RANK, RANKL, OPG, and M-CSF Expression in Stromal Cells during Corneal Wound Healing

Abstract

purpose. 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-κB 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.

In recent studies, Hamrah et al. ¹ detected bone marrow–derived monocytic dendritic cells that were CD45⁺, CD11c⁺, and CD11b⁺ in the stroma of the unwounded cornea. ¹ In addition, CD11C⁻, CD11b⁺ bone marrow–derived monocytes/macrophages were detected in the stroma in that study. Brissette-Storkus et al. ² identified a macrophage population in the normal mouse stroma. Other investigators have detected a large number of inflammatory cells in the corneal stroma after epithelial scrape injury or surgical procedures such as photorefractive keratectomy (PRK) or laser in situ keratomileusis (LASIK). ^{3 4} Thus, electron microscopy has been used to detect many cells that appear to be monocytes in the rabbit cornea after PRK or LASIK. ⁴  

The monocytes that are present in the cornea after epithelial injury or surgery could function to engulf microorganisms or fragments of cells that die by apoptosis or necrosis. ⁴ Monocytes have been found to have other critical functions unrelated to phagocytosis in some organs. In bone, for example, monocytes have been found to differentiate into osteoclasts in response to a juxtacrine-mediated interaction between osteoblasts and monocytes. ^{5 6 7 8} Because in recent studies performed by our laboratory we have noted the influx of large numbers of monocytes into the corneal stroma after epithelial injury, we have begun studies to determine how these monocyte cells contribute to the wound-healing response and whether they perhaps undergo transition to another cell type that participates in stromal remodeling after injury of the cornea. Direct cell–cell interactions involving monocytes in bone are mediated by receptor activator of NF-κB (RANK) expressed by monocyte/macrophage lineage cells and RANK ligand (RANKL) expressed by osteoblasts (Fig. 1) . ⁶ Osteoprotegerin (OPG) is a decoy receptor that is also produced by osteoblasts to downregulate monocyte differentiation into osteoclasts by binding cellular RANKL. Monocyte chemotactic and stimulating factor (M-CSF) produced by osteoblasts is also a critical modulator of monocyte-to-osteoclast transition. Thus, RANKL, OPG, and M-CSF produced by osteoblasts interact to regulate the transition of monocyte/macrophage cells to osteoclasts. Once formed, the bone-resorbing osteoclasts participate with the bone-producing osteoblasts in maintenance, formation, and remodeling of bone. RANKL- and OPG-mediated interactions have also been noted to be of importance in the physiology of skin and gingival fibroblasts. ^{9 10 11}  

In the present study, to better characterize corneal cytokine-mediated interactions involving monocytes, the mouse model was used to explore whether (1) RANK-expressing cells bearing the monocyte marker CD11b are present in mouse corneal stroma after corneal epithelial injury; (2) mouse stromal fibroblasts in culture and keratocytes or activated keratocytes in situ express the monocyte-regulatory cytokines and receptors RANKL, OPG, or M-CSF; and (3) more direct evidence of influx of bone marrow–derived cells into the cornea could be obtained. 

Materials and Methods

Mouse Stromal Fibroblast Cell Culture and Mouse Monocytes

A new enzymatic method for isolating pure populations of primary mouse stromal fibroblasts (MSFs) from the mouse corneas was developed. All reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Fresh eyes from Swiss Webster mice were obtained from Pel Freeze (Rogers, AR), in DMEM containing a 1% penicillin-streptomycin solution. The corneas were separated from the eyes and incubated in 3 mL of medium containing 1.2 U/mL Dispase II (Roche Applied Sciences, Indianapolis, IN) at 37°C for 45 minutes. The medium (MSF medium) was prepared by adding 10% fetal bovine serum, 1% essential amino acid, 1% nonessential amino acids, 1× l-glutamine, 1× MEM vitamins, 1× sodium pyruvate, and 1% antibiotic-antimycotic solution into the MEM and adjusting the pH to 7.2. After incubation, the epithelium and endothelium were removed from the corneas by gentle brushing with a no. 64 scalpel blade. The tissues were washed twice with Hanks’ balanced salt solution and MSF medium. Fifteen to 20 corneas were cut in small pieces and incubated in 4 mL of MSF medium containing 4 mg/mL collagenase (Roche Applied Sciences) at 37°C in a 5% CO₂-95% air incubator for 45 minutes. The resultant homogeneous stromal cell suspension was centrifuged, and the cell pellet was washed three times with MSF medium. The cells were resuspended in 5 mL MSF medium and plated into the 60 mm TC plates (Fisher Scientific, Hanover Park, IL). Seventy percent to 80% confluent MSF cultures were obtained after 10 to 12 days of incubation at 37°C in a moist 5% CO₂-95% air atmosphere. The cultures were fed with fresh medium at 3-day intervals. 

