AIMS Amniotic membrane (AM) transplantation reduces inflammation in a variety of ocular surface disorders. The aim of this study was to determine if AM stroma suppresses the expression of the IL-1 gene family in cultured human corneal limbal epithelial cells.
METHODS Human corneal limbal epithelial cells were cultured from limbocorneal explants of donor eyes on plastic or on the AM stroma. Transcript expression of IL-1α, IL-1β, IL-1 receptor antagonist (RA), and GAPDH was compared with or without addition of lipopolysaccharide to their serum-free media for 24 hours using RNAse protection assay (RPA). Their protein production in the supernatant was analysed by ELISA.
RESULTS Expression of IL-1α and IL-1β transcripts and proteins was significantly reduced by cells cultured on the AM stromal matrix compared with plastic cultures whether lipopolysaccharide was added or not. Moreover, expression of IL-1 RA by cells cultured in the lipopolysaccharide-free medium was upregulated by AM stromal matrix. The ratio between IL-1 RA and IL-1α protein levels in AM cultures was higher than in plastic cultures.
CONCLUSIONS AM stromal matrix markedly suppresses lipopolysaccharide induced upregulation of both IL-1α and IL-1β. These data may explain in part the effect of AM transplantation in reducing ocular surface inflammation, underscoring the unique feature of the AM as a substrate for tissue engineering.
- amniotic membrane
- interleukin 1
- corneal limbal epithelium
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The amniotic membrane (AM)—that is, the innermost layer of the placenta, consists of a prominent basement membrane and a subjacent avascular stroma. When appropriately procured and processed, AM can be preserved and used as a substrate for transplantation. In ophthalmology, transplantation of specially preserved AM was introduced by Kim and Tseng in 19951 for ocular surface reconstruction. Since then, such an AM matrix has been used for treating various ocular surface disorders including chemical or thermal burns,1-3 pterygium,4 persistent corneal ulcers of different aetiologies,5-7 symptomatic bullous keratopathy,8 removal of tumour, scar, or adhesion,9 ocular cicatricial pemphigoid, Stevens-Johnson syndrome, and other causes leading to limbal stem cell deficiency.21011 These studies generally noted that ocular surface inflammation is markedly reduced in the area covered by AM, although the exact mechanism of this anti-inflammatory effect remains unclear.
Chronic inflammation of the ocular surface may lead to several sequelae. At the stem cell containing limbus, chronic inflammation leads to limbal stem cell deficiency1213; at the conjunctiva it may lead to scarring and squamous metaplasia.14 One of prototypic potent cytokine systems that mediates inflammation and immune responses is the interleukin 1 (IL-1) gene family.15 This family is composed of three forms—that is, two proinflammatory forms, IL-1α and IL-1β, each having a precursor form, and an anti-inflammatory form: IL-1 receptor antagonist (IL-1 RA). IL-1β mRNA and protein are expressed by the corneal epithelium, stromal fibroblasts, and endothelium.16 The type 1 receptor for IL-1 is expressed by corneal stromal fibroblasts.17 IL-1 is elevated in tears of patients with chronic ocular surface inflammation such as rosacea,1819 aqueous tear deficiency, and meibomian gland dysfunction.2021
The purpose of this study was to test the hypothesis that one plausible mechanism for the anti-inflammatory effect exerted by the AM is mediated by the suppression of the expression and secretion of the IL-1 gene family. Here we provide evidence that human limbal epithelial cells cultured from limbal explants reduced the expression of IL-1α and IL-1β mRNA and protein upon direct contact with the AM stromal matrix.
Materials and methods
Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), HEPES buffer, and F12 (Ham) were from Life Technologies (Rockville, MD, USA). Tissue culture plates were from Becton Dickinson (Franklin Lakes, NJ, USA). Thirty mm diameter Millicell-CM 0.4 μm culture plate inserts were from Millipore (Bedford, MA, USA). Cholera toxin subunit A, epidermal growth factor (EGF), hydrocortisone, lipopolysaccharide (LPS) (derived from Serratia marcescens), were from Sigma (St Louis, MO, USA). IL-1β ELISA kits were from Cistron (Pine Brook, NJ, USA). IL-1α and IL-1 RA ELISA kits were from R&D systems (Minneapolis, MN, USA). The Oligotex Direct mRNA Isolation System was from Qiagen (Valencia, CA, USA). The Superscript II Reverse Transcription Kit was from Life Technologies. RNA lysis and ribonuclease protection kits were from Ambion (Austin, TX, USA). RNA Quick Spin columns were from Roche Molecular Biochemicals (Indianapolis, IN, USA). X-OMAT and Biomax films were from Eastman Kodak (Rochester, NY, USA). BCA protein assay kit was from Pierce (Rockford, IL, USA). All other reagents and intensifying screens came from Sigma (St Louis, MO, USA).
