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First published online June 1, 2006
Stem Cells Vol. 24 No. 9 September 2006, pp. 2130 -2139
doi:10.1634/stemcells.2005-0590; www.StemCells.com
© 2006 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Interleukin-1 Receptor Antagonist (IL-1RA) Prevents Apoptosis in Ex Vivo Expansion of Human Limbal Epithelial Cells Cultivated on Human Amniotic Membrane

Chi-Chin Suna,b,c, Jong-Hwei Su Pangb, Ching-Yi Chengc, Hsiao-Fen Chengc, Yun-Shien Leed,e, Wan-Chen Kua, Ching-Hsi Hsiaoa, Jen-Kan Chenc, Chuen-Mao Yangc

aDepartment of Ophthalmology, Chang Gung Memorial Hospital, Keelung, Taiwan;
bGraduate Institute of Clinical Medical Sciences, Chang Gung University, Kwei-shan, Taoyuan, Taiwan;
cDepartment of Physiology and Pharmacology, Chang Gung University, Kwei-shan, Taoyuan, Taiwan;
dGenomic Medicine Research Core Laboratory (GMRCL) of Chang Gung Memorial Hospital, Kwei-shan, Taoyuan, Taiwan;
eDepartment of Biotechnology, Ming-Chuan University, Kwei-shan, Taoyuan, Taiwan

Key Words. Limbal epithelial cells • Amniotic membrane • Interleulin-1 receptor antagonist • Cytokines • Extracellular matrix

Correspondence: Chuen-Mao Yang, Ph.D., Department of Physiology and Pharmacology, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan, Taiwan. Telephone: 03-2118800 (ext. 5123); Fax: 03-2118365; e-mail: chuenmao{at}mail.cgu.edu.tw

Received November 25, 2005; accepted for publication May 22, 2006.
First published online in STEM CELLS EXPRESS   June 1, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Stem cells of the corneal epithelium have been found to be located exclusively at the anatomical junction between the cornea and the conjunctiva, the limbus. Ex vivo expanded limbal epithelial cells on amniotic membrane (AM) are capable of restoring the corneal surface with limbal stem cell deficiency. Recent studies indicate that intact AM preserves the limbal epithelial phenotype and that distinct epithelial morphology is noted among various culture matrix. However, the factors in response to the interaction between limbal epithelial cells and AM were not well understood. Using Annexin V-fluorescein isothiocyanate staining, we found that human limbal epithelial cells expanded on intact human AM demonstrated fewer apoptotic cells as compared with those on plastic dishes. To identify the anti-apoptotic factors, we performed cDNA microarray analysis and showed that interleukin-1 receptor antagonist (IL-1RA) was overexpressed in cultures on intact AM, which was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (Q-PCR) and enzyme-linked immunosorbent assay. In addition, we also noted that the phenomenon of apoptosis detected in cultures on plastic dishes could be reversed by adding recombinant IL-1RA protein into the media, whereas apoptosis of limbal epithelial cells cultivated on intact AM could be induced by exogenous neutralizing IL-1RA neutralizing antibody. These results demonstrated that intact human AM may prevent cultured human limbal epithelial cells from undergoing apoptosis. IL-1RA might be a candidate mediator to exert as an anti-apoptotic molecule during the interaction between human limbal epithelial cells and intact human AM.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Accumulating evidence has indicated that corneal epithelial stem cells (SCs) reside in the basal layer of the limbal area, a transitional zone between the cornea and conjunctiva [1]. The limbal stem cell concept and the use of variable surgical techniques for limbal epithelial stem cell transplantation (for reviewing, see [2]) have brought the classification and treatment of ocular surface diseases to a new frontier. Recently, an innovative procedure using amniotic membrane (AM) as a carrier for ex vivo expansion of limbal epithelial SCs was utilized to treat limbal stem cell deficiency (LSCD) [3, 4]. This treatment modality, taking only a small piece of limbal tissue, theoretically minimizes the risk of LSCD to the donor eye and reduces the incidence of allograft rejection, because only epithelial cells rather than a limbal graft are transplanted [5].

The AM is the innermost membrane lining the placenta facing the fetus. This semitransparent membrane consists of a simple cuboidal epithelium, a thick basement membrane, and an avascular mesenchymal stroma. Transplantation of human AM to provide a substrate for regeneration of corneal epithelial cells has been proven to be effective in ocular surface reconstruction in human eyes [6–9]. Together with limbal auto- or allografts, AM transplantation (AMT) successfully reconstructed the ocular surface in patients with LSCD secondary to chemical or thermal burns, ocular cicatricial pemphigoid and Stevens-Johnson syndrome [10, 11]. A recent review by Grueterich et al. [12] indicates that AM itself may serve as an ideal "niche" to support ex vivo expansion of human limbal epithelial SCs, the use of which has recently gained popularity in treating the damaged ocular surface. Such a "composite graft" has been successfully applied in reconstructing the ocular surface in partial or total LSCD in several human studies [3, 4, 13–15].

