February 2011
Volume 52, Issue 2
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Cornea  |   February 2011
Cytokeratin 8 Is Expressed in Human Corneoconjunctival Epithelium, Particularly in Limbal Epithelial Cells
Author Affiliations & Notes
  • Stanislava Merjava
    From the Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders and
  • Kristyna Brejchova
    From the Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders and
  • Amanda Vernon
    the Cells for Sight Transplantation and Research Programme, Department of Ocular Biology and Therapeutics, University College London (UCL) Institute of Ophthalmology, London, United Kingdom.
  • Julie T. Daniels
    the Cells for Sight Transplantation and Research Programme, Department of Ocular Biology and Therapeutics, University College London (UCL) Institute of Ophthalmology, London, United Kingdom.
  • Katerina Jirsova
    From the Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders and
    the Ocular Tissue Bank, General Teaching Hospital and First Faculty of Medicine, Charles University in Prague, Czech Republic;
  • Corresponding author: Stanislava Merjava, Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, General Teaching Hospital and First Faculty of Medicine, Charles University, Unemocnice 2, 128 08, Prague, Czech Republic; [email protected]
Investigative Ophthalmology & Visual Science February 2011, Vol.52, 787-794. doi:https://doi.org/10.1167/iovs.10-5489
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      Stanislava Merjava, Kristyna Brejchova, Amanda Vernon, Julie T. Daniels, Katerina Jirsova; Cytokeratin 8 Is Expressed in Human Corneoconjunctival Epithelium, Particularly in Limbal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(2):787-794. https://doi.org/10.1167/iovs.10-5489.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: The purpose of this study was to investigate the expression of cytokeratin (CK) 8 in the corneoconjunctival epithelium.

Methods.: In 17 cadaveric corneoscleral discs and 3 other discs, the presence of CK8 alone or CK8, together with CK3, CK15, vimentin, and integrin α6, was investigated by using indirect immunohistochemistry on radial cryosections. Four corneoscleral discs stored in organ culture were used for the preparation of tangential sections of the limbus and for the isolation of limbal epithelial cells and their subsequent cultivation. CK8 expression was examined by RT-PCR in the corneal, limbal, and conjunctival epithelium.

Results.: Sixty percent of the cadaveric corneoscleral samples and all samples stored in organ culture revealed positivity for CK8 in the basal epithelial layer of the limbus. Positive basal cells formed a single line or separated clusters. The signal for CK8 became weaker toward the surface of the limbal epithelium. The colocalization of CK8 with vimentin and CK15 in the limbus was also found. CK3 showed only occasional positivity in some of the surface limbal cells. The expression of integrin α6 in the basal membrane was absent or decreased under the CK8-positive clusters. Cell cultures revealed strong positivity for CK8 in approximately 80% of the cultured cells, and CK8 expression in the cornea, limbus, and conjunctiva was determined by RT-PCR.

Conclusions.: The study demonstrates the strong expression of CK8 in limbal epithelial basal cells, which is maintained during the differentiation and migration of the limbal cells toward the central corneal epithelium.

