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Retinal neuronal MCP-1 induced by AGEs stimulates TNF-α expression in rat microglia via p38, ERK, and NF-κB pathways.

Dong N, Chang L, Wang B, Chu L - Mol. Vis. (2014)

Bottom Line: The ability of neuronal MCP-1 to stimulate microglia activation was examined by preexposing the retinal neurons to AGEs and an MCP-1 antibody or by pretreating microglia with AGEs and siRNA specific for CC-chemokine receptor 2 (CCR2) knockdowns.Additionally, we investigated the effects of microglial activation on neuronal MCP-1-induced nuclear factor-κB (NF-κB) activation and phosphorylation of mitogen-activated protein kinases (MAPKs).This study indicates that TNF-α was released from the activated microglia induced by retinal neuronal MCP-1 via the p38, ERK, and NF-κB pathways, but not c-Jun N-terminal kinase (JNK), which may be an important finding in diabetic retinopathy pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People's Republic of China.

ABSTRACT

Purpose: Retinal microglia can be activated by retinal neuronal monocyte chemoattractant protein-1 (MCP-1) and play a pivotal role in early retinal degeneration. The current study investigates the pathways via which retinal neuronal MCP-1 stimulates tumor necrosis factor-α (TNF-α) expression in rat microglia.

Methods: Primary rat retinal neurons and microglia were separated and cocultured in a Transwell apparatus. The levels of TNF-α mRNA and soluble TNF-α produced by the microglia in response to advanced glycation end product (AGE)-induced retinal neuronal MCP-1 were measured with real-time PCR and enzyme-linked immunosorbent assay (ELISA). The ability of neuronal MCP-1 to stimulate microglia activation was examined by preexposing the retinal neurons to AGEs and an MCP-1 antibody or by pretreating microglia with AGEs and siRNA specific for CC-chemokine receptor 2 (CCR2) knockdowns. Additionally, we investigated the effects of microglial activation on neuronal MCP-1-induced nuclear factor-κB (NF-κB) activation and phosphorylation of mitogen-activated protein kinases (MAPKs).

Results: Stimulation with AGEs significantly increased the expression of TNF-α mRNA and soluble TNF-α in the microglial cells. Retinal neurons that had been pretreated with AGEs and an MCP-1 antibody or microglia that were CCR2 knockdowns displayed greatly reduced TNF-α secretion. Using signaling pathway-specific inhibitors, we showed that blocking the p38, extracellular signal-regulated kinase (ERK), and NF-κB signaling pathways significantly reduced the expression of TNF-α by retinal neuronal MCP-1-stimulated microglia.

Conclusions: This study indicates that TNF-α was released from the activated microglia induced by retinal neuronal MCP-1 via the p38, ERK, and NF-κB pathways, but not c-Jun N-terminal kinase (JNK), which may be an important finding in diabetic retinopathy pathogenesis.