In our previous study, we have found that matrix metalloproteinase-9 (MMP-9) plays an important role in mediating the outgrowth of human limbal explants on intact AM [16]. In addition to different outgrowth rates among various culture conditions, the existence of AM as an underlying matrix or not also induced the expanded limbal epithelial cells toward distinct differentiated status. This phenomenon was evident in previous studies, which indicated that intact AM preserves limbal epithelial phenotype, whereas denuded AM promotes corneal phenotype [17, 18]. Indeed, during the culture period, we noted several morphological differences between limbal epithelial cells grown on intact AM and those cultured on plastic dishes. For example, the sizes of limbal epithelial cells expanded on intact AM were more uniform, whereas those grown on plastic dishes were irregular in size and shape with the cells near the leading edge much larger and disorganized than those adjacent to the limbal explants. Moreover, we also found that many pyknotic and apoptotic cells were readily seen in cultures of limbal explants seeded on plastic dishes from the second week in culture. These apoptotic cells, however, were very few or not observed in ex vivo expanded limbal epithelial cells cultivated on intact AM. Based on these morphological observations, we hypothesized that during the culture period, the factor(s) likely involved in maintaining limbal epithelial cells in a less apoptotic state was released from the interaction between limbal epithelial cells and AM.

Several lines of evidence have demonstrated that many growth factors and neurotrophic factors could be released from AM with or without amniotic epithelial cells [19–21]. These growth factors are believed to be involved in the epithelialization process, which is one of the action mechanisms exerted by AMT. On the other hand, AM has also been shown to suppress the expression of various cytokines such as transforming growth factor-ß (TGF-ß) isoforms, interleukin-1{alpha} (IL-1{alpha}), and IL-1ß in ocular surface epithelial and fibroblastic cells [22, 23]. These effects, in turn, illustrate the antifibrotic and anti-inflammatory properties of the AM. Despite of a lot of secreted growth factors mediating the actions of AM, however, there is no such "survival factor" proposed in the literature in this ex vivo expansion model of human limbal epithelial cells on AM as of today. Therefore, the present study was conducted to identify the candidate factor(s) responsible for keeping limbal epithelial cells cultivated on intact AM from undergoing apoptosis. In this study, with the use of cDNA microarray analysis, real-time polymerase chain reaction (RT-PCR), real-time quantitative PCR (Q-PCR), and enzyme-linked immunosorbent assay (ELISA), we successfully identified a naturally occurring cytokine, interleukin-1 receptor antagonist (IL-1RA), as an anti-apoptotic factor in this culture model.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
The tissue culture dishes (40 x 10 mm) were purchased from Orange Scientific (Waterloo, Belgium, http://www.orangesci.com). Dulbecco's modified Eagle's medium (DMEM)/F-12 medium and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, http://www.invitrogen.com). The detection kits for Annexin V-fluorescein isothiocyanate (FITC) apoptosis, ELISA detection kit for human IL-1RA, recombinant human IL-1RA protein, and anti-human IL-1 RA neutralizing antibody were purchased from R&D Systems (Minneapolis, Minneapolis, http://www.rndsystems.com). Mouse IgG2A isotype control for IL-1 RA neutralizing antibody was from eBioscience (San Diego, http://www.ebioscience.com). The ELISA detection kit for human IL-1ß was from Cayman (Ann Arbor, MI, http://www.caymanchemical.com). All studies were performed with the approval of the institutional ethics committee.

Human Limbal Explants Cultured on Human AM
Human tissue was handled according to the tenets of the Declaration of Helsinki. Corneoscleral buttons from human donor eyes, aged 15–65 years, were obtained from the Chang Gung Memorial Hospital Eye Bank. The tissue was rinsed three times with DMEM/F-12 containing 50 µg/ml gentamicin and 1.25 µg/ml amphotericin B. After careful removal of excessive sclera, iris, corneal endothelium, conjunctiva, and Tenon's capsule, the remaining tissue was placed in a culture dish and cut into cubes of approximately 1.5 x 2 x 3 mm3 by a scalpel. Human AM was obtained by elective cesarean section from Chang Gung Memorial Hospital (Keelung, Taiwan) with properly informed consent and was processed as described [24]. Briefly, the AM was aseptically washed three times in 200 ml of phosphate buffered saline containing 50 µg/ml penicillin, 50 µg/ml streptomycin, 2.5 µg/ml amphotericin B, and 25 ng/ml gentamicin. The AM was preserved sterile in DMEM/F-12 with 50% glycerin at –80°C for at least 6 months. Before use, the AM was thawed and placed on a culture dish with the basement membrane side up and incubated at 37°C in a humidified incubator under 95% air and 5% CO2 overnight. For experiments of Annexin V-FITC binding assay, square cover slides (2.2 x 2.2 cm in size, Assistent, Sondheim, Germany, http://www.hecht-assistent.de) placed on the center of culture dishes were paved with or without intact AM and incubated for at least 48 hours before culture. On the center of AM, a limbal explant was placed and cultured in a medium made of an equal volume of HEPES-buffered DMEM containing bicarbonate and F-12, and supplemented with 5% FBS, 0.5% dimethyl sulfoxide, 2 ng/ml mouse epidermal growth factor, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, 0.5 µg/ml hydrocortisone, 30 ng/ml cholera toxin, 50 µg/ml gentamicin, and 1.25 µg/ml amphotericin B. Cultures were incubated at 37°C under 5% CO2 and 95% air, and the medium was changed and saved for determination of IL-1ß and IL-1RA concentrations every 2–3 days, while the extent of each outgrowth was monitored with a phase contrast microscope. When addition of exogenous IL-1RA protein or IL-1RA neutralizing antibody was necessary to detect their anti-apoptotic or pro-apoptotic effects on limbal epithelial cells, various concentrations of IL-1RA protein or IL-1RA neutralizing antibody was added in the culture media every 2–3 days from day 14 in culture for another week, and then subjected to apoptosis detection and microscopic photography.