Murine macrophage complementary DNA samples were graciously provided by the laboratory of Chen Dong (Department of Immunology, University of Washington, Seattle, WA). Murine macrophages were isolated from the mice by a previously published method. ¹²  

Wounding and Fixation of Mouse Corneas

These studies were approved by the Institutional Animal Care and Use Committee at the University of Washington (Seattle, WA). All animals were treated in accordance with the tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Six-week-old BALB/c mice were used in all wounding experiments. Anesthesia and euthanasia were performed as previously described. ⁴ Epithelial scraping was performed in an anesthetized mouse with a no. 64 beaver blade under an operating microscope, so that all the corneal epithelium except approximately 0.5 mm at the limbus was removed. Whole control, 24-hour–, or 48-hour wounded eyes were removed and fixed for 4 hours with 4% paraformaldehyde and embedded in paraffin. Eight-micrometer-thick sections were then cut and mounted on slides (Superfrost Plus; Fisher Scientific). 

RT-PCR and Nucleic Acid Sequencing

RT-PCR was performed using previously reported methods ¹³ with the primers listed in Table 1 . The amplified PCR product was cloned into the PCR II cloning vector (Invitrogen, San Diego, CA). The DNA sequence of the cloned products was confirmed by DNA sequencing using a dye terminator cycle sequencing reaction (Prism; Applied Biosciences, [ABI], Foster City, CA) in an automated gene analyzer (model 310; ABI). 

RNase Protection Assay Analysis

The RNase protection assay was performed using a kit (RPA III; Ambion, Austin, TX) according to the manufacturer’s instructions. The ³²P-labeled antisense RNA probes were synthesized in vitro employing T7 or SP6 RNA polymerase enzyme from the linearized DNA constructs using an in vitro transcription kit (MAXIscript; Ambion). Appropriate DNA constructs were generated by cloning the PCR-amplified mouse-OPG, mouse-MCSF, mouse-RANKL, and mouse-β-actin gene products into the PCR II vector (Invitrogen). The nucleic acid sequence of the constructs was confirmed by DNA sequencing, using the dye terminator cycle sequencing reaction (Prism; ABI) in the automated analyzer (model 310; ABI). The vectors were linearized by the HindIII or the XbaI restriction enzyme. The sizes of the mouse OPG, mouse M-CSF, mouse RANKL, and mouse-β-actin transcripts were 503, 362, 568, and 245 bases, respectively. 

A multiprobe protection assay was performed with 5 and 10 μg of total cellular RNA in two separate microfuge tubes, according to the vendor’s instructions. The Total RNA obtained from the primary MSF cultures was hybridized simultaneously with 8 × 10⁴ cpm of ³²P-labeled mouse OPG, mouse M-CSF, mouse-RANKL probes and 1000 cpm of ³²P labeled mouse-β-actin probe. The probes and sample RNA were coprecipitated, and RNase digestion was performed as described in the protocol of the assay system (RPA III; Ambion). The protected RNA fragments were resolved by the SDS-PAGE on a 0.75-mm-thick, 20-cm wide and 15-cm long 5% acrylamide gel at 250 V constant voltage in 1× TBE buffer. The gel was transferred onto the filter paper, dried, and exposed to x-ray film (BioMax; Eastman-Kodak, Rochester, NY) for 8 to 10 hours at −80°C. 