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Related in: MedlinePlus

Tumor necrosis factor-α (TNF-α) was released from the activated microglia induced by retinal neuronal monocyte chemoattractant protein-1 (MCP-1) via the nuclear factor-κB pathways. A: Real-time PCR was used to measure TNF-α mRNA expression. Dose-dependent inhibition of the expression of TNF-α mRNA was induced by retinal neuronal MCP-1 in the retinal neuron–microglia Transwell culture system by nuclear factor-κB (NF-κB) inhibitors. B: Enzyme-linked immunosorbent assay (ELISA) was used to measure the soluble TNF-α concentration. Dose-dependent inhibition of the expression of soluble TNF-α was induced by NF-κB inhibitors. C: Western blot analysis of NF-κB p50 and p65 subunits in nuclear fractions of microglial cells treated with advanced glycation end products (AGEs; 750 μg/ml) in the presence or absence of anti-MCP-1. The levels of the p50 and p65 subunits of NF-κB from the microglial nuclear fractions increased by retinal neuronal MCP-1; however, anti-MCP-1 led to downregulation (**p<0.01). D: Purified microglia were stained with fluorescein isothiocyanate (FITC)-CD11b (green), and the expression of TNF-α was labeled with PE (red). The number of CD11b and TNF-α double-stained cells (activated microglia) decreased markedly after AGE treatment with NF-κB inhibitors (control: 11.33±3.56 cells/microscopic visual field; AGE: 26.36±4.52 cells/microscopic visual field; NF-κB inhibitors: 17.89±2.87 cells/microscopic visual field; p=0.015).
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f6: Tumor necrosis factor-α (TNF-α) was released from the activated microglia induced by retinal neuronal monocyte chemoattractant protein-1 (MCP-1) via the nuclear factor-κB pathways. A: Real-time PCR was used to measure TNF-α mRNA expression. Dose-dependent inhibition of the expression of TNF-α mRNA was induced by retinal neuronal MCP-1 in the retinal neuron–microglia Transwell culture system by nuclear factor-κB (NF-κB) inhibitors. B: Enzyme-linked immunosorbent assay (ELISA) was used to measure the soluble TNF-α concentration. Dose-dependent inhibition of the expression of soluble TNF-α was induced by NF-κB inhibitors. C: Western blot analysis of NF-κB p50 and p65 subunits in nuclear fractions of microglial cells treated with advanced glycation end products (AGEs; 750 μg/ml) in the presence or absence of anti-MCP-1. The levels of the p50 and p65 subunits of NF-κB from the microglial nuclear fractions increased by retinal neuronal MCP-1; however, anti-MCP-1 led to downregulation (**p<0.01). D: Purified microglia were stained with fluorescein isothiocyanate (FITC)-CD11b (green), and the expression of TNF-α was labeled with PE (red). The number of CD11b and TNF-α double-stained cells (activated microglia) decreased markedly after AGE treatment with NF-κB inhibitors (control: 11.33±3.56 cells/microscopic visual field; AGE: 26.36±4.52 cells/microscopic visual field; NF-κB inhibitors: 17.89±2.87 cells/microscopic visual field; p=0.015).

Mentions: Treatment with PDTC (an NF-κB inhibitor) significantly decreased the expression of TNF-α mRNA (Figure 6A) and inhibited soluble TNF-α release (Figure 6B) in a dose-dependent manner. The levels of the p50 and p65 subunits of NF-κB from the microglial nuclear fractions induced by AGEs increased in the retinal neuron–microglia Transwell culture system (Figure 6C). However, anti-MCP-1 led to attenuation of the nuclear translocation of NF-κB induced by retinal neuronal MCP-1 (Figure 6C). The NF-кB inhibitor downregulated the number of CD11b and TNF-α double-stained cells (Figure 6D).


Retinal neuronal MCP-1 induced by AGEs stimulates TNF-α expression in rat microglia via p38, ERK, and NF-κB pathways.

Dong N, Chang L, Wang B, Chu L - Mol. Vis. (2014)

Tumor necrosis factor-α (TNF-α) was released from the activated microglia induced by retinal neuronal monocyte chemoattractant protein-1 (MCP-1) via the nuclear factor-κB pathways. A: Real-time PCR was used to measure TNF-α mRNA expression. Dose-dependent inhibition of the expression of TNF-α mRNA was induced by retinal neuronal MCP-1 in the retinal neuron–microglia Transwell culture system by nuclear factor-κB (NF-κB) inhibitors. B: Enzyme-linked immunosorbent assay (ELISA) was used to measure the soluble TNF-α concentration. Dose-dependent inhibition of the expression of soluble TNF-α was induced by NF-κB inhibitors. C: Western blot analysis of NF-κB p50 and p65 subunits in nuclear fractions of microglial cells treated with advanced glycation end products (AGEs; 750 μg/ml) in the presence or absence of anti-MCP-1. The levels of the p50 and p65 subunits of NF-κB from the microglial nuclear fractions increased by retinal neuronal MCP-1; however, anti-MCP-1 led to downregulation (**p<0.01). D: Purified microglia were stained with fluorescein isothiocyanate (FITC)-CD11b (green), and the expression of TNF-α was labeled with PE (red). The number of CD11b and TNF-α double-stained cells (activated microglia) decreased markedly after AGE treatment with NF-κB inhibitors (control: 11.33±3.56 cells/microscopic visual field; AGE: 26.36±4.52 cells/microscopic visual field; NF-κB inhibitors: 17.89±2.87 cells/microscopic visual field; p=0.015).
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f6: Tumor necrosis factor-α (TNF-α) was released from the activated microglia induced by retinal neuronal monocyte chemoattractant protein-1 (MCP-1) via the nuclear factor-κB pathways. A: Real-time PCR was used to measure TNF-α mRNA expression. Dose-dependent inhibition of the expression of TNF-α mRNA was induced by retinal neuronal MCP-1 in the retinal neuron–microglia Transwell culture system by nuclear factor-κB (NF-κB) inhibitors. B: Enzyme-linked immunosorbent assay (ELISA) was used to measure the soluble TNF-α concentration. Dose-dependent inhibition of the expression of soluble TNF-α was induced by NF-κB inhibitors. C: Western blot analysis of NF-κB p50 and p65 subunits in nuclear fractions of microglial cells treated with advanced glycation end products (AGEs; 750 μg/ml) in the presence or absence of anti-MCP-1. The levels of the p50 and p65 subunits of NF-κB from the microglial nuclear fractions increased by retinal neuronal MCP-1; however, anti-MCP-1 led to downregulation (**p<0.01). D: Purified microglia were stained with fluorescein isothiocyanate (FITC)-CD11b (green), and the expression of TNF-α was labeled with PE (red). The number of CD11b and TNF-α double-stained cells (activated microglia) decreased markedly after AGE treatment with NF-κB inhibitors (control: 11.33±3.56 cells/microscopic visual field; AGE: 26.36±4.52 cells/microscopic visual field; NF-κB inhibitors: 17.89±2.87 cells/microscopic visual field; p=0.015).
Mentions: Treatment with PDTC (an NF-κB inhibitor) significantly decreased the expression of TNF-α mRNA (Figure 6A) and inhibited soluble TNF-α release (Figure 6B) in a dose-dependent manner. The levels of the p50 and p65 subunits of NF-κB from the microglial nuclear fractions induced by AGEs increased in the retinal neuron–microglia Transwell culture system (Figure 6C). However, anti-MCP-1 led to attenuation of the nuclear translocation of NF-κB induced by retinal neuronal MCP-1 (Figure 6C). The NF-кB inhibitor downregulated the number of CD11b and TNF-α double-stained cells (Figure 6D).