Detection of Apoptosis by Annexin V-FITC Binding
Human limbal explants with or without AM were grown on coverslides. After three weeks in culture, the explants were washed with 1x binding buffer provided in the TACS Annexin V-FITC apoptosis detection kit and then incubated in 1x binding buffer containing Annexin V-FITC and propidium iodide in the dark for 10 minutes at room temperature. Excess unbound Annexin V-FITC and propidium iodide were washed off with 1x binding buffer, and explants were mounted on slides for fluorescent microscopic observation. Cells stained with green fluorescence of FITC only were identified as apoptotic cells at early stage. Cells that stained with both green and orange fluorescence were in the late phase apoptotic state.

RNA Extraction
Total cellular RNA from limbal epithelial cells expanded on plastic dishes or intact AM or from intact AM alone (i.e. without overlying limbal explants) was isolated by lysis in a guanidinium isothiocyanate buffer followed by single step phenol-chloroform-isoamyl alcohol extraction [25]. Briefly, cells are harvested and lysed in solution D containing 4 M guanidium isothiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sodium sarkosine and 0.1 M ß-mercaptoethanol. Sequentially, one-tenth volume of 2 M sodium acetate (pH 4.0), one volume of phenol and one-fifth volume of chloroform-isoamyl alcohol (49:1, vol:vol) were added to the homogenate. After vigorous vortexing for 30 seconds, the solution was centrifuged at 10,000g for 15 minutes at 4°C. RNA in the aqueous phase was precipitated by the addition of 0.5 ml of isopropanol. RNA quality is confirmed by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, http://www.agilent.com) with RNA 6000 Nano Chips. On average, 32.5 µg of RNA from each culture on plastic dish could be harvested as contrast to 22.1 µg of RNA from that cultivated with intact AM. The difference was possibly due to the different outgrowth rates between these two conditions. Because the AM used in this study is cryopreserved for at least 6 months, we can hardly retrieve any RNA from intact AM alone. Therefore, the RNA obtained from limbal explants cultured on intact AM was derived from expanded limbal epithelial cells without contamination of the AM epithelial cells.

Microarray Procedures
In this study, we used the GMRCL Human 7K set, Version 2 chips as previously described [26]. Total RNA extracted from human limbal epithelial cells grown on intact AM was analyzed with cDNA microarray assays as tests against the same amount of RNA from those cultured on plastic dishes. The RNA was analyzed with the dye-swapping microarray design. We used 10 µg of RNA for labeling and hybridization using 3DNA Submicro EX Expression Array kit (Genisphere, Hatfield, PA, http://www.genisphere.com), and scanned slides with a confocal scanner ChipReader (Virtek Vision International Inc., Waterloo, Ontario, Canada, http://www.virtekvision.com). We acquired the spot and background intensities with GenePix Pro 4.1 software (Axon Instruments/Molecular Devices Corp., Union City, CA, http://www.moleculardevices.com) and carried out within-slide normalization with programs wrote with MATLAB 6.0 software (The MathWorks, Inc., Natick, MA, http://www.mathworks.com). The rank of each gene was determined by the average value of dye-swapping log-ratio from four independent paired primary cultures.