Immunoprecipitation and Western Blot Analysis

Protein lysates were prepared by incubating the cells on ice for 1 hour in 0.8 mL cell lysis buffer (50 mM Tris-HCl [pH 8.0], 0.5% Triton X-100, 10% glycerol, 0.2 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol [DTT], 0.5 mM phenylmethylsulfonyl fluoride [PMSF] and protease inhibitor cocktail; cat no. 1836153; Roche Applied Sciences). The cellular homogenate was passed through a 21-gauge needle 10 to 12 times to shear the DNA. Debris was removed by centrifugation at 4°C. The Bradford assay (Bio-Rad, Hercules, CA) was used to determine the protein concentration. Immunoprecipitation and Western blot analysis was performed with previously reported methods. ¹³ Immunoprecipitation was performed, using 500 μg of the total protein, except for OPG where 900 μg of total protein was used. Samples were precleared with 0.25 μg of mouse IgG together with 20 μL of protein A-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) by incubation at 4°C for 1 hour. After brief centrifugation in a tabletop microfuge, the supernatants were transferred into new tubes and incubated with 5 μg anti-mouse OPG (cat. no. AF459; R&D Systems, Minneapolis, MN), anti-mouse M-CSF (cat. no. 416; R&D Systems), or anti-mouse RANKL (cat no. AF462; R&D Systems) and 20 μL protein A-agarose overnight at 4°C. The reaction mixtures were centrifuged, washed, and heated for 5 minutes at 95°C in 25 μL Laemmli loading buffer. Samples were loaded on 4% to 20% Tris-glycine gel, and electrophoresis was performed. Separated proteins were electrophoretically transferred onto a nitrocellulose membrane using a blot module (Xcell-II; Novex, San Diego, CA). Nonspecific proteins were blocked by incubating membrane for 1 hour at room temperature in blocking buffer (5% nonfat dry milk in TBST). After the membrane was washed, it was probed with 10 μg anti-mouse OPG (cat. no. AF459; R&D Systems), anti-mouse M-CSF (cat. no. 416; R&D Systems), or anti-mouse RANKL (cat. no. AF462; R&D Systems) in 5 mL blocking buffer at 4°C for 16 to 18 hours. The membrane was washed in TBST, incubated with anti-goat AP conjugated secondary antibody at 1:5000 dilution for 1 hour at room temperature. The bands were visualized by nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate. Fifty or 100 ng of recombinant protein and 1 μg of antibody were run on gels at the same time as positive controls for antibody binding. 

Immunocytochemistry

Antigen retrieval for RANKL, M-CSF, OPG, and RANK proteins was performed by a proteolytic enzyme digestion method in which sections were incubated with 0.1% trypsin in 0.05 M Tris/HCl buffer (pH 7.8) containing 0.1% CaCl₂ for 15 minutes at 37°C. Antigen retrieval for CD11b was performed by incubating sections in 0.01 M citrate buffer (pH 6.0) for 15 minutes. After antigen retrieval, sections were incubated in several changes of phosphate-buffered saline before performing immunocytochemistry. 

Five different antibodies were used to label the sections. Biotin-conjugated rat anti-mouse CD11b (cat no. 01712A; PharMingen) was used to label heated citrate buffer–treated sections. The goat antibodies RANKL (C-20; cat. no. sc7627), M-CSF (N-16; cat. no. sc-1324), OPG (N-20; cat. no. sc-8468), and RANK (H-300; cat. no. sc-9072; all from Santa Cruz Biotechnology) were used to label sections. 

