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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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Effects of retinal neuronal monocyte chemoattractant protein-1 (MCP-1) on activated microglial cells. A: Tumor necrosis factor-α (TNF-α) mRNA from microglia was significantly stimulated by exogenous MCP-1 and CC-chemokine receptor 2 (CCR2) knockdown, which led to downregulation of TNF-α mRNA from microglia. B: The concentrations of soluble TNF-α were significantly stimulated by exogenous MCP-1. In addition, CCR2 knockdown led to downregulation of TNF-α release from microglia induced by exogenous MCP-1. C: The expression of TNF-α mRNA was significantly increased by neuronal MCP-1 in the retinal neuron–microglia Transwell culture system. In addition, TNF-α mRNA expression was reduced when the MCP-1 blocking peptide and CCR2 siRNA were used. However, the incubation of control immunoglobulin G (IgG) or control siRNA did not reduce the TNF-α production. D: Enzyme-linked immunosorbent assay (ELISA) was used to measure the soluble TNF-α concentration in the retinal neuron–microglia Transwell culture system. When cultured with advanced glycation end products (AGEs), the retinal neurons had a markedly increased effect on retinal microglial activation in the Transwell culture system. In addition, the concentration of soluble TNF-α was decreased when the MCP-1 blocking peptide and CCR2 siRNA were used. However, the incubation of control IgG or control siRNA did not reduce TNF-α production. Data shown are the mean±SD (**p<0.01).
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f2: Effects of retinal neuronal monocyte chemoattractant protein-1 (MCP-1) on activated microglial cells. A: Tumor necrosis factor-α (TNF-α) mRNA from microglia was significantly stimulated by exogenous MCP-1 and CC-chemokine receptor 2 (CCR2) knockdown, which led to downregulation of TNF-α mRNA from microglia. B: The concentrations of soluble TNF-α were significantly stimulated by exogenous MCP-1. In addition, CCR2 knockdown led to downregulation of TNF-α release from microglia induced by exogenous MCP-1. C: The expression of TNF-α mRNA was significantly increased by neuronal MCP-1 in the retinal neuron–microglia Transwell culture system. In addition, TNF-α mRNA expression was reduced when the MCP-1 blocking peptide and CCR2 siRNA were used. However, the incubation of control immunoglobulin G (IgG) or control siRNA did not reduce the TNF-α production. D: Enzyme-linked immunosorbent assay (ELISA) was used to measure the soluble TNF-α concentration in the retinal neuron–microglia Transwell culture system. When cultured with advanced glycation end products (AGEs), the retinal neurons had a markedly increased effect on retinal microglial activation in the Transwell culture system. In addition, the concentration of soluble TNF-α was decreased when the MCP-1 blocking peptide and CCR2 siRNA were used. However, the incubation of control IgG or control siRNA did not reduce TNF-α production. Data shown are the mean±SD (**p<0.01).

Mentions: The expression of TNF-α mRNA in the primary retinal microglia was analyzed with real-time PCR (Figure 2A,C), and the concentration of soluble TNF-α in the culture medium was measured with an ELISA kit (Figure 2B,D). A cultured retinal microglia model was developed to characterize the microglial response to recombinant rat MCP-1 (Peprotech, Rocky Hill, NJ) treatment. As shown in Figure 2A, TNF-α mRNA were set to 100%, when the cells were cultured with culture medium alone for 24 h and used as a control. With the use of this model, incubation with exogenous MCP-1 significantly stimulated TNF-α production from microglia (Figure 2A,B). In addition, CCR2 knockdown led to downregulation of TNF-α release from microglia induced by exogenous MCP-1 in this model (Figure 2B).


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)

Effects of retinal neuronal monocyte chemoattractant protein-1 (MCP-1) on activated microglial cells. A: Tumor necrosis factor-α (TNF-α) mRNA from microglia was significantly stimulated by exogenous MCP-1 and CC-chemokine receptor 2 (CCR2) knockdown, which led to downregulation of TNF-α mRNA from microglia. B: The concentrations of soluble TNF-α were significantly stimulated by exogenous MCP-1. In addition, CCR2 knockdown led to downregulation of TNF-α release from microglia induced by exogenous MCP-1. C: The expression of TNF-α mRNA was significantly increased by neuronal MCP-1 in the retinal neuron–microglia Transwell culture system. In addition, TNF-α mRNA expression was reduced when the MCP-1 blocking peptide and CCR2 siRNA were used. However, the incubation of control immunoglobulin G (IgG) or control siRNA did not reduce the TNF-α production. D: Enzyme-linked immunosorbent assay (ELISA) was used to measure the soluble TNF-α concentration in the retinal neuron–microglia Transwell culture system. When cultured with advanced glycation end products (AGEs), the retinal neurons had a markedly increased effect on retinal microglial activation in the Transwell culture system. In addition, the concentration of soluble TNF-α was decreased when the MCP-1 blocking peptide and CCR2 siRNA were used. However, the incubation of control IgG or control siRNA did not reduce TNF-α production. Data shown are the mean±SD (**p<0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4016805&req=5

f2: Effects of retinal neuronal monocyte chemoattractant protein-1 (MCP-1) on activated microglial cells. A: Tumor necrosis factor-α (TNF-α) mRNA from microglia was significantly stimulated by exogenous MCP-1 and CC-chemokine receptor 2 (CCR2) knockdown, which led to downregulation of TNF-α mRNA from microglia. B: The concentrations of soluble TNF-α were significantly stimulated by exogenous MCP-1. In addition, CCR2 knockdown led to downregulation of TNF-α release from microglia induced by exogenous MCP-1. C: The expression of TNF-α mRNA was significantly increased by neuronal MCP-1 in the retinal neuron–microglia Transwell culture system. In addition, TNF-α mRNA expression was reduced when the MCP-1 blocking peptide and CCR2 siRNA were used. However, the incubation of control immunoglobulin G (IgG) or control siRNA did not reduce the TNF-α production. D: Enzyme-linked immunosorbent assay (ELISA) was used to measure the soluble TNF-α concentration in the retinal neuron–microglia Transwell culture system. When cultured with advanced glycation end products (AGEs), the retinal neurons had a markedly increased effect on retinal microglial activation in the Transwell culture system. In addition, the concentration of soluble TNF-α was decreased when the MCP-1 blocking peptide and CCR2 siRNA were used. However, the incubation of control IgG or control siRNA did not reduce TNF-α production. Data shown are the mean±SD (**p<0.01).
Mentions: The expression of TNF-α mRNA in the primary retinal microglia was analyzed with real-time PCR (Figure 2A,C), and the concentration of soluble TNF-α in the culture medium was measured with an ELISA kit (Figure 2B,D). A cultured retinal microglia model was developed to characterize the microglial response to recombinant rat MCP-1 (Peprotech, Rocky Hill, NJ) treatment. As shown in Figure 2A, TNF-α mRNA were set to 100%, when the cells were cultured with culture medium alone for 24 h and used as a control. With the use of this model, incubation with exogenous MCP-1 significantly stimulated TNF-α production from microglia (Figure 2A,B). In addition, CCR2 knockdown led to downregulation of TNF-α release from microglia induced by exogenous MCP-1 in this model (Figure 2B).

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