RT-PCR and Real-Time Q-PCR Analysis
One µg of total RNA retrieved from limbal epithelial cells grown on plastic dish or intact AM was reverse-transcribed into cDNA by incubating with 200 units of reverse transcriptase in 20 µl of reaction buffer containing 0.25 µg of random primers and 0.8 mM dNTPs at 42°C for 1 hour. Two µl of the cDNA was used for the PCR reaction as templates. The PCR was performed in buffer containing 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 1 µM of each primer and 5 units of Taq DNA polymerase for 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 40 seconds and extension at 72°C for 30 seconds. The resulting PCR product was analyzed by 1.5% agarose gel electrophoresis. For real-time Q-PCR detection of RNA transcripts, the cDNA was analyzed in an ABI PRISM 7900 sequence detection system using the SYBR Green I PCR Master Mix (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). The data are calculated with {Delta}{Delta}CT. The sequences of specific PCR primers were IL-1RA (RT-PCR) sense, 5'-GCCGACCCTCTGGGAGAAAA-3'; anti-sense, 5'-GTCTGAGCGGATGAAGGCGA-3' (PCR product: 470 bp); IL-1RA (real-time Q-PCR) sense, 5'-GGCACTTGGAGACTTGTATGAAAGAT-3'; anti-sense, 5'-TCGCTGAGTACCTGCCAAGA-3' (PCR product: 152 bp); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (RT-PCR and real-time Q-PCR) sense, 5'-CTGCCCCCTCTGCTGATG-3'; and anti-sense, 5'-TCCACGATACCAAAGTTGTCATG-3' (PCR product: 170 bp).

Interleukin-1 RA and Interleukin-1ß ELISA Assay
The conditioned media saved from both culture conditions derived from six donor limbal explants at different time points during the culture period were collected, centrifuged, and stored at –20°C until assayed. The supernatants were subjected to quantitative sandwich immunoassay with ELISA detection kits for human IL-1RA (R&D Systems) or human IL-1ß (Cayman) and assayed spectrophotometrically according to the manufacturer's instructions. The amounts of extracted total RNA were used as an internal control of cellular mass.

Statistical Analysis
Data were analyzed with GraphPad Prism Program (GraphPad, San Diego, http://www.graphpad.com) and expressed as the mean ± SEM and analyzed with a two-tailed Student's t-test at a p < .05 level of significance.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
Apoptosis of Human Limbal Epithelial Cells Cultured on Intact AM Versus Those on Plastic Dish
To verify whether the AM possesses anti-apoptotic effects, human limbal explants were expanded on either intact AM or plastic dish for 3 weeks. The appearance of apoptotic cells and their morphological differences were photographed and confirmed by Annexin V-FITC binding assay. Under phase contrast microscopy, cells expanded on intact AM were about the same size with polygonal and cuboidal shape in morphology, which were typical for epithelial cells (Fig. 1B, 1D, and 1F). However, many pyknotic or irregular-shaped epithelial cells were noted and some were detaching from the underlying cell layer in human limbal epithelial cells expanded on plastic dishes. This phenomenon was evident at the end of second week in culture (Fig. 1A, 1C, and 1E). On the contrary, there were very few, if any, apoptotic cells in expanded limbal epithelial cells cultured on intact AM (Fig. 1B, 1D, and 1F). To further investigate the differences between these two culture conditions, we characterized the pyknotic cells on the plastic dish as apoptotic cells using a fluorescent staining with Annexin V-FITC binding assay (Fig. 1G). We also noted that cells at the outer edge of the expanded limbal epithelial cells remained uniform in size (Fig. 1F), whereas cells cultured on plastic dishes at the corresponding area showed a great variety in size and shape (Fig. 1E). These results suggested that intact AM may prevent cultured human limbal epithelial cells from undergoing apoptosis.


Figure 1
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  		Figure 1. Phase contrast photographs and fluorescent staining of human limbal epithelial cells expanded on plastic dishes, H and on intact amniotic membrane (AM), H/A at the end of third week in culture. Many pyknotic and irregularly shaped cells were found in cultures on plastic dishes (A, C, and E; arrows) as compared to those on intact AM (B, D, and F) at perilimbal, central, and edge areas. Fluorescent staining with Annexin V-fluorescein isothiocyanate at central area demonstrated that the irregularly shaped limbal epithelial cells cultured on plastic dishes were cells undergoing variable stages of apoptosis (G). In contrast, apoptotic cells were barely detected in cultures with intact AM (H). Green fluorescence: cells undergoing apoptosis. Orange fluorescence: cell nuclei counterstained by propidium iodide. Scale bar, 20 µm. Magnification: x200. Abbreviations: H, human limbal epithelial cells expanded on plastic dishes; H/A, human limbal epithelial cells expanded on intact AM.

 
Overexpression of IL-1RA Gene in Human Limbal Epithelial Cells Expanded on Intact AM Determined by cDNA Microarray Assay
On the basis of data obtained in Figure 1, we demonstrated that limbal epithelial cells expanded on intact human AM underwent little or no apoptosis through the culture period, implying that there may be a survival factor released in response to the interaction between limbal epithelial cells and intact AM. To investigate the candidate gene(s) responsible for this anti-apoptotic effect, we performed cDNA microarray assay to analyze the differential expression of mRNA in cells between two culture conditions (i.e. limbal epithelial cells cultured on either intact AM or plastic dishes). As shown in Table 1, a list of top 10 genes, which were overexpressed in limbal epithelial cells cultured on intact AM was generated from analyzing a total of 7,334 genes. From the Medline search for the potential function of each gene, we found that the IL-1RA, a naturally occurring cytokine and a member of the interleukin one family, has been shown to be capable of inhibiting apoptosis in various cell types [27–29]. Figure 2A shows two sets of representative dye-swapping results out of four microarray analyses using limbal specimens from different donors. It indicated that the IL-1RA gene (green or red spots in yellow square) was found to be overexpressed in human limbal epithelial cells cultured on intact AM compared with those on plastic dishes. The averaged log-ratio of IL-1RA gene overexpression in cells cultured on intact AM from four different microarray analyses was 2.61 (Fig. 2B). It is also noteworthy that half of the genes were extracellular matrix-related proteins (such as laminin, contactin, and fibrillin), implying a significant role of cell-cell matrix interaction in this model.