All sections were pretreated with blocking solution containing 5% donkey serum (cat. no. 017-000-121; Jackson ImmunoResearch, West Grove, PA), 1% BSA, and 0.3% Triton X-100. All five antibodies were diluted to 1 μg/mL in PBS containing 0.3% Triton X-100. The sections were incubated in primary antibodies overnight at 4°C. Preabsorbed controls were performed by incubating each antibody with a blocking peptide at 20× concentration of the antibody for 30 minutes at 37°C. The sections were then incubated with biotinylated anti-goat IgG secondary antibody at 1:200 (cat. no. BA-9500; Vector, Burlingame, CA) for 1 hour. Streptavidin-conjugated AlexaFluor 488 (1:100; cat. no. S11223; Molecular Probes, Eugene, OR) was finally applied, to visualize the labels. A confocal microscope system (510 LSM-MP NLO; Carl Zeiss Meditec, Dublin, CA) was used to produce scanned-image z-stacks of the specimens. An argon ion laser (488 nm) provided visible light illumination of the antibody fluorophores, and a helium neon laser (543 nm) provided illumination for the nuclear stain propidium iodide. Projections of the image stacks produced with the NIH Object Image program (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD), and figures were created with them (Photoshop; Adobe Systems, Mountain View, CA). 

Immunocytochemistry for macrophages and neutrophils in the corneas of enhanced green fluorescent protein (EGFP)-positive mice was performed by enucleating the eye 10 days after epithelial debridement. Five-micrometer frozen sections were stained for the macrophage-specific CD11b antigen using primary purified rat anti-mouse antibodies (MAC-1α chain; BD Biosciences-PharMingen) and neutrophil-specific F4/80 antigen, using purified rat anti-mouse antibody (Serotec Ltd., Oxford, UK). AlexaFluor 594 goat anti-rabbit IgG antibodies were used as secondary antibodies (Molecular Probes). 

Generation of EGFP Chimeric Mice

EGFP chimeric mice were generated by injecting 2 to 5 × 10⁵ bone marrow–derived cells from the tibia and femur of C57BL/6-TgN (ACTbEGFP; Jackson Laboratory, Bar Harbor, ME) into C57BL/6 recipient mice that have received a total of 1200 rads whole body radiation in two doses (800 and 400 rads) 3 hours apart. The injection of C57BL/6-TgN (ACTbEGFP) bone marrow cells into irradiated recipients reconstitute the thymus, spleen, bone marrow, and peripheral blood of these mice with EGFP-positive bone marrow–derived cells. Blood of chimeric mice was sampled 2 weeks after transplantation, and peripheralblood was analyzed by flow cytometry for the presence of EGFP-positive cells. 

In Vivo Stereomicroscopic Analysis of EGFP-Positive Cells in the Cornea after Wounding

The presence of EGFP-positive cells in the cornea was evaluated by using a fluorescent filter with a stereomicroscope (Leica Microsystems Inc., Bannockburn, IL) at different time points after epithelial debridement, sparing approximately 0.5 mm of limbus, and in unwounded controls. Images were captured with a digital camera (SpotCam RT KE; Diagnostic Instruments, Inc., Sterling Heights, MI), and quantification of EGFP expression in the surface area of the cornea was calculated on computer (Image Pro-Plus Software; MediaCybernetics, Carlsbad, CA). 

Results

RANKL (Fig. 1) , M-CSF (Fig. 2) , and OPG (Fig. 3) mRNA expression was detected in mouse stromal fibroblasts by RT-PCR. RANK mRNA expression was also detected, as expected, in mouse monocytes (Fig. 4) . Each amplified sequence was the expected size based on the known RNA sequences (see Table 1 ). All amplified sequences were confirmed by cloning into the PCR II cloning vector and sequencing. 

Simultaneous expression of RANKL, M-CSF, and OPG mRNAs in mouse stromal fibroblasts was further confirmed using an RNase protection assay (Fig. 5) . 

The corresponding RANKL (Fig. 6) , M-CSF (Fig. 7) , and OPG (Fig. 8) proteins were detected in cultured monocytes using immunoprecipitation and Western blot analysis. In each case, the detected protein was the expected size. 

CD11b-positive monocytes were detected in the corneal stroma in high numbers at 24 and 48 hours after wounding, by immunocytochemistry (Fig. 9) . The CD11b-positive cells were also detected in the unwounded control cornea (Fig. 9C) , but in a much lower number than in the wounded corneas (Figs. 9A 9B) . 

