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a Department of Ophthalmology, University of Aberdeen, Scotland, United Kingdom;
b Department of Neuroimmunology, Center for Brain Research, Medical University of Vienna, Austria
Key Words. Cornea • CD34 • Hemopoietic stem cell • Dendritic cell • Leukocytes
Correspondence: John V. Forrester, M.D., Department of Ophthalmology, University of Aberdeen, AB255ZD, Aberdeen, Scotland, United Kingdom. Telephone: 0044-122-455-3782; Fax: 0044-122-455-5955; e-mail: j.forrester{at}abdn.ac.uk
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ABSTRACT |
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Combined staining for F-actin and leukocyte differentiation markers clearly showed that the corneal stroma contains a population of CD45+ resident bone marrow–derived cells, whereas most cells were CD45-F-actin+ keratocytes. A significant proportion (two thirds) of CD45+ cells in the normal corneal stroma expressed CD34+, whereas no CD45– cells (i.e., keratocytes) coexpressed CD34. In addition, CD34+ cells were CD11c– and CD11b+. Fewer than 10% of the CD34+ cells also coexpressed Sca-1+, but no CD34+ cells coexpressed major histocompatibility complex (MHC) class II+. In contrast, the remaining population of CD45+CD34– cells in the corneal stroma expressed CD11b, MHC class II+ but not CD11c and were found mostly in the anterior and peripheral part of stroma. These cells are in intimate contact with corneal keratocytes, which stained only for F-actin and were negative for all leukocyte markers. Very few CD45+ cells expressed the B220 marker, suggesting a plasmacytoid dendritic cell phenotype. Flow cytometry analyses confirmed the morphometric data showing that 68% of CD45+ cells coexpress CD34 and CD11b, whereas 22% are CD11b+CD34–.
We conclude that the normal mouse cornea contains two populations of bone marrow–derived leukocytes, both of which are distinct from stromal keratocytes. The larger population resembles CD34+ hemopoietic stem cells, whereas the smaller population are CD34–CD11b+ MHC class II+ macrophages. A very small percentage comprises plasmacytoid dendritic cells.
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INTRODUCTION |
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The mechanisms for tissue and cell maintenance and renewal during adult life are considered to depend on pluripotent stem cells. Recently, a more general role has been proposed for bone marrow stem cells in tissue regeneration such as in cardiac muscle cells, Purkinje neurons in the brain, and liver cells [4]. In these studies it was suggested that bone marrow cells have the potential to form new tissue cells especially after injury. Hemopoietic cells thus may either transdifferentiate into a fully mature tissue-specific cell or more likely fuse with existing cells to form a multinucleated cell. HSCs are characterized by a set of discrete molecular markers, including CD34, CD133, and Sca-1 (Ly-6A/E) [5]. CD34 is also expressed on vascular endothelium.
Recent studies have suggested that corneal keratocytes express CD34 [6, 7]. The observations were made on single-stained immunohistochemical preparations of human corneal tissues containing stromal cells with the morphology of keratocytes. The cells also expressed the L-selectin ligand, CD62L. No CD34+ cells were found in corneal epithelium or endothelium [7]. In addition, CD34+ cells were very recently found in corneas with Mooren’s ulcer [8]. Intriguingly, culture of stromal keartocytes is associated with loss of the CD34 marker [9].
In work by others, the corneal stroma in the mouse has also been shown to contain a population of CD45+ leukocytes [10, 11], and thus the possibility exists that the CD34+ population of stromal cells may in fact be true HSCs and not keratocytes expressing CD34. In the present study, we decided to investigate more closely the resident cells in normal mouse cornea using dual and triple immunostaining, flow cytometric analysis, and short-term cell culture.
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MATERIALS AND METHODS |
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All rats were kept at the decentral facilities of the Institute for Biomedical Research (Vienna, Austria). Initially, the green fluorescent protein (GFP) transgene had been introduced into the Sprague-Dawley strain of rats [12]. It is now crossed back into Lewis rats. Throughout this study, animals of the 3.-5. backcross generation were used.
Preparation of Rat Chimeras
For the production of bone marrow chimeras, young adult wild-type rats were irradiated with 1,000 rads and then injected with bone marrow cells isolated from the femurs of GFP transgenic rats. Typically, cells derived from two femurs were used to reconstitute one irradiated wild-type rat.