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  		Table 1. Top 10 upregulated genes determined by cDNA microarray analysis Genomic Medicine Research Core Laboratory for human limbal epithelial cells cultured on intact amniotic membrane versus those on plastic dishes

 


Figure 2
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  		Figure 2. Upregualtion of IL1-RA transcripts in cells cultured on intact amniotic membrane (AM), H/A versus those on plastic dishes, H. (A): Figures were two sets of representative dye-swapping results out of four microarray analyses. The IL-1RA gene (green or red spots in yellow square) was found to be overexpressed in H/A compared with H. (B): The averaged log ratio of IL-1RA gene overexpression in H/A from four different microarray analyses was 2.61. (Data were analyzed and expressed in log ratio as the mean ± SEM of four independent experiments. *, p < .05). Abbreviations: H, human limbal epithelial cells expanded on plastic dishes; H/A, human limbal epithelial cells expanded on intact AM.

 
Upregulation of IL-1RA RNA Transcripts in Human Limbal Epithelial Cells Expanded on Intact AM Analyzed by RT-PCR and Real-Time Q-PCR
To further confirm the cDNA microarray results, we performed RT-PCR and real-time Q-PCR to compare the IL-1RA RNA transcripts in these two culture conditions. As shown in Figure 3A, when normalized to the level of GAPDH, the IL-1RA transcripts were threefold increased in limbal epithelial cells grown on intact AM as compared with those on plastic dishes (n = 6). The absence of IL-1RA RNA transcripts detected in AM ruled out the possibility that IL-1RA transcripts might originally exist in AM. Real-time Q-PCR (Fig. 3B) also demonstrated that the IL-1RA RNA transcripts in cultures on intact AM were threefold of those on plastic dishes (n = 8, *, p < .05, data were calculated with {Delta}{Delta}CT). Taken together, these results indicated that the expression of IL-1RA was upregulated at the transcriptional level when limbal epithelial cells were expanded on intact AM.


Figure 3
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  		Figure 3. Differential expression of IL-1RA RNA transcripts in limbal epithelial cells expanded on intact amniotic membrane (AM), H/A, on plastic dishes, H, or in intact AM alone without overlying limbal explant, A. (A): The total RNA was extracted and analyzed by reverse transcription-PCR at the end of third week culture. Data were representative results in one of six individual experiments. (B): The total RNA was extracted and analyzed by real-time quantitative-PCR assays at the end of 3rd week culture. Data were summarized and expressed as the mean ± SEM of eight independent experiments. *, p < .05, compared with those of human limbal explants cultured on plastic dishes. Abbreviations: H, human limbal epithelial cells expanded on plastic dishes; H/A, human limbal epithelial cells expanded on intact AM; IL-1RA, interleukin-1 receptor antagonist; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; M, molecular mass marker; PCR, polymerase chain reaction.

 
ELISA of IL-1RA and IL-1ß in Conditioned Media from Human Limbal Explants Cultivated with or Without AM
To identify whether IL-1RA was also overexpressed at the translational level in cultures grown on intact AM, we performed ELISA in these two different culture conditions. We found that after 3 weeks in culture, the IL-1RA protein with or without intact AM was 8.93 ± 3.55 and 3.01 ± 0.70 ng/µg RNA, respectively (n = 6, p < .05). In our previous study, we have demonstrated the different cell growth rate between these two culture conditions. The outgrowth areas in cultures on plastic dishes were usually larger than those on intact AM at the end of 3 weeks [16]. Since the concentrations of IL-1RA also paralleled with cellular mass, the IL-1RA protein concentrations should be corrected to reveal actual differences between these two culture conditions. We utilized the amount of extracted total RNA instead of total protein from each culture as an internal control of cellular mass at the end of the 3 weeks to avoid the potential problem of getting contamination from AM proteins during sample extraction. If not corrected with total RNA, the concentration of IL-1RA protein in cultures with intact AM was 288.8 ng/ml. Since the function of IL-1ß is mediated by the alteration of IL-1ß/IL-1RA balance, we wondered whether upregulation of IL-1RA in cultures on intact AM was secondary to IL-1ß overexpression, with the IL-1ß/IL-1RA balance unchanged. Previous study has also indicated that activation of IL-1ß signaling cascades may lead to cellular apoptosis [29]. Therefore, we performed ELISA of IL-1ß protein and noted that the concentrations of IL-1ß protein in cultures with or without intact AM was 2.71 ± 0.20 and 2.63 ± 0.07 pg/µg RNA, respectively (n = 6, p = .71), which were not significantly different. These results implied that overexpression of IL-1RA protein in cultures on intact AM was not secondary to concomitant production of IL-1ß protein. To correlate the critical point beyond which apoptosis of limbal epithelial cells was significantly increased in cultures on plastic dish, we analyzed the conditioned media for IL-1ßand IL-1RA from both culture conditions every 2–3 days. We found that there was a time-dependent increase in the ratio of IL-1RA to IL-1ß in cultures on intact AM as compared to those on plastic dishes (Fig. 4), indicating that upregulation of IL-1RA in H/A culture exerted its anti-apoptotic effect on limbal epithelial cells. It is also noteworthy that the ratio gradually increased at the beginning (1.5 at day 2) and reached a peak (4.9) at day 14 in culture, which was well-correlated with the onset of apoptosis in limbal epithelial cells expanded on plastic dishes.