RANKL protein was detected in stromal cells in the mouse cornea at 24 (Fig. 10A) and 48 (Fig. 10B) hours after corneal epithelial scrape injury using immunocytochemistry. The number of cells that were RANKL⁺ was greatest at the 24-hour time point. Most RANKL appeared to be cell associated, but RANKL also appeared to be present in the stromal matrix (Figs. 10A 10B) , the epithelial basement membrane (Fig. 10A) , and Descemet’s membrane (Fig. 10B) , depending on the time after injury. Only small amounts, if any, of RANKL protein was detected in unwounded mouse cornea (Fig. 10C) . 

OPG protein was detected in stromal cells in the mouse cornea at 24 (Fig. 11A) and 48 (Fig. 11B) hours after corneal epithelial scrape injury, using immunocytochemistry. More stromal cells were RANKL⁺ at the 48-hour time point than at the 24-hour time point. OPG, a soluble protein, was cell associated, but also appeared to be present in extracellular areas of the stroma, especially in the posterior stroma (Fig. 11B) . Only small amounts, if any, of OPG protein was detected in unwounded mouse cornea (Fig. 11C) . 

M-CSF protein was detected in the mouse corneal stroma at 24 (Fig. 12A) and 48 (Fig. 12B) hours after corneal epithelial scrape injury using immunocytochemistry. More stromal cells, especially keratocytes in the posterior stroma, were M-CSF–positive at the 24-hour time point than at the 48-hour time point. At the 24-hour time point, the corneal endothelial cells (Fig. 12A , arrowheads) also appeared to produce large amounts of M-CSF protein. M-CSF is also a soluble protein. By 48 hours after corneal epithelial injury, most M-CSF was detected in the anterior stroma and appeared to be extracellular (Fig. 12B , arrows). Only small amounts, if any, of M-CSF protein were detected in unwounded mouse corneas (not shown). 

RANK protein, a marker for monocyte/macrophage cells, was detected in occasional cells in the mouse corneal stroma at 24 hours (Fig. 13A) and 48 hours (Fig. 13B) after epithelial scrape injury. At 24 hours after injury, most RANK-expressing cells were detected in the midstroma (Fig. 13A) . RANK-expressing cells were also detected in the midstroma at 48 hours after epithelial injury, but RANK was also detected in the anterior stroma (Fig. 13B , asterisks) at 48 hours after epithelial injury. Some or all of this RANK protein in the anterior stroma at 48 hours appeared to be extracellular. 

Wounding experiments in the corneas of EGFP chimeric mice demonstrated conclusively that large numbers of bone marrow–derived cells migrate into the corneal stroma after epithelial debridement (Fig. 14A) . Influx of EGFP-positive cells was detected by 1 day after wounding, and the total fluorescence in the cornea continued to increase up to the maximum 10-day observation in this study. Immunocytochemistry demonstrated that a large number of CD11b⁺ monocytes were present in the cornea at 10 days after wounding, relative to the unwounded control (Fig. 14B) . F480⁺ neutrophils were also increased at 10 days relative to the control, but the difference was less (Fig. 14B) . 

Discussion

It has long been suspected that a variety of cell types participate in the wound-healing response. ^{4 14 15} Differences in cellular morphology and protein expression in the corneal stroma after various injuries have provided important clues. For example, after epithelial scrape injury or photorefractive keratectomy “activated” corneal fibroblastshave been noted through changes in morphology with the light microscope and electron microscopy. ^{4 18} More recent studies have confirmed the involvement of myofibroblasts, ^{4 16 17 18} inflammatory cells, ^{2 3 4} and dendritic cells ¹ in the normal and healing cornea. In large part, the contributions of the individual cell types to the wound-healing response have been poorly characterized. 

