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Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages.

Lu P, Li L, Wu Y, Mukaida N, Zhang X - Mol. Vis. (2008)

Bottom Line: To evaluate the roles of CCL3 and its specific chemokine receptors, CCR1 and CCR5, in alkali-induced corneal neovascularization (CNV).Moreover, CCL3 enhanced VEGF expression by murine peritoneal macrophages at both the mRNA and the protein level.In alkali-induced CNV, CCL3 induced macrophages to infiltrate and produce VEGF by binding to CCR5 but not to CCR1 and eventually promoted angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Clinical Immunology Key Laboratory of Jiangsu Province, the First Affilated Hospital of Soochow University, Suzhou, China.

ABSTRACT

Purpose: To evaluate the roles of CCL3 and its specific chemokine receptors, CCR1 and CCR5, in alkali-induced corneal neovascularization (CNV).

Methods: Chemical denudation of corneal and limbal epithelium was performed on wild-type (WT) BALB/c mice and CCL3-, CCR1-, and CCR5-deficienct (knockout [KO]) counterparts. Two weeks after injury CNV was quantified by immunostaining with anti-CD31. Angiogenic factor expression and leukocyte accumulation in the early phase after injury were quantified by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical analysis, respectively.

Results: Alkali injury augmented the intraocular mRNA expression of CCL3 and its receptors, CCR1 and CCR5, together with a transient infiltration of F4/80 positive macrophages and Gr-1 positive neutrophils. Compared with WT mice, CCL3-KO and CCR5-KO mice but not CCR1-KO mice exhibited reduced CNV two weeks after injury both macroscopically and microscopically as evidenced by CD31 positive areas. Concomitantly, the infiltration of F4/80 positive macrophages but not Gr-1 positive neutrophils was significantly attenuated in CCL3-KO mice compared with WT mice. Intracorneal infiltration of CCR5 expressing cells was significantly impaired in CCL3-KO mice compared with WT mice. Alkali injury induced a massive increase in the intraocular mRNA expression of a potent angiogenic factor, vascular endothelial growth factor (VEGF), in WT mice whereas these increments were severely retarded in CCL3-KO mice. Moreover, CCL3 enhanced VEGF expression by murine peritoneal macrophages at both the mRNA and the protein level. Furthermore, topical CCL3 application restored CNV, which was macroscopically and microscopically reduced in CCL3-KO mice after two weeks to levels similar to those found in WT mice.

Conclusions: In alkali-induced CNV, CCL3 induced macrophages to infiltrate and produce VEGF by binding to CCR5 but not to CCR1 and eventually promoted angiogenesis.

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Related in: MedlinePlus

The expression of CCL3 and its receptors in cornea after alkali injury. A: Semi-quantitative RT–PCR was performed to assess mRNA expression of CCL3 and its receptors, CCR1 and CCR5, and the ratios of target gene expression to β-actin were determined. All values represent the mean±SEM of three to five independent measurements. B: Whole eyes were obtained at 0, 2, 4, and 7 days after alkali injury and processed for immunohistochemical analysis using anti-CCL3 (upper panels) or anti-CCR1 antibodies (lower panels). Representative results from five individual animals are shown. Original magnifications, 400X. Scale bar, 50 μm.
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f1: The expression of CCL3 and its receptors in cornea after alkali injury. A: Semi-quantitative RT–PCR was performed to assess mRNA expression of CCL3 and its receptors, CCR1 and CCR5, and the ratios of target gene expression to β-actin were determined. All values represent the mean±SEM of three to five independent measurements. B: Whole eyes were obtained at 0, 2, 4, and 7 days after alkali injury and processed for immunohistochemical analysis using anti-CCL3 (upper panels) or anti-CCR1 antibodies (lower panels). Representative results from five individual animals are shown. Original magnifications, 400X. Scale bar, 50 μm.

Mentions: We first examined the expression of CCL3, a ligand for CCR5 and CCR1, in corneas after alkali-induced injury. CCL3 mRNA was barely detectable in untreated eyes but was markedly increased after alkali injury (Figure 1A). Concomitantly, CCL3 protein was immunohistochemically detected in epithelial cells and infiltrating cells after alkali injury but not in untreated eyes (Figure 1B). Moreover, alkali injury markedly augmented the mRNA expression of specific receptors for CCL3, CCR1, and CCR5 (Figure 1A). Furthermore, immunohistochemical analysis demonstrated the infiltration of CCR1 expressing leukocytes, which started two days after the injury and increased thereafter (Figure 1B). These observations suggest that alkali injury induced intracorneal production of CCL3, which in turn attracted CCR1 expressing or CCR5 expressing leukocytes into the cornea.


Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages.

Lu P, Li L, Wu Y, Mukaida N, Zhang X - Mol. Vis. (2008)

The expression of CCL3 and its receptors in cornea after alkali injury. A: Semi-quantitative RT–PCR was performed to assess mRNA expression of CCL3 and its receptors, CCR1 and CCR5, and the ratios of target gene expression to β-actin were determined. All values represent the mean±SEM of three to five independent measurements. B: Whole eyes were obtained at 0, 2, 4, and 7 days after alkali injury and processed for immunohistochemical analysis using anti-CCL3 (upper panels) or anti-CCR1 antibodies (lower panels). Representative results from five individual animals are shown. Original magnifications, 400X. Scale bar, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2529469&req=5

f1: The expression of CCL3 and its receptors in cornea after alkali injury. A: Semi-quantitative RT–PCR was performed to assess mRNA expression of CCL3 and its receptors, CCR1 and CCR5, and the ratios of target gene expression to β-actin were determined. All values represent the mean±SEM of three to five independent measurements. B: Whole eyes were obtained at 0, 2, 4, and 7 days after alkali injury and processed for immunohistochemical analysis using anti-CCL3 (upper panels) or anti-CCR1 antibodies (lower panels). Representative results from five individual animals are shown. Original magnifications, 400X. Scale bar, 50 μm.
Mentions: We first examined the expression of CCL3, a ligand for CCR5 and CCR1, in corneas after alkali-induced injury. CCL3 mRNA was barely detectable in untreated eyes but was markedly increased after alkali injury (Figure 1A). Concomitantly, CCL3 protein was immunohistochemically detected in epithelial cells and infiltrating cells after alkali injury but not in untreated eyes (Figure 1B). Moreover, alkali injury markedly augmented the mRNA expression of specific receptors for CCL3, CCR1, and CCR5 (Figure 1A). Furthermore, immunohistochemical analysis demonstrated the infiltration of CCR1 expressing leukocytes, which started two days after the injury and increased thereafter (Figure 1B). These observations suggest that alkali injury induced intracorneal production of CCL3, which in turn attracted CCR1 expressing or CCR5 expressing leukocytes into the cornea.

Bottom Line: To evaluate the roles of CCL3 and its specific chemokine receptors, CCR1 and CCR5, in alkali-induced corneal neovascularization (CNV).Moreover, CCL3 enhanced VEGF expression by murine peritoneal macrophages at both the mRNA and the protein level.In alkali-induced CNV, CCL3 induced macrophages to infiltrate and produce VEGF by binding to CCR5 but not to CCR1 and eventually promoted angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Clinical Immunology Key Laboratory of Jiangsu Province, the First Affilated Hospital of Soochow University, Suzhou, China.

ABSTRACT

Purpose: To evaluate the roles of CCL3 and its specific chemokine receptors, CCR1 and CCR5, in alkali-induced corneal neovascularization (CNV).

Methods: Chemical denudation of corneal and limbal epithelium was performed on wild-type (WT) BALB/c mice and CCL3-, CCR1-, and CCR5-deficienct (knockout [KO]) counterparts. Two weeks after injury CNV was quantified by immunostaining with anti-CD31. Angiogenic factor expression and leukocyte accumulation in the early phase after injury were quantified by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical analysis, respectively.

Results: Alkali injury augmented the intraocular mRNA expression of CCL3 and its receptors, CCR1 and CCR5, together with a transient infiltration of F4/80 positive macrophages and Gr-1 positive neutrophils. Compared with WT mice, CCL3-KO and CCR5-KO mice but not CCR1-KO mice exhibited reduced CNV two weeks after injury both macroscopically and microscopically as evidenced by CD31 positive areas. Concomitantly, the infiltration of F4/80 positive macrophages but not Gr-1 positive neutrophils was significantly attenuated in CCL3-KO mice compared with WT mice. Intracorneal infiltration of CCR5 expressing cells was significantly impaired in CCL3-KO mice compared with WT mice. Alkali injury induced a massive increase in the intraocular mRNA expression of a potent angiogenic factor, vascular endothelial growth factor (VEGF), in WT mice whereas these increments were severely retarded in CCL3-KO mice. Moreover, CCL3 enhanced VEGF expression by murine peritoneal macrophages at both the mRNA and the protein level. Furthermore, topical CCL3 application restored CNV, which was macroscopically and microscopically reduced in CCL3-KO mice after two weeks to levels similar to those found in WT mice.

Conclusions: In alkali-induced CNV, CCL3 induced macrophages to infiltrate and produce VEGF by binding to CCR5 but not to CCR1 and eventually promoted angiogenesis.

Show MeSH
Related in: MedlinePlus