The corneal epithelium is a rapidly regenerating, nonkeratinized, stratified squamous epithelium that is continuously renewed throughout life from basal cells of the epithelium and from the population of limbal epithelial stem cells (LESCs), which proliferate and migrate centripetally to the corneal epithelium. 1 5 LESCs exhibiting high proliferative capacity are located in the basal layer of the limbus, a highly vascularized and innervated transition zone between the cornea and conjunctiva. Unipotent LESCs undergo asymmetric self-renewal cell division, in which one cell remains undifferentiated as a stem cell, while the fast-dividing progenitor cell, referred to as the transit amplifying cell (TAC), begins to divide and differentiates into the suprabasal and superficial cells of the corneal epithelium. 6 9  
LESCs are involved in the renewal of the corneal epithelium, which is important, not only for sustaining the integrity of the ocular surface, but also for the maintenance of visual function. 2,5 Despite extensive investigative effort, no definitive marker that may be helpful in identifying and isolating LESCs is known with certainty. Although cytokeratin (CK) expression alone is not sufficient to identify stem cells or progenitor TACs, the expression profile of several key cytokeratins (CK19, CK15), together with other known potential markers (ABCG2, p63), can be used to identify LESCs. 9 12 Adhesion molecules such as integrins can also play a role in the identification of LESCs and TACs (especially a lack of α6 and β4 integrins in the limbal area). 13  
CKs are intermediate filaments typical of epithelial cells, which are expressed in a tissue-specific, differentiation-dependent manner. 14 A broad spectrum of CKs has been detected in the corneal and limbal epithelium. 15 17 Limbal basal cells do not express CK3/12, which is a typical marker for advanced corneal epithelial differentiation, 14,18 although CK5/14, CK19, and vimentin have been detected in such cells. 13,16,18 21 CK15 was proposed to be a putative marker of stem cells in the hair follicle bulge 22 and a potential marker for LESCs and early TACs. 23,24  
CK8 (together with CK18) is the major component of the intermediate filaments of the simple and single-layered epithelia found in the liver and mammary gland, among other tissues. 25,26 CK8 and CK18 are the first cytokeratins expressed during embryogenesis, and the absence of CK8 causes midgestational lethality or colorectal hyperplasia and inflammation in mice. 27 29 The total amount of cellular CK8 and CK18 is kept at a stable level under physiological conditions, 14 whereas the expression of both often increases during carcinogenesis. 26,30 CK8 and -18 have a cytoprotective role against chemical insults 31 and modulate the cellular response to proapoptotic signals. 32 36 Their role in the regulation of the cell cycle has already been described. 37 40  
Recently, it was shown that besides the presence of CK8 and -18 in the corneal epithelium, 16 both CKs are expressed in the corneal endothelium. 41 Moreover, CK8 has been found in human superficial conjunctival cells as well as scattered throughout the mammalian conjunctival epithelium. 42,43 In this study, we report the strong expression of CK8 in the basal cells of the limbus, which is also retained during the differentiation and migration of the limbal cells toward the central cornea. The possible function of CK8 in the activation, proliferation, and/or migration of limbal cells is not clear and requires further investigation. 
Materials and Methods
Samples
The study was conducted according to the standards of the Ethics Committee of the General Teaching Hospital and Charles University, Prague, and adhered to the tenets set forth in the Declaration of Helsinki. In total, 29 cadaveric corneoscleral discs (11–17 mm in diameter; mean age, 57.5 ± 17.4 years; range, 16–83) that were not acceptable for transplantation because of low endothelial cell density or a positive serology of the donor obtained from the Ocular Tissue Bank, Prague, were used. 
Twenty corneoscleral discs (mean age, 56.8 ± 19.3 years) were dissected, snap frozen in liquid nitrogen, embedded in OCT, and stored at −70°C. The time between death and storage in liquid nitrogen did not exceed 24 hours. Tissues were cryosectioned radially at a thickness of 7 μm, to evaluate the whole structure of the cornea, and four sections were mounted per slide. 
Four corneoscleral discs (mean age, 51.3 ± 7.3 years) were stored in organ culture in minimum essential medium with 2% fetal calf serum, as described elsewhere, 44 at 31°C for 8 to 9 days. After storage, the corneas were divided, and half of each cornea was used for the isolation of limbal epithelial cells and their subsequent cultivation. The second half was divided into two quarters, which were frozen (−70°C) and sectioned (7 μm) as tangential sections of the limbus, allowing evaluation of the complex architecture of the limbal crypts. 45  
Five corneoscleral discs (mean age, 65.4 ± 11.4 years) were used for the preparation of corneal epithelial, limbal epithelial, and perilimbal conjunctival samples. Lamellar rectangular dissections of the limbal and central corneal epithelial tissue (2 mm in diameter), both containing a minimum of underlying stromal tissue, were prepared with the use of a diamond knife (depth, ∼100 μm). The samples of perilimbal conjunctiva were cut using corneal scissors. Immediately after preparation, the samples were placed in RNA stabilizer (RNAlater; Qiagen GmbH, Hilden, Germany); mRNA was isolated and subsequently used for semiquantitative reverse transcription polymerase chain reaction (RT-PCR). 
Primary Limbal Epithelial Cell Isolation and Culture: Single-Cell Suspension Culture
Limbal epithelial cells (LECs) were cultured in corneal epithelial culture medium (CECM) consisting of DMEM F12 (1:1) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic solution (Invitrogen), 0.1 nM cholera toxin (Sigma-Aldrich Corp., St. Louis, MO), 5 μg/mL human recombinant insulin (Sigma-Aldrich) and 10 ng/mL epidermal growth factor (Invitrogen). 