Raza et al. ¹ were the first to detect resident dendritic cells in the corneal stroma. Brissette-Storkus et al. ² also identified a novel macrophage population in the normal mouse corneal stroma. Similarly, two studies ^{4 5} demonstrated significant numbers of inflammatory cells in wounded corneas, including some that appear to be of monocyte/macrophage lineage. In the present study monocyte/macrophage cells (expressing CD11b and RANK) were present in the central corneal stroma in high numbers at 24 and 48 hours after corneal epithelial scrape injury and persisedt beyond 10 days after injury. The EGFP chimeric mouse experiments performed in this study demonstrated conclusively that bone marrow–derived cells migrate into the mouse cornea after epithelial scrape injury and that these cells include both monocytes and neutrophils. 

Recent landmark studies performed in bone have conclusively demonstrated that cells of monocyte lineage give rise to osteoclasts involved in bone remodeling through cytokine-mediated interactions with osteoblasts. ^{5 6 7 8} The cytokines involved in the transition from monocyte to osteoclast include RANKL, monocyte chemotactic and stimulating factor (M-CSF), and osteoprotegerin (OPG) expressed by the osteoblast and RANK expressed by the monocyte. The cytokine interactions involved in this transition in bone include paracrine and juxtacrine mechanisms. The presence of monocyte-lineage cells in the corneal stroma and migration of bone marrow–derived cells into the cornea after epithelial injury led to the current investigations into the possibility that analogous cytokine-mediated interactions occur in the cornea. 

This study demonstrates that cultured mouse stromal fibroblasts express RANKL, M-CSF, and OPG mRNAs and proteins. It also shows that cells expressing RANKL, M-CSF, OPG, and RANK are present in the stroma at 24 and 48 hours after corneal epithelial scrape injury and that RANKL, M-CSF, and OPG proteins are markedly upregulated in corneal cells in response to epithelial injury. Presumably, the RANK-expressing cells are monocyte lineage cells that also express CD11b. Double-labeling experiments are being pursued to examine this. We have not detected RANKL, M-CSF, or OPG expression in monocytes. Both M-CSF and RANK proteins were also found extracellularly in the corneal stroma after epithelial wounding, either in the soluble form or associated with extracellular matrix. Both OPG and M-CSF have been detected in extracellular matrix in other tissues. ^{6 7 8 9 10 11}  

The in vitro experiments in this study suggest that the RANKL, M-CSF, and OPG proteins are probably expressed by keratocyte-derived cells after epithelial injury, although we cannot exclude expression by other cell types, since the cellular response in the stroma after epithelial injury has not been well characterized. In the authors’ opinion, considering the early expression of these markers, it is most likely that they are expressed in keratocytes that are activated in the stroma in response to the epithelial injury. Expression of these chemokines is noted within 12 to 24 hours of the epithelial injury, and they are not expressed in the unwounded normal cornea. In addition, other cells of the cornea may participate in these cell–cell interactions. Thus, corneal endothelial cells appear to contribute to M-CSF production at 24 hours after epithelial injury (Fig. 12A) . Soluble or organelle-associated M-CSF appears to accumulate in the anterior stroma by 48 hours after epithelial injury (Fig. 12B) . 

Expression in the corneal stroma of the key cytokines involved in the monocyte to osteoclast transition in bone suggests that a similar transition of monocytes into a cell type involved in stromal remodeling in the cornea may occur. We have speculated that bone-derived cells may persist in the corneal stroma after epithelial injury and contribute to stromal remodeling. Our experiments with chimeric EFGP mice in which bone marrow–derived cells are labeled demonstrate that these cells persist for at least 10 days. Further studies are in progress to determine how long cells expressing bone marrow–derived cell markers persist in the cornea after wounding and whether they retain monocyte morphology or transform into another cell type that participates in the late wound-healing response. Further work is also needed to define the functions regulated by RANK/RANKL, OPG, and M-CSF in the cornea during the healing response. 

 Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2003.

 Supported in part by National Eye Institute Grants EY10056 and EY01730, National Institutes of Health, Bethesda, Maryland, and an unrestricted grant from Research to Prevent Blindness, New York, New York.

 Submitted for publication October 22, 2003; revised March 3, 2004; accepted March 20, 2004.