PREPARATION OF HUMAN AMNIOTIC MEMBRANE
Human placenta, kindly procured and provided by Bio-Tissue (South Miami, FL, USA), was from caesarean section delivery of a healthy mother free of human immunodeficiency virus, hepatitis B and C viruses, and syphilis. AM was prepared following a previously described method,9 adhered onto a nitrocellulose paper, and stored in DMEM medium at −80°C for at least 48 hours before use. In preparation for cell culture, AM was thawed, removed from its nitrocellulose paper, cut into smaller pieces, and sutured with a 4-0 nylon suture to the outer aspect of a 30 mm Millicell-CM culture plate insert with the basement membrane side facing upwards, towards the inner aspect of the insert ring. The insert was then placed in the well of a six well plate, and immersed in medium until cultured.
CULTURES OF HUMAN LIMBOCORNEAL EXPLANTS ON PLASTIC OR AMNIOTIC MEMBRANE
After corneal transplantation, the remaining limbocorneal ring was removed from the endothelial layer and iris remnants, treated with dispase II (1.2 U/ml in Hank's balanced salt solution) for 15 minutes, and cut into 12 equal pieces. Two such pieces were applied on the stromal side of the AM sutured to the ring insert, which was mounted in a well of a six well plastic dish, and covered with a drop of FBS overnight, following a previously described method.22 For comparison, human limbal explants from the same donor prepared in the same manner were placed directly on the plastic surface in six well plates. The explants were cultured in supplemented hormonal epithelial medium (SHEM)23 with modification. This medium contained equal amounts of DMEM and Ham's F12 medium, supplemented with 5% FBS, 0.5% dimethyl sulphoxide, 2 ng/ml EGF, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium, 0.5 μg/ml hydrocortisone, 30 ng/ml cholera toxin A, 50 μg/ml gentamicin, and 1.25 μg/ml amphotericin B. Cultures were incubated at 37°C under 95% humidity and 5% carbon dioxide. The medium was changed every 2–3 days. Cultures were maintained for 10–14 days until confluence, and then switched to the same medium as described above without FBS for a period of 24 hours. At that time, some cultures were added with 10 μg/ml LPS for an additional 24 hours, while the rest were maintained for the same period in the same serum-free medium. Then the culture supernatant was collected from each well, centrifuged, and stored in −80°C until assayed by enzyme linked immunosorbent assay (ELISA). The cells were subjected to Direct Protect Lysis Buffer (Ambion, Austin, TX), and total RNA was isolated for RNAse protection assay (RPA).
RPA TEMPLATE CONSTRUCTION AND RNA PROBES
Poly-A+ RNA was isolated from cultured human corneal epithelial cells using oligo-dt coated beads. Partial cDNAs for human IL-1α, IL-1β, IL-1RA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were prepared by reverse transciption-polymerase chain reaction (RT-PCR) using 200 ng mRNA as template and gene specific primers (see Table 1) prepared for human IL-1α (GenBank accession No X02531), IL-1β (GenBank accession No X02532), IL-1RA (GenBank accession NoM63099), and GAPDH (GenBank accession No NM002046). The resultant first strand cDNA was used for polymerase chain reaction using a gene specific upstream primer and the same downstream primer No 2 for reverse transcription. An aliquot of the initial PCR reaction (except for the GAPDH probe which required only a single round of PCR) was reamplified using the same upstream primer and a third gene specific primer, downstream primer No 1.
Radiolabelled antisense RNA was transcribed using the Ambion Maxiscript T7 Kit, labelling with [α-32P]-CTP (800 Ci/mmol). Plasmids were digested at a unique BamHI site upstream of the cloned cDNAs. RNA probes were generated for IL-1α, IL-1β, and IL-1RA, and GAPDH. Following transcription, probes were DNAse treated to remove template DNA and unincorporated nucleotides were removed using RNA Quick Spin columns.
RNASE PROTECTION ASSAY FOR IL-1α, IL-1β, IL-1 RA
Ribonuclease protection assays were performed using the Ambion Direct Protect system. Briefly, cultured human corneal epithelial cells were resuspended and lysed in direct protect lysis buffer at ∼107 cells/ml. Assays were performed using 50 μl of cell lysate, 105 cpm of each cytokine probe (specific activity 5 × 105 cpm/μg) and 4 × 104 cpm of the GAPDH probe (specific activity 5 × 104 cpm/μg) for each sample. Samples were allowed to hybridise overnight at 37°C and then treated for 30 minutes at 37°C with ribonuclease solution followed by inactivation by proteinase K. Protected RNA fragments were precipitated and separated on a 6% polyacrylamide Urea-TBE sequencing gel. RPAs for IL-1α, IL-1β, and IL-1 RA were repeated five times on primary cultures derived from five different donor corneas, respectively. Autoradiographs from these gels were scanned and analysed using the Gel-Pro image analysis software (Media Cybernetics, Silver Springs, MD, USA). The digitised data for each band were plotted, and the area under the curve for each peak was calculated with a statistical software package (GraphPad Prism, GraphPad Software, San-Diego, CA, USA). The value for each cytokine band was divided by the corresponding value of the GAPDH band in the same lane in order to calculate the relative mRNA amount for each gene. Results are shown as means (SEM) of relative mRNA amounts from five different experiments.