LECs were isolated from one-half of each corneal button or rim after incubation in a 1.2 U/mL Dispase II (Stem Cell Technologies, Inc., Vancouver, BC, Canada) solution at 37°C for 30 minutes. After Dispase treatment, the tissue was transferred into a Petri dish on a drop (200 μL) of nonamimal trypsin enzyme (Tryple Select; Invitrogen-Gibco), epithelial side down, and then immediately inverted so that the epithelial side was uppermost. The epithelial cells were gently scraped from the limbal area with fine-pointed forceps. The cells were collected in 1 mL CECM. The cell suspension was pipetted up and down on the tissue segment to disperse the cells and then transferred into a T-25 tissue culture flask (Fisher Scientific, Loughborough, UK) containing growth-arrested 3T3 mouse fibroblasts plated at a cell density of 7 × 105 cells/mL. The fibroblasts had been growth arrested by treatment for 2 hours in Dulbecco's modified Eagles' medium (DMEM; Invitrogen) supplemented with 10% newborn calf serum (Invitrogen), 1% antibiotic-antimycotic solution (Invitrogen), and 4 μg/mL mitomycin C (Sigma-Aldrich). The cultures were incubated at 37°C and 5% CO2 in air. The culture medium was changed three times a week, and the cultures were passaged on reaching 80% to 90% confluence. At each passage six-well plates and eight-chamber slides containing growth-arrested 3T3 mouse fibroblasts (2.6 × 105 cells/mL per well or 2.8 × 104 cells/mL per chamber) were seeded by LECs (700 LECs/well and 500 LECs/chamber, respectively). 
Indirect Immunofluorescence on Radial Sections
Slices obtained from all corneoscleral specimens were stained with anti-CK8 antibody, and each staining was performed in duplicate. A negative control (primary antibody omitted) was included in every slide. Immunohistochemistry on cryosections was performed, as described previously. 41 The mouse monoclonal antibody anti-CK8 (clone 4.1.18, 1:400; Millipore, Bedford MA) was used, and subsequently the sections were incubated with the appropriate secondary antibody (fluorescein isothiocyanate (FITC)–conjugated anti-mouse IgG; Jackson ImmunoResearch Laboratories, West Grove, PA). 
Double-Staining on Radial and Tangential Sections
Double-staining was performed on six different corneoscleral samples (three were used for radial and three for tangential sections). After the samples were fixed in cold acetone (10 minutes) and rinsed in phosphate-buffered saline (PBS), 5% donkey serum was applied. A mixture of mouse anti-CK8 antibody (Millipore) and goat anti-CK3 (clone C-14, 1:50), anti-CK15 (clone A-13, 1:400), anti-integrin α6 (clone C-18, 1:40), or anti-vimentin (clone C-20, 1:100; all from Santa Cruz Biotechnology, Santa Cruz, CA) was applied to the sections in one step, followed by a mixture of FITC-conjugated donkey anti-mouse IgG and TRITC-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories). All antibodies were diluted in PBS as described by the manufacturers. 
Indirect Immunofluorescence on Cultivated Cells
Immunocytochemical staining was performed to localize CK8. In brief, confluent corneal epithelial cultures were fixed in 4% paraformaldehyde in PBS at room temperature for 10 minutes and treated in blocking solution (5% normal goat serum, Jackson ImmunoResearch Laboratories). The blocking solution used for immunocytochemistry was supplemented with 0.33% Triton X100 (Sigma-Aldrich) to permeabilize the cell membranes. The primary antibody (monoclonal mouse anti-human CK8; Millipore) was applied for 1 hour at room temperature, followed by incubation with a secondary antibody conjugated with fluorescent dye (Alexa Fluor 488 conjugated goat anti-mouse IgG, Invitrogen) and mounted with antifade medium (Vectastain; Vector Laboratories, Inc., Burlingame, CA). 
Immunohistochemistry Assessment
Sections were examined by fluorescence microscopy (model BX51; Olympus Co., Tokyo, Japan) and by an inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany) at a magnification of 100× to 400×. The images were obtained with one of two cameras (VDS CCD-1300QF; VDS Vosskühler GmbH, Osnabrück, Germany, or Jenoptik ProgRes C12plus; Jenoptik; Laser.Optik.Systeme GmbH, Jena, Germany) and produced with image-management software (NIS Elements; Laboratory Imaging, Prague, Czech Republic). At least 300 cells of the central corneal, limbal, and perilimbal conjunctival epithelium were examined, and the percentage of positive cells was calculated. The following scale was used to grade the intensity of cell staining: N, negative; 1, weak; 2, moderate; 3, intense; and 4, very intense. The mean range was calculated from three sections and two experiments. 
Semiquantitative Reverse Transcription–Polymerase Chain Reaction
Total RNA was isolated from the corneal, limbal, and perilimbal conjunctival epithelium of five individual corneoscleral discs (RNeasy Plus Microkit; Qiagen). Six microliters of total RNA were reverse transcribed into cDNA in a 20-μL reaction mixture (SuperScript III/RNase OUT Enzyme Mix; Invitrogen), according to the manufacturer's instructions. Subsequently, equal amounts of cDNA from five individual samples were amplified with the following specific oligonucleotides for CK8 and the housekeeping gene glyceraldehyde-3-phospate dehydrogenase (GAPDH): human CK8, sense primer 5′-ATCAGCTCCTCGAGCTTCTC-3′, anti-sense primer 5′-TCCAGGAACCGTACCTTGTC-3′; and human GAPDH, sense primer 5′-AGCCACATCGCTCAGACAC-3′, anti-sense primer 5′-GCCCAATACGACCAAATCC-3′. The number of PCR cycles for the different primer pairs was 35 cycles for both CK 8 and GAPDH. Each cycle consisted of denaturation for 25 seconds at 95°C, annealing for 30 seconds at 65°C for CK 8, 60°C for GAPDH and elongation for 45 seconds at 72°C. PCR products were analyzed by ethidium bromide–stained 2% agarose gel electrophoresis. 
Results
Indirect Immunofluorescence on Cadaveric Corneoscleral Discs
The presence of CK8-positive cells in various locations of the corneoscleral discs assessed using indirect immunohistochemical fluorescence is shown in Table 1
Table 1.
 
