RANK, RANKL, OPG, and M-CSF expression in stromal cells during corneal wound healing.
OBJECTIVE
To examine the influx of monocytes into the cornea after epithelial scrape injury and the expression of chemokines that potentially regulate monocyte phenotype in cultured corneal fibroblasts and keratocytes in situ.


METHODS
Monocytes were detected by immunocytochemistry for the monocyte-specific antigen CD11b, in unwounded and epithelial scrape-wounded mouse corneas. The receptor activator of NF-kappa B ligand (RANKL), osteoprotegerin (OPG), and monocyte chemotactic and stimulating factor (M-CSF) mRNAs were detected in cultured mouse stromal fibroblasts by RT-PCR and RNase protection assay. RANKL, OPG, and M-CSF proteins were detected in cultured mouse stromal fibroblasts by immunoprecipitation and Western blot analysis. RANKL, RANK, M-CSF, and OPG proteins were detected in unwounded and wounded mouse corneas by immunocytochemistry. Chimeric mice with green fluorescent protein-labeled bone marrow-derived cells underwent corneal scrape injury and were monitored by fluorescence microscopy and immunocytochemistry.


RESULTS
A small number of cells expressing the monocyte-specific CD11b antigen were detected in the stromas of unwounded mouse corneas. A larger number of CD11b-positive cells was detected in the stroma at 24 or 48 hours after epithelial scraping injury. Experiments with chimeric mice with fluorescent green protein-labeled, bone marrow-derived cells demonstrated conclusively the origin of these CD11b(+) cells. RANKL, OPG, and M-CSF mRNAs and proteins were detected in cultured mouse stromal fibroblasts. RANKL, M-CSF, and OPG proteins were detected in unwounded corneas, but were expressed at higher levels in stromal cells during the 24- to 48-hour interval after epithelial scrape injury. RANK was detected in stromal cells presumed to be monocytes at 24 and 48 hours after epithelial injury.


CONCLUSIONS
Cells expressing the CD11b monocyte-specific antigen appear in the corneal stroma in high numbers by 24 hours after epithelial injury and persist beyond 10 days after wounding. Cultured corneal fibroblasts and keratocytes in situ express RANKL, OPG, and M-CSF cytokines involved in regulating osteoclast differentiation from monocytes in bone. Cells expressing RANK were detected in the stroma at 24 and 48 hours after epithelial injury. The cytokine systems that regulate monocyte transition to osteoclast in bone are upregulated in the cornea in response to epithelial injury and may participate in regulating monocyte phenotype during corneal stromal wound healing.
Wilson
Steven E
SE
Cole Eye Institute, The Cleveland Clinic Foundation, Ohio 44195, USA. wilsons4@ccf.org
Mohan
Rajiv R
RR
Netto
Marcelo
M
Perez
Victor
V
Possin
Dan
D
Huang
Jing
J
Kwon
Robert
R
Alekseev
Andrei
A
Rodriguez-Perez
Juan P
JP
D000818:Animals; D015153:Blotting, Western; D039481:CD11b Antigen / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism*; D003319:Corneal Stroma / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000502:physiology; D053244:Osteoprotegerin; D011233:Precipitin Tests; D053245:RANK Ligand; D012333:RNA, Messenger / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014945:Wound Healing
D039481:CD11b Antigen; D002352:Carrier Proteins; D018932:Chemokine CCL2; D006023:Glycoproteins; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D053244:Osteoprotegerin; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; C505687:Tnfrsf11a protein, mouse; C506031:Tnfrsf11b protein, mouse; C505689:Tnfsf11 protein, mouse; D049452:Green Fluorescent Proteins