Figure 4
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  		Figure 4. Differential IL-1RA/IL-1ß protein ratios between limbal explants expanded on intact amniotic membrane (AM), H/A, and on plastic dishes, H, at different time points in culture. The relative ratio: [(IL-1RA/IL-1ß)H/A/(IL-1RA/IL-1ß)H] gradually increased from the beginning (1.5 at day 2) and reached a peak (4.9) at day 14 in culture, which was well-correlated with the onset of apoptosis in limbal epithelial cells expanded on plastic dishes. Data were analyzed by enzyme-linked immunosorbent assay and expressed in relative ratio as the mean ± SEM of four independent experiments. *, p < .05, compared with the relative ratio at day 2. Abbreviations: H, human limbal epithelial cells expanded on plastic dishes; H/A, human limbal epithelial cells expanded on intact AM; IL-1RA, interleukin-1 receptor antagonist.

 
IL-1RA Protein Prevents Apoptosis in Human Limbal Epithelial Cells Cultured on Plastic Dishes
To confirm whether IL-1RA could indeed function as a surviving factor, we added 200 ng/ml IL-1RA protein in the medium of limbal epithelial cells cultured on plastic dishes to see whether the apoptosis observed in Figure 1 could be prevented. The amount of exogenous IL-1RA protein added to the culture was determined according to the ELISA result. As shown in Figure 5, there were almost no apoptotic cells detected in limbal epithelial cells grown on plastic dishes supplemented with IL-1RA protein (B, D, and F) as compared to those without exogenous IL-1RA protein (A, C, and E). These pyknotic and irregularly shaped cells were further confirmed as cells in variable stages of apoptosis determined by fluorescent staining with Annexin V-FITC binding assay (insets in Fig. 5A, 5C, and 5E). These results showed that IL-1RA protein might prevent limbal epithelial cells expanded on plastic dishes from apoptosis, as compared to those without IL-1RA protein.


Figure 5
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  		Figure 5. Phase contrast and Annexin V-fluorescein isothiocyanate fluorescent staining photographs of human limbal epithelial cells expanded on plastic dishes with or without adding 200 ng/ml IL-1RA protein in culture medium through the 3rd week in culture. Many apoptotic cells were found in cultures on plastic dishes without IL-1RA protein ([A], [C], and [E]; arrows) at perilimbal, central, and edge areas. As compared to cultures without exogenous IL-1RA protein, culture dishes supplemented with IL-1 RA protein successfully prevented the limbal epithelial apoptosis (B, D, and F) at the corresponding areas. Inlets in A, C, and E indicated cells undergoing variable stages of apoptosis. Green fluorescence: cells undergoing apoptosis. Orange fluorescence: cell nuclei counterstained by propidium iodide. White dashed lines delineated the boundary of the outgrowth. Scale bar: 20 µm. Magnification: x200. Abbreviation: IL-1RA, interleukin-1 receptor antagonist.