The Average Percentage of CK8-Positive Cells and the Intensity of the Signal in the Corneal, Limbal, and Perilimbal Conjunctival Epithelium of 20 Corneoscleral Discs
Table 1.
 
The Average Percentage of CK8-Positive Cells and the Intensity of the Signal in the Corneal, Limbal, and Perilimbal Conjunctival Epithelium of 20 Corneoscleral Discs
Sample Age (y) Basal Cells of the Limbus* Suprabasal and Superficial Cells of the Limbus* Corneal Epithelium* Perilimbal Conjunctiva*
1 16 N 50/2 50/2 50/1
2 26 N N N N
3 29 N N N N
4 30 N 30/1 s 55/1 s 25/1 s
5 38 Line/3 60/2 80/3† 80/3
6 49 Line/3 50/2 85/3 s 50/2
7 49 Clusters/1 5/1 40/1 s 20/1 s
8 51 N N N N
9 59 N 5/3 s 40/1 N
10 61 Line/3 30/1 50/2† 80/2
11 66 Line/1 65/1 25/1† 25/1†
12 67 Line/1 50/1 35/1† 30/1†
13 68 N N N N
14 70 N N N N
15 73 Clusters/3 30/1 30/1 75/2
16 74 Line + clusters/3 20/2 10/2 s N
17 74 Line + clusters/4 50/2 d 80/3
18 77 Line/1 35/1 40/1 s 30/1
19 77 Clusters/3 60/2 70/2 80/3
20 82 Clusters/4 35/1 60/2† 80/3
Sixty percent of the samples revealed weak to strong positivity for CK8 in the cytoplasm of cells of the limbal basal layer. Most of the positive basal cells were small, with a high nuclear–cytoplasmic ratio, and formed clusters (Figs. 1a–c) or showed continuous staining (positivity in a single line; Figs. 1d–f). In addition, positive elongated cells in the near vicinity of CK8-positive clusters were detected in the suprabasal layers of the limbal epithelium (Figs. 1d, 1e). Weak to moderate positivity was detected in approximately 40% of the surface cells of the limbus. In most specimens, the intensity of the signal in the superficial cells was weaker than the signal in the basal limbal layer. The central corneal epithelium was positive for CK8, predominantly in the superficial and suprabasal layers (Fig. 1h), but some heterogeneous positivity was detected in the basal layer of several samples as well (Table 1). In each specimen that contained positive limbal basal cells, the epithelium of the cornea was positive as well (Figs. 1g, 1h; Table 1). Similarly, in most specimens in which CK8 was absent from the limbal basal cells, the epithelium of the central cornea was negative (Figs. 1i, 1j). No signal was seen in any of the negative controls (Fig. 1k). 
Figure 1.
 