 Disclosure: S.E. Wilson, None; R.R. Mohan, None; M. Netto, None; V. Perez, None; D. Possin, None; J. Huang, None; R. Kwon, None; A. Alekseev, None; J.P. Rodriguez-Perez, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

RANKL mRNA was detected in MSFs by RT-PCR. The appropriate-sized amplification product of 568 bp was detected in three independent mouse corneal fibroblast cultures, but not in the water target control. Lane L: a 100-bp sizing ladder with the sizes of some bands provided on the left.

Table 1.

 

PCR Primers

Gene	Accession Number	Upstream Primer Sequence	Downstream Primer Sequence	Size (bp)
OPG	U94331	AAC CCC AGA GCG AAA CAC AGT	GGC TCT CCA TCA AGG CAA GAA	503
MCSF	M21149	TTG GCT TGG GAT GAT TCT CAG	GCC CTG GGT CTG TCA GTC TC	362
RANKL	AF013170	CCA GCA TCA AAA TCC CAA GTT	TCA AGG TTC TCA GTG GCA CAT	568
RANK	AF019046	CCA TCA TCT TCG GCG TTT ACT	ACT GTC GTT CTC CCC CAC TT	417

Figure 2.

 

M-CSF mRNA was detected in MSFs by RT-PCR. The appropriate-sized amplification product of 362 bp was detected in three independent mouse corneal fibroblast cultures, but not in the water target control. Lane L: as in Figure 1 .

Figure 3.

 

OPG mRNA was detected in MSFs by RT-PCR. The appropriate-sized amplification product of 503 bp was detected in three independent mouse corneal fibroblast cultures, but not in the water target control. Lane L: as described in Figure 1 .

Figure 4.

 

RANK mRNA was detected in mouse monocyte-macrophage cells (M’phage) by RT-PCR. The appropriate-sized amplification product of 417 bp was detected in isolated monocytes, but not in the water target control. Lane L: as described in Figure 1 .

Figure 5.

 

RANKL, M-CSF, and OPG mRNAs were detected in MSFs by RNase protection assay with either 5 or 10 μg of total cellular RNA. Protected RNA sizes were appropriate for each of the mRNAs. β-Actin detection was included as a positive control.

Figure 6.

 

RANKL protein was detected in MSFs using immunoprecipitation and Western blot analysis. The appropriate-sized native processed protein of 35 kDa (arrows) was detected in two independent cultures, but not in the antibody-alone control (Ab). Lane L: sizing control protein. rbP is recombinant control RANKL protein, to confirm detection by the antibody. The control protein did not undergo posttranslational processing and was of the expected size of 19 kDa.

Figure 7.

 

M-CSF protein was detected in MSFs using immunoprecipitation and Western blot analysis. The appropriate-sized native processed protein of 70 kDa (arrowheads) was detected in two independent cultures (each run in duplicate), but not in the antibody-alone control. Lane L: sizing control protein. Ab is antibody alone. rbP is recombinant control M-CSF protein to confirm detection by the antibody. The control protein did not undergo posttranslational processing and was of the expected size of 26 kDa. This control protein size is very close to the size of the antibody light chain, but note the intensity of the heavy-chain IgG in these lanes. The light chain’s contribution to the band at 26 kDa in the control would be similar.

Figure 8.

 

OPG protein was detected in MSFs using immunoprecipitation and Western blot analysis. The appropriate-sized native processed protein of monomeric OPG (46 kDa, arrows) and dimeric OPG (92 kDa, arrowheads) were detected at low levels in two independent cultures, but not in the antibody alone control. Lane L: sizing control protein. Ab is antibody alone. rbP is recombinant control OPG protein to confirm detection by the antibody. The control protein was generated from a hybrid translational product of the expected size of 71 kDa. There were also several other bands in the control protein lane (rbP) that may indicate some degradation of the control protein.

Figure 9.

 

Immunocytochemistry for monocyte-specific antigen CD11b in mouse corneas at 24 and 48 hours after epithelial scrape injury. A higher number of CD11b⁺ cells was noted in the corneal stroma at 24 (A, arrows) than at 48 (B, arrows) hours after injury. Only rare CD11b⁺ cells were detected in the unwounded cornea (C), with none present in the section depicted. (D) A 24-hour scrape control section, with primary antibody omitted, in which the anterior stroma remained devoid of cells due to apoptosis that occurred after epithelial scraping. Magnification, ×400.