Bottom Line: The ability of neuronal MCP-1 to stimulate microglia activation was examined by preexposing the retinal neurons to AGEs and an MCP-1 antibody or by pretreating microglia with AGEs and siRNA specific for CC-chemokine receptor 2 (CCR2) knockdowns.Additionally, we investigated the effects of microglial activation on neuronal MCP-1-induced nuclear factor-κB (NF-κB) activation and phosphorylation of mitogen-activated protein kinases (MAPKs).This study indicates that TNF-α was released from the activated microglia induced by retinal neuronal MCP-1 via the p38, ERK, and NF-κB pathways, but not c-Jun N-terminal kinase (JNK), which may be an important finding in diabetic retinopathy pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People's Republic of China.

ABSTRACT

Purpose: Retinal microglia can be activated by retinal neuronal monocyte chemoattractant protein-1 (MCP-1) and play a pivotal role in early retinal degeneration. The current study investigates the pathways via which retinal neuronal MCP-1 stimulates tumor necrosis factor-α (TNF-α) expression in rat microglia.

Methods: Primary rat retinal neurons and microglia were separated and cocultured in a Transwell apparatus. The levels of TNF-α mRNA and soluble TNF-α produced by the microglia in response to advanced glycation end product (AGE)-induced retinal neuronal MCP-1 were measured with real-time PCR and enzyme-linked immunosorbent assay (ELISA). The ability of neuronal MCP-1 to stimulate microglia activation was examined by preexposing the retinal neurons to AGEs and an MCP-1 antibody or by pretreating microglia with AGEs and siRNA specific for CC-chemokine receptor 2 (CCR2) knockdowns. Additionally, we investigated the effects of microglial activation on neuronal MCP-1-induced nuclear factor-κB (NF-κB) activation and phosphorylation of mitogen-activated protein kinases (MAPKs).

Results: Stimulation with AGEs significantly increased the expression of TNF-α mRNA and soluble TNF-α in the microglial cells. Retinal neurons that had been pretreated with AGEs and an MCP-1 antibody or microglia that were CCR2 knockdowns displayed greatly reduced TNF-α secretion. Using signaling pathway-specific inhibitors, we showed that blocking the p38, extracellular signal-regulated kinase (ERK), and NF-κB signaling pathways significantly reduced the expression of TNF-α by retinal neuronal MCP-1-stimulated microglia.

Conclusions: This study indicates that TNF-α was released from the activated microglia induced by retinal neuronal MCP-1 via the p38, ERK, and NF-κB pathways, but not c-Jun N-terminal kinase (JNK), which may be an important finding in diabetic retinopathy pathogenesis.

Show MeSH
Related in: MedlinePlus