Immunolocalization of CK8 in radial sections of the limbus and cornea. CK8 expression in the limbus: positive limbal basal cells forming clusters (circles; ac) or lines with clearly visible elongated CK8-positive cells projecting from the basal layer (arrows; d, e); limbal specimen in which, besides a basal line, CK8 is abundantly present throughout the suprabasal and superficial limbal layers (f). If the limbal basal cells were positive for CK8 (g), the central corneal epithelium of the same specimen was positive as well (h). If CK8 staining was absent from the limbal epithelium (i), the central corneal epithelium of the same specimen was negative also (j). Negative control of the limbus (k). Scale bar, 10 μm.
Figure 1.
 
Immunolocalization of CK8 in radial sections of the limbus and cornea. CK8 expression in the limbus: positive limbal basal cells forming clusters (circles; ac) or lines with clearly visible elongated CK8-positive cells projecting from the basal layer (arrows; d, e); limbal specimen in which, besides a basal line, CK8 is abundantly present throughout the suprabasal and superficial limbal layers (f). If the limbal basal cells were positive for CK8 (g), the central corneal epithelium of the same specimen was positive as well (h). If CK8 staining was absent from the limbal epithelium (i), the central corneal epithelium of the same specimen was negative also (j). Negative control of the limbus (k). Scale bar, 10 μm.
Cells in the basal layer of the limbus showed positive staining for CK8/CK15 and CK8/vimentin in double-immunostained radial sections (Fig. 2). On the other hand, only a few CK3-positive cells were scattered in the superficial layer of the limbus, and no CK3-positive cells were present in the basal layer of the limbus. In the CK3-positive central corneal epithelium, CK8-positive cells were found as well (Fig. 3). Positive staining for integrin α6 in the basal membrane decreased or was absent in the area of the clusters of CK8-positive cells (Fig. 2). 
Figure 2.
 
Immunolocalization of CK8 with CK15, vimentin, CK3 or integrin α6 in the basal layer of the limbus on radial sections. CK8 (green, FITC) colocalized with CK15 (red, TRITC) and with vimentin (red, TRITC). CK3 (red, TRITC), in contrast to CK8, was completely absent from the basal cells of the limbus, whereas a few suprabasal and superficial cells were CK3-positive. The expression of integrin α6 (red, TRITC) decreased in the areas where CK8-positive clusters occurred (arrows). Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm.
Figure 2.
 
Immunolocalization of CK8 with CK15, vimentin, CK3 or integrin α6 in the basal layer of the limbus on radial sections. CK8 (green, FITC) colocalized with CK15 (red, TRITC) and with vimentin (red, TRITC). CK3 (red, TRITC), in contrast to CK8, was completely absent from the basal cells of the limbus, whereas a few suprabasal and superficial cells were CK3-positive. The expression of integrin α6 (red, TRITC) decreased in the areas where CK8-positive clusters occurred (arrows). Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm.
Figure 3.
 
Immunolocalization of CK8 (green, FITC) and CK3 (red, TRITC) in the central corneal epithelium. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm.
Figure 3.
 
Immunolocalization of CK8 (green, FITC) and CK3 (red, TRITC) in the central corneal epithelium. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm.
Indirect Immunofluorescence Staining of Corneas Stored in Organ Culture
Strong positivity for CK8 in the basal cells of the limbus located in the limbal crypts was detected in all tangential sections of all corneoscleral samples. Elongated CK8-positive cells were detected in the suprabasal layer of the limbus, and a strong signal was observed in some superficial cells. Storage in organ culture for 8 to 9 days led to a decrease in the number of epithelial layers to one to three. 
Double immunofluorescence staining of tangential sections showed positive staining for CK8/CK15 and CK8/vimentin of cells in the basal layer of the limbal crypts. No immunostaining for CK3 was present in the limbal crypts. The most intensive staining for integrin α6 was observed in the basal membrane of the limbus. The difference between CK8-positive and integrin α6-negative areas was less apparent than in radial sections (Fig. 4). 
Figure 4.
 
The immunohistochemical localization of CK8 in tangential sections of corneas after storage in organ culture. CK8 (green, FITC) colocalized with CK15 (red, TRITC) and with vimentin (red, TRITC) in the limbal crypts. The limbal epithelial cells were CK8 positive, whereas no CK3 (red, TRITC) signal was detected in these cells. The difference between CK8-positive and integrin α6 (red, TRITC)-negative areas was less apparent than in radial sections. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm.
Figure 4.
 