ELISA OF IL-1α, IL-1β, AND IL-1 RA
The conditioned media of the corneal limbal epithelial cell cultures from five independent primary cultures, derived from five different donor corneas were collected, centrifuged, and stored at −80°C until assayed. The concentrations of IL-1β, IL-1α, and IL-1 RA were assayed using commercial ELISA kits according to the respective manufacturer's protocols.
The total cellular protein content in the cell lysate was determined by the micro-BCA protein assay reagent kit (Pierce). The protein concentration of IL-1β, IL-1α, and IL-1 RA in the culture supernatants was adjusted by its corresponding total cellular protein content for any possible difference in cultured cell numbers. Thus, all ELISA results are expressed in picograms per milligrams of total protein.
Results are expressed as mean (SEM) of five experiments performed on cultures derived from five different donor corneas, respectively. Statistical analysis was performed using Student'st test; p <0.05 was considered significant.
SUPPRESSION OF IL-1α AND IL-1β mRNA TRANSCRIPTS IN LPS STIMULATED CELLS BY AM
When adjusted to the level of GAPDH transcript of each loading sample, a similar amount of IL-1α and IL-1β mRNA was expressed by the control cells that were not exposed to LPS when they were grown on plastic or AM (Fig 1A and 1B). Nevertheless, LPS stimulated cells grown on plastic showed increased expression of both IL-1α and IL-1β mRNA (6.25-fold for IL-1α, p=0.023; 5.5-fold for IL-1β, p=0.045). Compared with the levels of these two transcripts in LPS stimulated cultures on plastic, those of LPS treated cultures on AM were significantly decreased down to the baseline level of the non-stimulated cultures (3.5-fold for IL-1α, p=0.029; 2.9-fold for IL-1β, p=0.054).
UPREGULATION OF IL-1 RA mRNA IN NON-STIMULATED CELLS BY AM
When adjusted to the level of GAPDH mRNA, the level of IL-1 RA mRNA expressed by the control cells not exposed to LPS was dramatically increased when grown on AM when compared to those cultured on plastic (3.4-fold, p=0.0002, Fig 1A and 1B). The level of IL-1 RA transcript in LPS stimulated cells grown on plastic was also higher than that of the control cells (3.8-fold, p=0.008). Such an upregulation of IL-1 RA transcript by LPS was also evident in AM cultures (2.7-fold, p=0.018).
SUPPRESSION OF THE IL-1α AND IL-1β PROTEIN EXPRESSION BY THE AM
The levels of IL-1α and IL-1β protein in culture supernatants were decreased in cultures grown on AM when compared with those grown on plastic (Fig 2A and 2B, respectively). While the decrease of IL-1α protein was not significant in the non-stimulated control cells, there was a dramatic decrease of this protein in AM cultures following LPS treatment (from 24.76 (7.12) pg/mg protein on plastic to 6.08 (0.95) pg/mg protein on AM, fourfold decrease, p=0.009, Fig 2A). Likewise, a significant decrease in the IL-1β protein levels was observed in AM cultures, in both non-stimulated (53.45 (16.64) pg/mg protein on plastic compared with 3.36 (1.19) pg/mg protein on AM, 16-fold, p=0.017, Fig 2B) and LPS stimulated cells (from 136.79 (35.08) pg/mg protein on plastic to 25.72 (12.88) pg/mg protein on AM, fivefold, p=0.017, Fig 2B). The IL-1 RA protein was expressed at similar levels in LPS stimulated and non-stimulated cultures, and such expression was not changed by AM (Fig 2C). The IL-1 RA/IL-1α ratio, a measurement of anti-inflammatory state, increased for all AM cultures, and was especially significant for LPS stimulated cells (p=0.012) (Fig 2D).
This study demonstrated a direct suppressive effect of AM stromal matrix on the expression of two of the most potent proinflammatory cytokines, IL-1α and IL-1β, at both protein and mRNA levels, and an upregulatory effect on the expression of the anti-inflammatory cytokine IL-1RA. Such a suppressive effect was pronounced, especially when limbal epithelial cells were challenged by bacterial LPS. The expression of IL-1RA mRNA but not protein was upregulated by the AM matrix under both LPS stimulated and non-stimulated conditions. As a result the ratio IL-1RA/IL-1α was increased in amniotic cultures.