Preparation of Tissues
Whole-Mount Corneas Mice were euthanatized, and the entire cornea was excised at the limbus under the operating microscope. For staining of the corneal stroma, the epithelium was removed after 20 minutes of incubation at 37°C in phosphate-buffered saline (PBS) containing 20 mM EDTA. Corneas were then fixed for 30 minutes at 4°C in 1% paraformaldehyde PBS. After fixation, stromal tissue was washed in PBS and ready for staining. For staining of corneal epithelium, mice were euthanatized and corneas were immediately fixed in situ with 4% paraformaldehyde in PBS for 20–30 minutes. After that, corneas were excised and fixed for 1 hour in 4% paraformaldehyde. The tissues were then washed (five times, 5 minutes each) in PBS and stained.
Rats were euthanatized and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.2 (PFA/PBS). The whole eyes were then removed and postfixed in 4% PFA/PBS for an additional 24 hours and then transferred to PBS. The entire cornea was excised at the limbus under the microscope and stained.
Single-Cell Suspension of Corneal Cells Seventy normal corneas were removed as above under the operating microscope, dissected into small pieces, and then incubated in Hanks’ balanced salt solution containing 0.2% collagenase A for 1 hour with rotation (70 rpm) at 37°C. There action was stopped by adding 2-mercaptoethanol, and the solution was strained through a 70-micron flow cytometry filter and the filtrate collected. The cell sample was centrifuged at 1,200 rpm for 10 minutes at 4°C, and the cells were resuspended in fluorescence-activated cell sorter (FACS) buffer (1% BSA/PBS/10 mM NaN3) and aliquots were prepared for further staining and flow cytometric analysis.
Corneal Stromal Cell Culture Corneas from 10 normal mice were excised and incubated at 37°C in PBS containing 20 mM EDTA for 30 minutes. The epithelial and endothelial layers were then peeled off. Corneal stromal samples were washed in PBS, chopped into small pieces, and incubated in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, penicillin 100 U/ml, streptomycin 100 µg/ml, and gentamicin 50 µg/ml and cultured on tissue culture–treated 24-well plates in a humidified atmosphere containing 5% CO2. Nonadherent cells were harvested after 3 days, and cytospins were prepared.
Antibodies
The immunochemical staining procedures were performed with the following antibodies: purified rat anti-mouse CD34 (RAM34) (BD Pharmingen, San Diego), hamster anti-mouse CD11c (HL3) (BD Pharmingen), rat anti-mouse CD16/CD32 (2.4G2) (BD Pharmingen), rat anti-mouse CD45 (30-F11) (BD Pharmingen), biotinylated rat anti-mouse CD11b (M1/70) (BD Pharmingen), biotinylated mouse anti-mouse IAb (AF6-120.1) (BD Pharmingen), directly conjugated rat anti-mouse CD34 fluorescein iso-thiocyanate (FITC) (MEC 14.7) (Serotec, Oxford, U.K.), rat anti-mouse Ly6A/E (Sca-1) FITC (D7) (BD Pharmingen), rat anti-mouse CD45 phycoerythrin (PE), CD45 FITC (30-F11) (BD Pharmingen), rat anti-mouse CD11b Per CPCy5.5, 11b PE (M 1/70) (BD Pharmingen), hamster anti-mouse CD11c FITC (HL3) (BD Pharmingen), mouse anti-mouse IAb PE (AF6-120.1) (BD Pharmingen), rat anti-mouse Ly6G and Ly6C (GR-1) allophycocyanin (APC) (RB6-8C5) (BD Pharmingen), and rat anti-mouse CD45R/B220 PE (RA3-6B2) (BD Pharmingen). Secondary antibodies used were biotinylated goat anti-hamster immunoglobulin G (G70-204, G94-56) (BD Pharmingen) and biotinylated rabbit anti-rat immunoglobulins (DakoCytomation, Glostrup, Denmark). Streptavidin conjugated with Rhodamine (TRITC), with FITC, and with APC were purchased from Jackson Immunoresearch Laboratories, West Grove, PA. For F-actin staining, BODIPY 558/568 phalloidin was used (Molecular Probes, Inc., Eugene, OR). For G-actin staining, DNase I Alexa Fluor 488 conjugate was used (Molecular Probes, Inc.). For immunostaining of rat corneas, the following antibodies were used: purified mouse anti-rat CD45 (MRC OX-1) (Serotec), purified mouse anti-rat major histocompatibility complex (MHC) class II (MRC OX-6) (Serotec), and biotinylated rabbit anti-mouse immunoglobulins (DakoCytomation).