The immunohistochemical localization of CK8 in tangential sections of corneas after storage in organ culture. CK8 (green, FITC) colocalized with CK15 (red, TRITC) and with vimentin (red, TRITC) in the limbal crypts. The limbal epithelial cells were CK8 positive, whereas no CK3 (red, TRITC) signal was detected in these cells. The difference between CK8-positive and integrin α6 (red, TRITC)-negative areas was less apparent than in radial sections. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm.
A strong signal for CK8 was detected in approximately 80% of the cultured LECs. Most of these positive cells were small, with a high nuclear–cytoplasmic ratio, but the signal was retained in elongated cells as well (Fig. 5). 
Figure 5.
 
The immunohistochemical localization of CK8 in cell cultures obtained from limbal epithelial cells (a, b). Scale bar, 10 μm.
Figure 5.
 
The immunohistochemical localization of CK8 in cell cultures obtained from limbal epithelial cells (a, b). Scale bar, 10 μm.
Figure 6.
 
Expression of cytokeratin 8 in five samples of limbal epithelium (lim) and in three representative samples of corneal (cor) and perilimbal conjunctival epithelium (con) determined by RT-PCR. GAPDH was used as an internal control. NCo-negative control (reaction without sample cDNA), a marker for internal contamination, L-50-bp DNA ladder (25–1000 bp).
Figure 6.
 
Expression of cytokeratin 8 in five samples of limbal epithelium (lim) and in three representative samples of corneal (cor) and perilimbal conjunctival epithelium (con) determined by RT-PCR. GAPDH was used as an internal control. NCo-negative control (reaction without sample cDNA), a marker for internal contamination, L-50-bp DNA ladder (25–1000 bp).
CK8 mRNA was found in the corneal, limbal, and perilimbal conjunctival epithelium of all five corneoscleral discs, using semiquantitative RT-PCR, as was GAPDH mRNA, which served as an internal control. Representative results are shown in Figure 6
Discussion
Our results clearly showed CK8 expression in the corneal, limbal, and conjunctival epithelium. A strong signal for CK8 was detected, especially in the basal layer of the limbal epithelium. Similar staining with respect to this location had been shown for CK15 and -19. 13,24 Most of our radial and all our tangential sections revealed an intense signal for CK8 located in the basal layer of the limbal crypts, which extended into the suprabasal and superficial epithelial layers of the limbus and cornea, where the signal intensity gradually decreased. The location of the CK8-positive cells, which is in accordance with the X, Y, Z hypothesis, 4 indicates that the expression of CK8 probably persists during the differentiation process from LESCs through TACs up to terminally differentiated corneal epithelial cells. The explanation for the absence of CK8 in some specimens could lie in the fact that radial sections from cadaveric corneas were not obtained solely from the superior or inferior areas (areas rich in limbal crypts) 46 or that its absence reflects a situation in which the basal limbal cells are in a quiescent state and thus do not proliferate. 
In the central cornea, CK8 immunostaining was located predominantly in the superficial and suprabasal layers, where it partially colocalized with the corneal differentiation marker CK3. Occasional positivity in the basal layer of the central cornea may be explained by the fact that these basal cells proliferate, differentiate, and, in this way, participate in the renewal of the central corneal epithelium. 1,3 Moreover, we have found CK8 in all layers of the perilimbal conjunctiva (Table 1) and bulbar conjunctiva (data not shown). All the obtained data support our idea that CK8 may be expressed in activated basal cells that are ready to divide and differentiate. 
Our hypothesis that CK8 expression persists during differentiation was confirmed by its detection in cultured LECs obtained from corneoscleral buttons after long-term storage. It was found that most cells in LEC cultures are limbal rather then corneal in phenotype (expressing CK19, β1 integrin, and p63). 47 This finding does not, however, prove that any of these cultured cells are stem cells. It is likely that LEC cultures consist of a heterogeneous population of LESCs and a gradient of differentiated cells. Our results showed that both cells with a high nucleocytoplasmic ratio as well as more differentiated elongated cells with a larger cytoplasm in LEC sheets were positively stained for CK8. 
Regarding the localization of CK8 in the basal layers of the limbus, we wanted to determine the phenotype of the CK8-positive basal cells of the limbal epithelium using double-staining immunohistochemistry. CK8 in the limbus was clearly localized in the same cells as CK15 and vimentin, which are accepted as potential stem cell markers. 19,24 An inverse staining gradient was seen between CK8 and cornea-specific CK3 protein as well as between CK8 and integrin α6 in the limbus. Integrin α6 (together with integrin β4) is a component of hemidesmosomes, localized specifically to the basal membrane of basal cells. 48 It has been suggested that the lack of its expression is an inherent feature of LESCs, reflecting their need for independence, and could facilitate the migration of cells derived from LESCs. 9 Although we agree with this interpretation, it is at odds with the human study by Kim et al., 49 and with the study by Pajoohesh-Ganji et al. 50 in the mouse, which showed that more adherent cells expressing elevated integrin levels are more stemlike than cells expressing lower integrin levels. 
On the basis of all these facts, the possibility that CK8 is a newly found marker for LESCs could be considered, but CK8 is present in abundance in half of the basal limbal epithelial cells; moreover, it is still present in elongated cells projecting from CK8-positive clusters up to the cells in the central corneal epithelium. As stem cells represent less than 10% of the total limbal basal cell population, 51 it is clear that CK8 cannot be considered as a potential marker specific only for LESCs. 
Much more interesting would be to elucidate the exact function of CK8 in the human cornea, especially in the limbal epithelium. Providing mechanical strength in the single layered epithelia and interacting with desmosomes are the basic, but not the only, functions of CK8 and -18. The expression of both CKs increases the migratory and invasive ability of transfected cells. 52 They bind DNA, RNA as well as molecules that are important in signal transduction. 53 56 The CK8/18 pair plays an important role in cell-cycle regulation through phosphoserine-binding protein 14-3-3, which is a key regulator in signal-transduction/cell-cycle checkpoint control. 57 During the S/G2/M phases, when cytokeratins become hyperphosphorylated, 14-3-3 binds to CK8/18 and, due to cdc25 (dual-specificity phosphatase), remains free to dephosphorylate the cdc2/cyclin B complex, thus cells come through the M-phase checkpoint. 54,58 60 The key role of CK8 in cell-cycle regulation was demonstrated and confirmed in CK8-null mice, where hepatic cells exhibited an altered cellular redistribution of 14-3-3 protein into the nuclei and its binding to cdc25. All this leads to cell-cycle deregulation and finally, in the G2-phase, to cell-cycle arrest. 38,40  
Although the expression of CK8 is very important for normal cell signaling and cell-cycle regulation, 38,40 as well as for the migratory and invasive ability of cells, 52 its exact function in the cells of the corneoscleral discs is not known yet. The obtained data support our hypothesis that CK8 could play some still unidentified role in the activation of cells and their proliferation and migration. The signal for CK8 is retained in cells during their differentiation, but the exact relation between CK8 expression and the renewal of cells in the corneoconjunctival area remains to be elucidated. 
Footnotes
 Supported by the research project of the Ministry of Education, Youth, and Sports of the Czech Republic MSM0021620806 and by Project 260501 from Charles University in Prague.
Footnotes
 Disclosure: S. Merjava, None; K. Brejchova, None; A. Vernon, None; J.T. Daniels, None; K. Jirsova, None
The authors thank Viera Vesela and Sarka Kalasova for excellent technical assistance with the preparation of the specimens. 
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Figure 1.
 
