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

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.

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).

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.

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.