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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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Intracorneal CCR5 positive cell infiltration. A: A double-color immunofluorescence analysis of CCR5-expressing cells is illustrated. Corneas were obtained from WT mice 0 and 4 days after the injury. The samples were immunostained with a combination of anti-CCR5 and anti-CCR2 antibodies as described in Methods and observed with fluorescence microscopy (original magnification, 400X). Signals were digitally merged in the right panels. Arrows indicate the double, positively stained cells. Representative results from three independent experiments are shown. B: Corneal tissues from WT mice (left panel) or CCL3-KO mice (right panel) obtained four days after the injury were stained with anti-CCR5 Ab. Scale bar, 100 μm. C: The numbers of intracorneal CCR5 positive cells four days after the injury were determined as described in Methods, and the mean and SEM are shown here (n=5).
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f4: Intracorneal CCR5 positive cell infiltration. A: A double-color immunofluorescence analysis of CCR5-expressing cells is illustrated. Corneas were obtained from WT mice 0 and 4 days after the injury. The samples were immunostained with a combination of anti-CCR5 and anti-CCR2 antibodies as described in Methods and observed with fluorescence microscopy (original magnification, 400X). Signals were digitally merged in the right panels. Arrows indicate the double, positively stained cells. Representative results from three independent experiments are shown. B: Corneal tissues from WT mice (left panel) or CCL3-KO mice (right panel) obtained four days after the injury were stained with anti-CCR5 Ab. Scale bar, 100 μm. C: The numbers of intracorneal CCR5 positive cells four days after the injury were determined as described in Methods, and the mean and SEM are shown here (n=5).

Mentions: We previously revealed that the CCL2/CCR2 interactions could induce VEGF expression [18]. Hence, we next examined whether intraocularly infiltrating CCR5 expressing cells also expressed CCR2. A double-color immunofluorescence analysis demonstrated that CCR5 expressing cells also expressed CCR2 (Figure 4A). Moreover, alkali injury-induced increases in intracorneal CCR5 positive cell numbers were attenuated in CCL3-KO mice (Figures 4B,C). Thus, it is likely that CCL3 can regulate intraocular infiltration of CCR5 expressing macrophages, which can express VEGF by the CCL2/CCR interactions.


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)

Intracorneal CCR5 positive cell infiltration. A: A double-color immunofluorescence analysis of CCR5-expressing cells is illustrated. Corneas were obtained from WT mice 0 and 4 days after the injury. The samples were immunostained with a combination of anti-CCR5 and anti-CCR2 antibodies as described in Methods and observed with fluorescence microscopy (original magnification, 400X). Signals were digitally merged in the right panels. Arrows indicate the double, positively stained cells. Representative results from three independent experiments are shown. B: Corneal tissues from WT mice (left panel) or CCL3-KO mice (right panel) obtained four days after the injury were stained with anti-CCR5 Ab. Scale bar, 100 μm. C: The numbers of intracorneal CCR5 positive cells four days after the injury were determined as described in Methods, and the mean and SEM are shown here (n=5).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Intracorneal CCR5 positive cell infiltration. A: A double-color immunofluorescence analysis of CCR5-expressing cells is illustrated. Corneas were obtained from WT mice 0 and 4 days after the injury. The samples were immunostained with a combination of anti-CCR5 and anti-CCR2 antibodies as described in Methods and observed with fluorescence microscopy (original magnification, 400X). Signals were digitally merged in the right panels. Arrows indicate the double, positively stained cells. Representative results from three independent experiments are shown. B: Corneal tissues from WT mice (left panel) or CCL3-KO mice (right panel) obtained four days after the injury were stained with anti-CCR5 Ab. Scale bar, 100 μm. C: The numbers of intracorneal CCR5 positive cells four days after the injury were determined as described in Methods, and the mean and SEM are shown here (n=5).
Mentions: We previously revealed that the CCL2/CCR2 interactions could induce VEGF expression [18]. Hence, we next examined whether intraocularly infiltrating CCR5 expressing cells also expressed CCR2. A double-color immunofluorescence analysis demonstrated that CCR5 expressing cells also expressed CCR2 (Figure 4A). Moreover, alkali injury-induced increases in intracorneal CCR5 positive cell numbers were attenuated in CCL3-KO mice (Figures 4B,C). Thus, it is likely that CCL3 can regulate intraocular infiltration of CCR5 expressing macrophages, which can express VEGF by the CCL2/CCR interactions.

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