 
IL-1RA-Neutralizing Antibody Induces Apoptosis in Human Limbal Epithelial Cells Cultured on Intact AM
If upregulation of IL-1 RA in cultures on intact AM was critically associated with the prevention of apoptosis of limbal epithelial cells, downregulation of IL-1RA in cultures on intact AM was expected to induce apoptosis of expanded limbal cells. As shown in Figure 6, we then performed ablative experiments to neutralize IL-1RA protein by exogenous IL-1RA antibody (100 µg/ml). This IL-1RA-neutralizing antibody [human IL-1RA/IL-1F3 monoclonal antibody (Clone 10309) from R&D Systems] was selected for its ability to neutralize the biological activity of human IL-1RA. No significant cross-reactivity was observed with recombinant IL-1{alpha}, IL-1ß, or IL-1 receptors I and II. As shown in Figure 6C and 6D, pretreatment with IL-1RA-neutralizing antibody induced apoptosis of limbal epithelial cells on intact AM. To exclude the potential nonspecific effect of the antibody used in this assay, an isotype-matched (IgG2A) antibody at 100 µg/ml was added into limbal cultures as control. Data in Figure 6E and 6F demonstrated that the isotype-matched antibody had no effect on limbal epithelial cell apoptosis. To justify this effect of IL-1RA-neutralizing antibody on limbal epithelial cell apoptosis on intact AM, IL-1RA antibody (1, 10, 50, and 100 µg/ml) was used and analyzed. As shown in Figure 6G, IL-1RA-neutralizing antibody increased the number of apoptotic cells on intact AM in a concentration-dependent manner, as determined by Annexin V-FITC staining. In fact, IL-1 RA antibody at 10 µg/ml is capable of inducing limbal epithelial cell apoptosis on intact AM. Taken together, we concluded that IL-1RA was critical for the survival of limbal epithelial cells grown on intact AM.


Figure 6
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  		Figure 6. Phase contrast and Annexin V-fluorescein isothiocyanate fluorescent staining photographs of human limbal epithelial cells expanded on intact amniotic membrane with or without adding 100 µg/ml IL-1RA neutralizing antibody in culture medium through the 3rd week in culture at a central area. Many apoptotic cells were found in cultures supplemented with IL-1RA neutralizing antibody (C, D; arrow) as compared to the control (A, B). Isotype-matched IgG2A antibody at 100 µg/ml served as internal control, however, did not induce limbal epithelial apoptosis (E, F). There was a positive dose-dependent correlation between the concentrations of IL-1RA neutralizing antibody used and the amount of apoptotic cells induced as determined by Annexin-V staining (G). *, p < .05, compared with the control. Green fluorescence: cells undergoing apoptosis. Orange fluorescence: cell nuclei counterstained by propidium iodide. Scale bar: 20 µm. Magnification: x200. Abbreviation: IL-1RA, interleukin-1 receptor antagonist.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
The clinical outcome of transplantation of cultivated limbal epithelial progenitor cells using AM as a supporting matrix relies predominantly on two major factors. One is the severity of subjacent stromal inflammation. Actually, intense stromal inflammation associated with disease chronicity has been shown to negatively affect the outcome of limbal transplantation in a rabbit model of LSCD [30]. AMT has been demonstrated to possess the properties of anti-inflammation and anti-angiogenesis [31], both of which are paramount in suppressing the stromal inflammation. The anti-inflammatory effect of AM has been thoroughly investigated in several studies [23, 32–34]. In fact, such an effect of AM transplantation has been utilized in treating patients with acutely inflamed ocular surface diseases such as acute chemical burns [35] or herpetic necrotizing keratitis [36]. The second factor that affects the outcome of ex vivo limbal epithelial transplantation is the number of preserved limbal epithelial progenitor cells transplanted onto the cornea [37]. Several lines of evidence have demonstrated that the AM is not only a good substrate for ocular surface reconstruction but also is suitable for ex vivo expansion of limbal epithelial progenitor cells (for review, see [12]).

In the present study, we have demonstrated that a naturally occurring cytokine, IL-1RA, was upregulated in human limbal epithelial cells expanded on intact AM as compared to those on plastic dishes at both the transcriptional and translational levels by cDNA microarray, RT-PCR, real-time Q-PCR analysis, and ELISA. Moreover, as shown by Annexin V-FITC apoptosis studies, we found that IL-1RA was potentially involved in preventing limbal epithelial cells expanded on intact AM from apoptosis. Collectively, these results indicated that in addition to promoting re-epithelialization and inhibiting inflammation and fibrosis, transplantation of AM might protect limbal epithelial cells from apoptosis and the function of which might, at least in part, be mediated by IL-1RA.

Upregulation of IL-1RA protein without concurrent stimulation of IL-1ß protein in the conditioned medium of limbal epithelial cells expanded on intact AM was further confirmed by ELISA. This naturally occurring protein is reported to inhibit the effects of IL-1{alpha} and IL-1ß by competing for type I and type II IL-1 receptors, resulting in significantly attenuated inflammation [38, 39]. Previous studies have indicated that corneal epithelial cells are capable of producing IL-1RA, which may be important in regulating IL-1 or lipopolysaccharide (LPS)-induced corneal inflammation [40, 41]. A beneficial anti-inflammatory effect of IL-1RA administration has also been demonstrated in various experimental models such as corneal alkali injury [42] and corneal transplant [43]. Although there is evidence demonstrating that IL-1RA protein could be produced by cells in fresh AM [32, 44], it seems not to be the same in our study. By using RT-PCR and ELISA assay, we hardly detected any IL-1RA mRNA or protein in intact AM or conditioned media from cultures with intact AM alone, respectively. We also found that the apoptosis of limbal epithelial cells cultured on plastic dishes could not be prevented by adding conditioned media saved from cultures with intact AM alone (data not shown), excluding the possibility of IL-1RA released from AM alone. Moreover, the intact AM used in our study has been cryopreserved at –80°C for at least 6 months, and no viable amniotic epithelial cells could be detected under this condition [45]. Therefore, we concluded that, in our study, IL-1RA protein could only be induced from limbal epithelial cells during the interaction with intact AM.