Immunohistology
Immunostaining of Whole-Mount Corneal Tissue Corneas were prepared for staining as whole mounts as described above. To block nonspecific staining, corneas were first incubated for 20 minutes at 37°C in strain-specific serum diluted in PBS containing 3% bovine serum albumin, 0.25% gelatin, 5 mM EDTA, and 0.025% Nonodet-P40, a nonionic detergent (PBS-BGEN). Fc block was used before staining with purified anti-CD11c antibody and also when biotinylated or directly conjugated antibodies were used. After blocking, corneas were incubated overnight at 4°C with 100-µl primary antibodies or isotype-matched control antibodies diluted in PBS-BGEN. The tissue was then washed five times for 5 minutes each in PBS. Staining continued then according to the primary antibodies either with 100 µl of fluorescently labeled streptavidin diluted in PBS-BGEN for 1 hour at room temperature (RT) or with biotinylated secondary antibody diluted in PBS-BGEN for 2 hours at RT, washed five times for 5 minutes each in PBS, and incubated with fluorescently labeled streptavidin diluted in PBS-BGEN for 1 hour. This was followed by five washes for 5 minutes each in PBS and fixation in 1% paraformaldehyde for 30 minutes at 4°C. Four radial cuts were performed by a sharp razor blade, and corneas were then mounted in mounting medium (Vectashield or Vectashield-PI) in 18 x 18-mm wells made of nail polish on glass slides and covered with coverslip. For combined staining for actin, immediately after first fixation, corneas were washed in PBS and incubated with BODIPY 558/568-phalloidin for F-actin or DNase I for G-actin for 2 hours at room temperature, washed five times for 5 minutes each in PBS, and then stained as described previously. At least four different corneas were examined for each experiment. All experiments were repeated at least twice. For negative controls, we used isotype-matched immunoglobulins.
Immunostaining of Cytospins Cytospins were prepared and left to dry overnight. They were then fixed with acetone for 10 minutes at room temperature, and nonspecific staining was blocked with strain-specific 10% serum for 20 minutes. Cell cytospins were stained with primary mono-clonal antibodies or isotype-matched controls for 1 hour, washed five times for 5 minutes each in PBS, and incubated with secondary biotinylated antibody for 1 hour, washed again five times for 5 minutes, and incubated with fluorescently labeled streptavidin for 30 minutes. This was followed by final wash (five times for 5 minutes), and slides were mounted in mounting medium Vecta-shield or Vectashield-DAPI and covered with coverslip. All steps were performed at room temperature.
Confocal Microscopy
Whole-mount corneas and cell cytospins were analyzed using a confocal Laser Scanning Microscope (LSM Meta; Zeiss, Gottingen, Germany). Dry objective (x10, x20) and oil-immersion objective (x40) were used to obtain individual images. To count the number of positively labeled cells in corneal whole mounts, series of multiple Z-sections were generated, single images were created, and positive cells were manually counted. In some experiments, different regions of the cornea were analyzed; namely, the central, paracentral, and peripheral regions, as described previously [10].
Flow Cytometric Analysis
Directly conjugated monoclonal antibodies specific for mouse cell-surface markers and monochrome-isotype controls were purchased from Pharmingen BD and Serotec. Single-cell suspensions were incubated with directly conjugated primary antibodies for 30 minutes, washed twice in FACS buffer, and analyzed. Negative controls and single fluorochrome controls were performed to allow accurate compensation.
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RESULTS |
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Overview of Corneal Cell Populations
Normal mouse cornea consists of three cellular layers and two interfaces: epithelium, Bowman’s layer, stroma, Descemet’s membrane, and endothelium. The thickness of mouse cornea is approximately 100 µm [13]. The epithelium is comprised of several layers of squamous epithelial cells overlying a layer of small hexagonal, uniformly sized basal cells (Figs. 1A, 1B
). In the mouse, the corneal epithelium represents more than one third of normal corneal thickness. The corneal endothelium comprises a single layer of polygonal cells (Fig. 1C).
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). This contrasted with the characteristic scattered, spindle-shaped cell forms seen in conventional sections of corneal stroma (Fig. 1E). To estimate the full complement of cells in normal mouse corneal stroma, epithelium-denuded corneas were stained with propidium iodide as a general stain for cell nuclei (Fig. 1F). Using confocal microscopy, multiple Z-sections were obtained through the entire stromal thickness and a single image was created. It was thus possible to determine the number of nuclei per unit area (mm2) for the entire thickness of corneal stroma. Our data reveal that there are 3,710 ± 907 cells per mm2 in the normal mouse corneal stroma (Fig. 1F).