Immunolocalization of CK8 in radial sections of the limbus and cornea. CK8 expression in the limbus: positive limbal basal cells forming clusters (circles; ac) or lines with clearly visible elongated CK8-positive cells projecting from the basal layer (arrows; d, e); limbal specimen in which, besides a basal line, CK8 is abundantly present throughout the suprabasal and superficial limbal layers (f). If the limbal basal cells were positive for CK8 (g), the central corneal epithelium of the same specimen was positive as well (h). If CK8 staining was absent from the limbal epithelium (i), the central corneal epithelium of the same specimen was negative also (j). Negative control of the limbus (k). Scale bar, 10 μm.
Figure 1.
 
Immunolocalization of CK8 in radial sections of the limbus and cornea. CK8 expression in the limbus: positive limbal basal cells forming clusters (circles; ac) or lines with clearly visible elongated CK8-positive cells projecting from the basal layer (arrows; d, e); limbal specimen in which, besides a basal line, CK8 is abundantly present throughout the suprabasal and superficial limbal layers (f). If the limbal basal cells were positive for CK8 (g), the central corneal epithelium of the same specimen was positive as well (h). If CK8 staining was absent from the limbal epithelium (i), the central corneal epithelium of the same specimen was negative also (j). Negative control of the limbus (k). Scale bar, 10 μm.
Figure 2.
 
Immunolocalization of CK8 with CK15, vimentin, CK3 or integrin α6 in the basal layer of the limbus on radial sections. CK8 (green, FITC) colocalized with CK15 (red, TRITC) and with vimentin (red, TRITC). CK3 (red, TRITC), in contrast to CK8, was completely absent from the basal cells of the limbus, whereas a few suprabasal and superficial cells were CK3-positive. The expression of integrin α6 (red, TRITC) decreased in the areas where CK8-positive clusters occurred (arrows). Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm.
Figure 2.
 