A similar study has been reported by Solomon et al. [23], which also demonstrated a transcriptional regulation of IL-1{alpha}, IL-1ß, and IL-1RA in limbal epithelial cells cultured on stromal matrix of AM compared with those on plastic dishes. In their study, the decrease of IL-1{alpha},ß/IL-1RA ratio at the protein level was mainly resulted from the downregulation of IL-1{alpha},ß with IL-1RA unchanged, particularly when cultures were challenged by LPS. This finding could therefore potentially be one of the mechanisms responsible for the anti-inflammatory effect of AMT. However, by culturing the limbal epithelial cells on the opposite side of AM as in our study, we demonstrated the significant increase of IL-1RA both at the transcriptional and translational levels without change in IL-1ß. Most importantly, this anti-apoptotic effect of IL-1RA on the limbal epithelial cells cultured on intact AM was confirmed in our study. Whether the same protecting effect could also exist when cells were cultured on the stromal matrix side of AM has not been discussed in Solomon's report [23]. To further understand the different changes triggered by interaction between limbal epithelial cells and different sides of AM, more detailed studies are certainly required.

It has been shown that extracellular matrix (ECM) may function as a survival factor and suppress the apoptosis of mammary epithelial and human umbilical vein endothelial cells [46, 47]. In our study, the interaction of limbal epithelial cells with ECM of the intact AM might be critical for the suppression of apoptosis. Due to the complex composition of ECM in the intact AM, it is not clear yet how the IL-1RA gene could be induced in limbal epithelial cells by interacting with intact AM, not to mention exactly which ECM protein is responsible for triggering this signal. ECM-cell interactions are primarily mediated by the integrins, a family of more than 20 different membrane {alpha}ß heterodimers [48]. It has been demonstrated that functional ß1-integrin blocking antibody can inhibit the survival protection offered by ECM [49]. It may be speculated that ECM in the AM may trigger an integrin-dependent signaling pathway in limbal epithelial cells to produce such a survival factor. In our previous study, we reported that the upregulation of MMP-9 gene and activity in the limbal epithelial cells cultured on intact AM is essential for the cell outgrowth [16]. The importance of MMP-9 is known to function on the cleavage of ECM proteins such as collagen, elastin, and gelatin, mainly involved in the migration process at the beginning of tissue remodeling (for review, see [50]). From the cDNA microarray data, many of the upregulated genes such as laminin, contactin, and fibrillin were ECM-related proteins, again suggesting the importance of tissue remodeling during the growth of limbal epithelial cells on intact AM.

Although AMT has been proposed to possess an anti-apoptotic function [11, 34, 51], the factor(s) involved in this effect has never been addressed. In the present study, for the first time, we have demonstrated that IL-1RA is capable of preventing the apoptosis of limbal epithelial cells cultured on plastic dishes. Downregulation of IL-1RA protein by IL-1RA-neutralizing antibody could lead to the apoptosis of limbal epithelial cells grown on intact AM. The finding that IL-1RA may function as an anti-apoptotic molecule, has been reported in different in vivo culture models [27–29]. IL-1RA has been shown to attenuate the IL-1{alpha}- and IL-1ß-induced apoptosis. However, due to the highly complicated interaction between limbal epithelial cells and intact AM, we can not rule out the possibility that there could be other growth factors or cytokines apart from IL-1RA also involved in regulating the function of expanded limbal epithelial cells in this model. Further study is mandatory to elucidate these factors and to verify the connection network between these growth factors/cytokines or the interactions of these factors and specific cell surface molecules, such as integrins [46, 47].

In summary, we have demonstrated that human AM may prevent apoptosis in ex vivo expanded human limbal epithelial cells cultivated on intact AM. The identification of a naturally occurring protein IL-1RA, which exerts itself as an anti-apoptotic molecule during the interaction between limbal epithelial cells and intact AM, might disclose a new therapeutic target for tissue engineering in the treatment of ocular surface disorders.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
References
 
We thank Dr. Ray R. F. Tsai for technical support. The authors also thank Min-Li Wei (Genomic Medicine Research Core Laboratory of Chang Gung Memorial Hospital) for excellent techniques in analyzing real-time quantitative PCR. This work was supported by Grants CMRPG23002 (C.C.S.) and CMRP1371 (C.M.Y.) from Chang Gung Medical Research Foundation, and NSC94-2320-B182-003 from National Science Council and EMRPD2E11 from Ministry of Education (C.M.Y.), Taiwan.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

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