Characterization of Corneal Stromal Cells
We next wished to determine the level of heterogeneity in the stromal cell population, because previous studies have shown that, in addition to keratocytes, there are discrete populations of leukocytes in the stroma [10, 11]. Because keratocytes in the human are reportedly CD34+ [7], we investigated CD34 antigen expression in the mouse corneal stroma. Single immunostaining showed significant numbers of CD34+ cells (Fig. 2A), which were distributed throughout the cornea. In the periphery, they numbered 122 ± 33/mm2, whereas in the center there were somewhat fewer (88 ± 13/mm2). They thus represented approximately 2.4%–3.3% of the total population of stromal cells depending on location to periphery or center of the cornea. No CD34+ cells were observed in epithelium or on endothelium. The stromal CD34+ cells had a generally rounded, variable morphology, with several fine processes; they measured approximately 20 µm in diameter, which was significantly smaller that the more frequent stellate, scallop-edged stromal cells (compare Figs. 2B and 1D)
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) throughout the central, paracentral, and peripheral parts of the corneal stroma as described previously [10, 11]. Morphologically, there were two different subsets of CD45+ cells: a round compact mononuclear-type cell found throughout the corneal stroma (Fig. 2D) and a second cell type restricted to the peripheral cornea and anterior stroma, which appeared much larger with very long dendriform processes (Figs. 2E, 2F). The number of CD45+ cells in the normal mouse stroma was 215 ± 45/mm2 in the peripheral part and 138 ± 26/mm2 in the central part of the cornea, representing 5.8% and 3.7%, respectively, of the total cell population. Immunostaining of corneal stroma cells with the macrophage integrin marker CD11b showed similar results as with CD45 (173 ± 19 cells/mm2 for peripheral cornea and 164 ± 38 cells/mm2 for central region of the cornea). To investigate the possible presence of dendritic cells in the corneal stroma, corneas were stained for the dendritic cell marker CD11c. Only a few positive cells were found in the peripheral parts of cornea; no CD11c+ cells were detected in the central parts (data not shown).
To characterize additionally the resident stromal cells, normal corneal stromas were double- and triple-stained with antibodies to various leukocyte differentiation markers in combination with phalloidin to visualize F-actin. Simultaneous two-color staining with antibodies to CD45 and CD11b indicated that 100% of CD45+ cells were also CD11b+ (Table 1). As in the single immunostaining studies above, both markers were expressed on two different subsets of cells, distinguished by the expression of MHC class II antigen. MHC class II was coexpressed on 40 ± 10% of CD45+ cells, mostly restricted to the large dendriform cells in the anterior third and the periphery of the corneal stroma (Table 1, Fig. 3A). Only occasional MHC class II+ cells could be found in the central area of the corneal stroma.
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, Fig. 3B). Furthermore, as predicted from the above data, 100% of the CD34+ cell population coexpressed CD11b+. However, no CD34+ cells expressed MHC class II or CD11c. In addition, less than 10% of CD34+ cells coexpressed the stem cell marker Sca-1 (Table 1).
Most murine dendritic cell populations are CD11c+ [15]. However, there is a small population of dendritic cells recently characterized that are CD11clo or negative [15]. These cells, termed plasmacytoid dendritic cells (pDCs), are B220+ and variably expressive of Gr-1, the neutrophil marker. Accordingly, we examined mouse cornea for the presence of these cells by dual and triple staining for CD45, B220, and Gr-1. A very small population of CD45+ B220+Gr-1+ cells was found predominantly in the peripheral cornea (Fig. 4). Occasional CD45+ cells in this sub-population also either expressed only the B220 marker or the Gr-1 marker, suggesting an intermediate phenotype (Fig. 4).
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) and were distributed throughout the stroma. Most of these cells stained only for F-actin, whereas a small percentage also stained for G-actin, indicating their motile nature (Fig. 7). In contrast, in the anterior stroma, the large CD34–, CD45+ (Fig. 6B), CD11b+ (Fig. 6C), and MHC class II+ (Fig. 6D) cells that are restricted to the anterior and peripheral stroma adopted dendriform-shaped cells, which seemed to be predicated upon by their contact with the F-actin+CD34– stromal keratocytes. Indeed, their intercellular contacts seemed to follow precisely the contours of the keratocyte, presumably thus ensuring maximum cell–cell contact interface.