To investigate our hypothesis that the AM stroma contains a unique component (or factor) that is responsible for downregulation of inflammation, we cultured limbal epithelial cells directly on the stromal side of the AM. Recent models of bioengineered corneal tissue have cultured corneal epithelial cells directly on various matrix surfaces lacking a basement membrane.24-26 In these studies, epithelial cells were cultured on an artificial biomatrix prepared from collagen with or without stromal cells2425or in vivo on the denuded stroma of rabbit corneas.26Interestingly, the expression of IL-1α by corneal epithelial cells grown on such a matrix was higher than that of the normal corneal epithelium.24 Using a similar approach—that is, culturing the epithelium directly on the stromal side of the amniotic membrane, we noted that expression of IL-1 was significantly suppressed. This finding further underscores the unique feature of amniotic membrane compared with other methods of bioengineered corneas in normalising the expression of IL-1. Thus we conclude that the amniotic membrane matrix is superior to other bioengineered matrix replacements for ocular surface reconstruction.
We have reported that the expression of TGF-β2, TGF-β3 and TGF-βRI, TGF-βRII, and TGF-βRIII are downregulated in cultured human corneal and limbal fibroblasts22 and in conjunctival and pterygium body fibroblasts27 by the AM matrix. Our recent in vivo rabbit studies have also demonstrated that AM stromal matrix trapped leucocytes and rendered them into a state of rapid apoptosis.2829 Furthermore, the AM contains a number of protease inhibitors such as α1-antitrypsin, α2-macroglobulin, inter-α-trypsin inhibitor, α2-plasmin inhibitor, and α2-antichymotrypsin.30 The following preliminary studies have also provided some information concerning the anti-inflammatory effect of the AM matrix. The expression of IL-8, Gro-alpha, and epithelial cell derived neutrophil attractant (ENA) was suppressed in keratocytes cultured on AM stromal matrix.31Application of AM to mouse corneas with HSV-1 stromal keratitis resulted in reduced mRNA levels and protein expression of IL-1, IL-2, interferon-γ, and TNF-α.32 Expression of bFGF and PDFG was downregulated in limbal epithelial cells, while that of IL-1β and TGF-α was suppressed in conjunctival epithelial cells when cultured on AM basement membrane.33 The expression of inducible NO synthase was decreased in human corneal fibroblasts cultured with tumour necrosis factor α (TNFα) when AM extract was added to the culture.34 In that study, the expression and activity of MMP-1 and MMP-2 was also markedly decreased by the AM extract, suggesting that putative factors may exist in the AM matrix, which are capable of downregulating a series of inflammatory genes and gene products. Analysis of human cytokine and cytokine receptor genes expressed in corneal limbal epithelial cells cultured on AM basement membrane indicated a more than 10-fold decrease in VEGF and ENA-78 when compared with those cultured on plastic.35
The exact mechanism by which the AM suppresses the expression of the various genes described above remains unclear. Studies of mammary epithelial cells have disclosed that gene expression leading to proliferation, differentiation, and apoptosis is modulated by the extracellular matrix (ECM).36 Bidirectional communication between the matrix microenvironment and the nucleus is mediated via membrane receptors, resulting in selective gene expression. For example, overexpression of c-myc, TGF-α, fos, jun, and TGF-β in a two dimensional culture is suppressed upon contact with ECM, while that of growth inhibitor genes (p21, p27) is suppressed when cultured on plastic.37 In this model, signalling from ECM is mediated through such receptors as β1-integrin and EGFR, which are coordinately downregulated upon contact with the ECM. Therefore, future studies are needed to see if a similar mechanism may be involved in the suppression of IL-1α and IL-β.
The other possibility is implied in the following studies. In human and monkey placenta, hyaluronic acid (HA) is thought to be the only glycosaminoglycan found,38 but this finding has not been verified in AM. The synthesis of an HA enriched matrix may play a part in scarless fetal wound healing.39 Recently, high molecular weight HA was found to inhibit the expression of the transcription factor NF-κB and, as a result, to suppress the synthesis of NF-κB regulated cytokines such as IL-1α, IL-6, and tumour necrosis factor α, all of which are potent proinflammatory cytokines.40 Future studies are thus also needed to explore whether AM stromal matrix may utilise the same strategy in suppressing the expression of the IL-1 gene family. These studies may help unravel other new applications in the use of amniotic membrane transplantation.
Proprietary interest: SCGT has obtained a patent on the preparation and clinical uses of amniotic membrane.
Supported in part by Public Health Service Research Grant No EY06819 to SCGT from Department of Health and Human Services, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA.