Inhibitory effect of CCR3 signal on alkali-induced corneal neovascularization

Abstract

Aim: To investigate the effect of CC chemokine receptor 3 (CCR3) signal on corneal neovascularization (CRNV) induced by alkali burn and to explore its mechanism.

Methods: Specific pathogen-free male BALB/C mice (aged 6-8 weeks) were randomly divided into CCR3-antagonist treated group (experimental group) and control group. CRNV was induced by alkali burn in mice. The time kinetic CCR3 expression in injured corneas was examined by reverse transcription polymerase chain reaction (RT-PCR). CCR3-antagonist (SB-328437 at different concentration of 125µg/mL, 250µg/mL, and 500µg/mL) was locally administrated after alkali injury. The formation of CRNV was assessed by CD31 corneal whole mount staining at two weeks after injury. Monocyte chemotactic protein 1 (MCP-1), monocyte chemotactic protein 3 (MCP-3) expressions in the early phase after injury were quantified and compared by RT-PCR. Macrophage intracorneal accumulation in the early phase after injury was evaluated and compared by immunohistochemistry.

Results: Alkali injury induced the time kinetic intracorneal CCR3 expression. 500µg/mL of CCR3-antagonist treatment in the early phase but not the late phase resulted in significant impaired CRNV as compared to control group (P<0.05). CCR3-antagonist treatment in the early phase significantly reduced the intracorneal MCP-1 and MCP-3 enhancement compare to control group at day 2 and day 4 (P<0.05). Moreover, the number of intracorneal macrophage infiltration in the experimental group was reduced than those in control group at day 4 (P<0.05).

Conclusion: CCR3 signal is involved in alkali-induced CRNV. CCR3-antagonist can inhibit alkali-induced CRNV by reducing the intracorneal MCP-1 and MCP-3 mRNA expression and the intracorneal macrophage infiltration.

Keywords: CCR3; corneal neovascularization; macrophage; monocyte chemotactic protein 1; monocyte chemotactic protein 3.

Figure 1. Expression of CCR3 in corneas after alkali injury.

A: Semi-quantitative RT-PCR to assess mRNA expression of CCR3. Corneas were harvested at the indicated time points, and five corneas at each time point were pooled to extract total RNAs. RT-PCR was performed using the obtained total RNAs; B: The ratios of CCR3 to GAPDH were determined. All values represent mean±SEM of the three to five independent measurements. ^aP<0.05, ^bP<0.01 vs day 0.

Figure 2. Alkali-induced CRNV after CCR3 antagonist treatment in early phase.

A: Macroscopic appearance of different concentration of CCR3-antagonist (125µg/mL, 250µg/mL, 500µg/mL in 2g/L HA) treated and 2g/L HA treated mice eyes 2 weeks after alkali injury in early phase; B: Corneal neovascularization was assessed by CD31 corneal whole mount staining. The percentages of the corneal covered by vessels which were normalized to the total corneas were calculated; C: The relative neovascular areas were compared between treated groups and control group, the 500µg/mL CCR3-antagonist treated mice exhibited significantly decreased CRNV area compared to the control (n=10; t=2.715, ^aP<0.05 vs control group).

Figure 3. Intracorneal MCP-1 and MCP-3 mRNA expression

A: Representative RT-PCR results from three independent experiments of MCP-1 and MCP-3 mRNA expression of 500µg/mL CCR3-antagonist treated and control mice on day 2, day 4 and day 7 after alkali injury; B: Ratios of MCP-1 to GAPDH of CCR3-antagonist treated and control mice determined by RT-PCR at the indicated time intervals (^bP<0.01 vs control group); C: The relative level of MCP-3 mRNA expression at the indicated time between treated group and control. (^bP<0.01 vs control group).

Figure 4. Macrophage intracorneal infiltrations after alkali injury

The numbers of infiltrated F4/80-positive macrophages of 500µg/mL CCR3-antagonist treated mice and control mice at day 2 and day 4 were determined as described in materials and methods and the mean and SEM are shown there. (n=9, ^aP<0.05 vs untreated mice).