Figure 10.

 

Immunocytochemistry for detection of RANKL in mouse corneas at 24 (A) and 48 (B) hours after epithelial scrape injury. (C) An unwounded control cornea and (D) a control cornea at 24 hours after epithelial scraping in which the primary antibody was preabsorbed with RANKL antigen. A large number of cells expressing RANKL (arrows) were present in the corneal stroma of 24- or 48-hour wounded corneas, but not unwounded control corneas. Background staining was performed with 4′,6′-diamino-2-phenylindole (DAPI). Magnification, ×400.

Figure 11.

 

Immunocytochemistry for detection of OPG in mouse corneas at 24 (A) and 48 (B) hours after epithelial scrape injury. (C, D) Control mouse corneas at 24 and 48 hours, respectively, after epithelial scraping in which the primary antibody was preabsorbed with OPG antigen. A large number of cells expressing OPG were present in the corneal stroma in 48-hour wounded corneas (B, arrows). OPG is a soluble receptor for RANKL. There appeared to be noncellular OPG protein present in the posterior stroma at 48 hours after epithelial injury (*) and staining in Descemet’s membrane at 48 hours. Only a few cells have OPG protein at 24 hours (A, arrows). Preabsorption control sections have little detectable signal. Background staining was performed with 4′,6′-diamino-2-phenylindole (DAPI). Magnification, ×400.

Figure 12.

 

Immunocytochemistry for detection of M-CSF in mouse corneas at 24 (A) and 48 (B) hours after epithelial scrape injury. (C, D) Control mouse corneas at 24 and 48 hours, respectively, after epithelial scraping in which the primary antibody was preabsorbed with M-CSF antigen. A large number of cells expressing M-CSF were present in the corneal stroma in 24-hour wounded corneas (A, arrows). M-CSF was also associated with the epithelial basement membrane at 24 hours after epithelial scrape injury (A, *). Also note heavy expression of M-CSF in the endothelial cells (A, arrowheads) at 24 hours after epithelial scraping. M-CSF is a soluble cytokine. There appeared to be noncellular M-CSF present in the anterior stroma at 48 hours after epithelial injury (B, arrows). The preabsorption control sections (C, D) had little detectable signal. Background staining was performed with 4′,6′-diamino-2-phenylindole (DAPI). Magnification, ×400.

Figure 13.

 

Immunocytochemistry for RANK in mouse corneas at 24 (A) and 48 (B) hours after epithelial scrape injury. (C, D) Control mouse corneas at 24 and 48 hours, respectively, after epithelial scraping in which the primary antibody was preabsorbed with RANK antigen. Significant numbers of RANK-expressing cells (arrows) were detected in the corneal stroma at 24 and 48 hours after epithelial injury. There appeared to be some RANK antigen in the anterior stroma at 48 hours after injury that was not associated with cells (B, *). This antibody does not bind OPG and, therefore, this could be soluble RANK receptor from cells in the stroma or even the epithelium. Background staining was performed with 4′,6′-diamino-2-phenylindole (DAPI). Magnification, ×400.

Figure 14.

 

Corneal scrape injury in mice with GFP-labeled bone marrow–derived cells. (A) Fluorescent cells were detected by 1 day after epithelial scraping and continued to increase up to the final observation point of 10 days. No fluorescence was noted in unwounded control corneas. Bottom: Images taken with a fluorescence microscope in an unwounded control cornea and wounded cornea at 3 and 10 days after wounding. (B) Immunocytochemistry revealed some F480-positive and CD11b-positive cells (arrows) in the stroma of unwounded corneas (con). CD11b⁺ cells were markedly increased at 10 days after epithelial scrape injury (Scr). F480-positive cells also seemed increased relative to the control corneas at 10 days after epithelial scrape injury (scr), but the difference was much lower than for CD11b cells. Magnification, ×400.