production from microglia when given 24 h after the triggering signals ifn g and lipopolysaccharide chao et al 1993 we found that il 4 was more neuroprotective when added 2 h after lipopolysaccharide than before lipopolysaccharide.
5962IL10interleukin 10il 101.0besides il 4 il 10 and transforming growth factor beta tgf b are two other major anti inflammatory cytokines.
11766TGFB1transforming growth factor, beta 1transforming growth factor beta1.0besides il 4 il 10 and transforming growth factor beta tgf b are two other major anti inflammatory cytokines.
6014IL4interleukin 4il 41.0besides il 4 il 10 and transforming growth factor beta tgf b are two other major anti inflammatory cytokines.
5962IL10interleukin 10il 101.0we had detected the effects of il 10 and tgf b in our mn + mc coculture system as well as il 4.
6014IL4interleukin 4il 41.0we had detected the effects of il 10 and tgf b in our mn + mc coculture system as well as il 4.
5962IL10interleukin 10il 101.0we found that il 4 had stronger protective effects on motoneurons than il 10.
6014IL4interleukin 4il 41.0we found that il 4 had stronger protective effects on motoneurons than il 10.
5962IL10interleukin 10il 101.0like il 4 il 10 inhibited inos expression and nitric oxide production from microglia data not shown .
6014IL4interleukin 4il 41.0like il 4 il 10 inhibited inos expression and nitric oxide production from microglia data not shown .
5962IL10interleukin 10il 101.0however il 10 increased microglial release and also decreased rather than increased igf 1 release from microglia data not shown .
5962IL10interleukin 10il 101.0further studies are clearly necessary to define the mechanism of il 10 effects.
6014IL4interleukin 4il 41.0one potential source of the neuroprotective signal il 4 is th2 lymphocytes.
6014IL4interleukin 4il 41.0our present data suggest that il 4 may be one of the significant signals in t cell mediated neuroprotection.
6014IL4interleukin 4il 41.0accordingly immunological strategies to increase the presence of th2 lymphocytes and the release of il 4 would be a promising direction for treatment of neurodegenerative disorders.
6014IL4interleukin 4il 41.0in vivo studies will clearly be required to determine whether il 4 achieves neuroprotection by mitigating the neurotoxic effects of microglia.
6014IL4interleukin 4interleukin 41.0anti inflammatory agents|inflammation mediators|lipopolysaccharides|neuroprotective agents|receptors interleukin 4|nitric oxide|superoxides|interleukin 4|insulin like growth factor i|