Immunolocalization of CK8 with CK15, vimentin, CK3 or integrin α6 in the basal layer of the limbus on radial sections. CK8 (green, FITC) colocalized with CK15 (red, TRITC) and with vimentin (red, TRITC). CK3 (red, TRITC), in contrast to CK8, was completely absent from the basal cells of the limbus, whereas a few suprabasal and superficial cells were CK3-positive. The expression of integrin α6 (red, TRITC) decreased in the areas where CK8-positive clusters occurred (arrows). Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm.
Figure 3.
 
Immunolocalization of CK8 (green, FITC) and CK3 (red, TRITC) in the central corneal epithelium. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm.
Figure 3.
 
Immunolocalization of CK8 (green, FITC) and CK3 (red, TRITC) in the central corneal epithelium. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm.
Figure 4.
 
The immunohistochemical localization of CK8 in tangential sections of corneas after storage in organ culture. CK8 (green, FITC) colocalized with CK15 (red, TRITC) and with vimentin (red, TRITC) in the limbal crypts. The limbal epithelial cells were CK8 positive, whereas no CK3 (red, TRITC) signal was detected in these cells. The difference between CK8-positive and integrin α6 (red, TRITC)-negative areas was less apparent than in radial sections. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm.
Figure 4.
 
The immunohistochemical localization of CK8 in tangential sections of corneas after storage in organ culture. CK8 (green, FITC) colocalized with CK15 (red, TRITC) and with vimentin (red, TRITC) in the limbal crypts. The limbal epithelial cells were CK8 positive, whereas no CK3 (red, TRITC) signal was detected in these cells. The difference between CK8-positive and integrin α6 (red, TRITC)-negative areas was less apparent than in radial sections. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm.
Figure 5.
 
The immunohistochemical localization of CK8 in cell cultures obtained from limbal epithelial cells (a, b). Scale bar, 10 μm.
Figure 5.
 
The immunohistochemical localization of CK8 in cell cultures obtained from limbal epithelial cells (a, b). Scale bar, 10 μm.
Figure 6.
 
Expression of cytokeratin 8 in five samples of limbal epithelium (lim) and in three representative samples of corneal (cor) and perilimbal conjunctival epithelium (con) determined by RT-PCR. GAPDH was used as an internal control. NCo-negative control (reaction without sample cDNA), a marker for internal contamination, L-50-bp DNA ladder (25–1000 bp).
Figure 6.
 
Expression of cytokeratin 8 in five samples of limbal epithelium (lim) and in three representative samples of corneal (cor) and perilimbal conjunctival epithelium (con) determined by RT-PCR. GAPDH was used as an internal control. NCo-negative control (reaction without sample cDNA), a marker for internal contamination, L-50-bp DNA ladder (25–1000 bp).
Table 1.
 
The Average Percentage of CK8-Positive Cells and the Intensity of the Signal in the Corneal, Limbal, and Perilimbal Conjunctival Epithelium of 20 Corneoscleral Discs
Table 1.
 
The Average Percentage of CK8-Positive Cells and the Intensity of the Signal in the Corneal, Limbal, and Perilimbal Conjunctival Epithelium of 20 Corneoscleral Discs
Sample Age (y) Basal Cells of the Limbus* Suprabasal and Superficial Cells of the Limbus* Corneal Epithelium* Perilimbal Conjunctiva*
1 16 N 50/2 50/2 50/1
2 26 N N N N
3 29 N N N N
4 30 N 30/1 s 55/1 s 25/1 s
5 38 Line/3 60/2 80/3† 80/3
6 49 Line/3 50/2 85/3 s 50/2
7 49 Clusters/1 5/1 40/1 s 20/1 s
8 51 N N N N
9 59 N 5/3 s 40/1 N
10 61 Line/3 30/1 50/2† 80/2
11 66 Line/1 65/1 25/1† 25/1†
12 67 Line/1 50/1 35/1† 30/1†
13 68 N N N N
14 70 N N N N
15 73 Clusters/3 30/1 30/1 75/2
16 74 Line + clusters/3 20/2 10/2 s N
17 74 Line + clusters/4 50/2 d 80/3
18 77 Line/1 35/1 40/1 s 30/1
19 77 Clusters/3 60/2 70/2 80/3
20 82 Clusters/4 35/1 60/2† 80/3
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