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). There seemed to be two morphological types, one form adopting a spindle-shaped phenotype and the other being more rounded (Fig. 8A). Staining with phalloidin clearly indicated that they were a separate population of cells from the stromal keratocytes (Fig. 8B). They also uniformly expressed CD45+ (Fig. 8A, inset 1). A small percentage of the spindle-shaped cells also expressed MHC class II (Fig. 8A, inset 2). Examination of chimeric rats at different ages indicated that the numbers of CD45+ cells remained constant between 2 months and 1 year after reconstitution (data not shown). No GFP+ cells were found in corneal epithelium or endothelium.
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). To confirm the bone marrow origin of the CD34+ cells, we dual stained some cytospin preparations with CD45. All CD34+ cells after short-term cell culture coexpressed CD45 (Fig. 9C). No CD11c+ cells were observed in these preparations, and negative controls did not show any staining (Fig. 9D).
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DISCUSSION |
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). Approximately 3%–4% of corneal stromal cells were F-actin+CD45+ cells, which were uniformly CD11b+ and thus represented in part the novel macrophage-type cell recently described by Brissette-Storkus et al. [10]. In addition, approximately two thirds of these cells were CD34+, whereas none of the CD45– stellate cells expressed CD34. These data indicate that, at least in the mouse, the population of CD34+ cells are all bone marrow–derived cells because they are exclusively contained within the CD45+ cell population. In the mouse, therefore, F-actin+ corneal stromal keratocytes do not seem to express CD34. This contrasts with data from studies of human keratocytes, which have been reported to be CD34+ [6, 7, 9]. Keratocytes are mesenchymal cells of neural crest origin, which are considered to have low cell turnover during life but have the potential to undergo transdifferentiation to active myofibroblasts during injury and repair. Expression of the HSC marker CD34 by resident keratocytes therefore would provide indirect supportive evidence that normal keratocytes may exist in a semiprogenitor state, which can be triggered by certain stimuli such as transforming growth factor ß to full fibroblast maturity [9]. Although the data from the present study cannot exclude this possibility in the human, in the mouse there is no evidence for such a mechanism. It must also be emphasized that definitive evidence excluding a hemopoietic origin for human keratocyte demonstrating coexpression of either CD45 with CD34 or keratan sulphate with CD34 has not so far been presented [16]. Our data also show that CD34+ HSCs are not the only leukocyte subtype in the normal mouse cornea. Interestingly, there seem to be at least two subsets of CD45+ cells based on their expression of CD34. Although 66% of the CD45+ cells were CD34+, the remaining CD45+CD34– cells could also be differentiated on the basis of their morphology and expression of MHC class II. CD45+CD34+ corneal cells tended to be round and widely distributed, whereas the CD45+CD34– cells were larger, possessed many dendriform processes, expressed MHC class II, and were restricted to the periphery and the anterior stroma. They failed to express CD11c and resembled most closely the stromal macrophages referred to above [10]. These data indicated, therefore, that there were no myeloid dendritic cells in the normal cornea. However, a small proportion of the CD45+CD34– cells were CD11c–B220+, suggesting that they may represent pDCs.
The CD45+CD34+ leukocyte thus seemed to be the larger subset of CD45+ cells in the stroma. These cells also presented an interesting phenotype. Classically, CD45+CD34+ cells represent a late stage of HSC differentiation, which have become activated when they have migrated from the bone marrow to the secondary tissues [17]. In the earliest progenitor stage in the bone marrow, HSCs are CD45+CD34–Sca1+CD11b–. After this stage, they populate various secondary lymphoid tissues and downregulate Sca1 while upregulating CD34 and CD11b [17]. Intriguingly, a small proportion of corneal CD45+CD34+ cells were Sca1+, suggesting that they represented an intermediate stage in the differentiation of the HSC, possibly in arrest in the corneal stroma. A small proportion of these cells also expressed significant levels of G-actin, which is prominent in highly motile cells and suggests that they are rapidly transiting the tissues. An alternative possibility is that although most CD34+ progenitor HSCs entering the corneal stroma may differentiate into myeloid CD11b+ MHC class II+ stromal macrophages previously described [10], the few Sca1+CD34+ stromal cells develop into pDC.
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CONCLUSION |
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ACKNOWLEDGMENTS |
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REFERENCES |
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