Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages.
OBJECTIVE
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.
Lu
Peirong
P
Clinical Immunology Key Laboratory of Jiangsu Province, the First Affilated Hospital of Soochow University, Suzhou, China.
Li
Longbiao
L
Wu
Yu
Y
Mukaida
Naofumi
N
Zhang
Xueguang
X
D000468:Alkalies; D043925:Angiogenesis Inducing Agents / Q000378:metabolism; D000818:Animals; D002465:Cell Movement / Q000187:drug effects; D054405:Chemokine CCL3 / Q000378:metabolism* / Q000494:pharmacology; D018946:Chemokine CCL5 / Q000172:deficiency; D003315:Cornea / Q000187:drug effects / Q000473:pathology; D016510:Corneal Neovascularization / Q000139:chemically induced / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D019707:Receptors, Chemokine / Q000172:deficiency; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis*
D000468:Alkalies; D043925:Angiogenesis Inducing Agents; D054405:Chemokine CCL3; D018946:Chemokine CCL5; D019707:Receptors, Chemokine; D042461:Vascular Endothelial Growth Factor A
Invest Ophthalmol Vis Sci. 2002 Mar;43(3):639-46
11867578
Invest Ophthalmol Vis Sci. 1999 May;40(6):1112-23
10235544
J Leukoc Biol. 2001 Apr;69(4):513-21
11310836
Invest Ophthalmol Vis Sci. 2003 Aug;44(8):3578-85
12882810
Exp Eye Res. 2002 Dec;75(6):625-34
12470964
Invest Ophthalmol Vis Sci. 1999 Aug;40(9):1891-8
10440240
Immunity. 2000 Feb;12(2):121-7
10714678
Cancer Res. 2006 Jan 1;66(1):198-211
16397233
Drug Discov Today. 2002 Feb 1;7(3):171-2
11815230
Am J Pathol. 2003 Apr;162(4):1249-58
12651617
Cytokine Growth Factor Rev. 1996 Dec;7(4):355-76
9023058
PLoS Med. 2006 Aug;3(8):e310
16903779
Mol Vis. 2007 Sep 28;13:1802-12
17960132
Cornea. 2007 Feb;26(2):199-206
17251813
Int Ophthalmol Clin. 1983 Spring;23(1):27-39
6186626
J Exp Med. 1997 Jun 2;185(11):1959-68
9166425
Immunity. 2003 Jul;19(1):71-82
12871640
J Immunol. 2008 Mar 15;180(6):4283-91
18322241
Int J Cancer. 2006 Apr 15;118(8):1869-76
16284949
J Leukoc Biol. 2003 Jul;74(1):25-32
12832439
Curr Opin Ophthalmol. 2001 Aug;12(4):242-9
11507336
Invest Ophthalmol Vis Sci. 2003 Aug;44(8):3586-92
12882811
Mol Vis. 2002 Apr 21;8:119-26
11979237
FASEB J. 2002 Jul;16(9):963-74
12087057
Nat Immunol. 2003 Feb;4(2):154-60
12524535
Invest Ophthalmol Vis Sci. 2002 Jul;43(7):2264-71
12091426
Invest Ophthalmol Vis Sci. 2003 Feb;44(2):590-3
12556387
Cell Mol Immunol. 2004 Apr;1(2):95-104
16212895
J Atheroscler Thromb. 2004;11(1):15-21
15067194
Invest Ophthalmol Vis Sci. 2004 Apr;45(4):1117-24
15037577
Invest Ophthalmol Vis Sci. 2003 Feb;44(2):581-9
12556386
J Clin Invest. 2007 Jan;117(1):185-94
17200718
Nature. 2006 Oct 26;443(7114):993-7
17051153
Am J Pathol. 2007 Mar;170(3):843-54
17322370
Prog Retin Eye Res. 2007 Jan;26(1):1-37
17074526
Trends Cell Biol. 2005 Nov;15(11):599-607
16202600
Circulation. 2006 Aug 22;114(8):807-19
16908772
Cornea. 2003 Jul;22(5):465-7
12827053
Cornea. 1987;6(4):250-7
2446823
Physiol Rev. 2003 Jul;83(3):835-70
12843410