November 2002
Volume 43, Issue 11
Free
Cornea  |   November 2002
UVB-Mediated Induction of Interleukin-6 and -8 in Pterygia and Cultured Human Pterygium Epithelial Cells
Author Affiliations
  • Nick Di Girolamo
    From the Inflammation Research Unit, School of Medical Sciences, Department of Pathology, Faculty of Medicine, University of New South Wales, Sydney, Australia; and the
  • Rakesh K. Kumar
    From the Inflammation Research Unit, School of Medical Sciences, Department of Pathology, Faculty of Medicine, University of New South Wales, Sydney, Australia; and the
  • Minas T. Coroneo
    Department of Ophthalmology, Prince of Wales Hospital, Sydney, Australia.
  • Denis Wakefield
    From the Inflammation Research Unit, School of Medical Sciences, Department of Pathology, Faculty of Medicine, University of New South Wales, Sydney, Australia; and the
Investigative Ophthalmology & Visual Science November 2002, Vol.43, 3430-3437. doi:
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Nick Di Girolamo, Rakesh K. Kumar, Minas T. Coroneo, Denis Wakefield; UVB-Mediated Induction of Interleukin-6 and -8 in Pterygia and Cultured Human Pterygium Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2002;43(11):3430-3437.

      Download citation file:


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

      ×
  • Supplements
Abstract

purpose. Pterygia are common ocular surface lesions that are thought to be induced by exposure to ultraviolet (UV) radiation. The hypothesis tested in the current study is that UV radiation modulates the expression of interleukin (IL)-6 and -8, which could promote the neovascularization and chronic inflammation regularly observed in pterygia.

methods. Immunohistochemical analysis was performed on 10 pterygia and 14 specimens of normal conjunctiva (4 of which contained limbus), to identify the cellular source of these cytokines. Pterygium epithelial cells were exposed to UVB (0–100 mJ/cm2) and the expression of cytokine mRNA and protein was determined by reverse transcription–polymerase chain reaction (RT-PCR), RNase protection assay (RPA), and enzyme immunoassay. Similarly, pterygium tissue in organ culture was UVB irradiated and the supernatants analyzed for cytokine production.

results IL-6 and -8 proteins were abundantly expressed, predominantly by the pterygium epithelium, with additional IL-8 immunoreactivity associated with the vascular endothelium. In contrast, significantly less staining for both cytokines was observed in normal conjunctiva, cornea, and limbus. Expression of both IL-6 and -8 mRNA and protein was induced in UVB-irradiated pterygium epithelial cells in a time- and dose-dependent manner. Similarly, IL-6 and -8 proteins were significantly elevated in UVB-treated compared with nonirradiated pterygia.

conclusions. This study provides the first direct experimental evidence that implicates UV in the pathogenesis of pterygia. The two proinflammatory cytokines that are induced by UV radiation may play a key role in the development of pterygia, by initiating blood vessel formation, cellular proliferation, tissue invasion, and inflammation. Strategies aimed at reducing ocular exposure to UV light may decrease the incidence and recurrence of pterygia.

Pterygia are wing-shaped inflammatory fibrovascular lesions that invade the cornea. They are characterized by cellular proliferation, tissue remodeling, and neovascularization. Immunohistologic features of pterygia suggest that these lesions may be derived from altered limbal epithelial stem cells. 1 Injury or activation of these stem cells may initiate development of pterygium. 
Although the pathogenesis of pterygia is still incompletely understood, there is considerable epidemiologic evidence implicating ultraviolet (UV) radiation as an initiating environmental factor. 2 3 In addition, light-focusing experiments in the eye have provided an explanation for the location and shape of pterygia. 4 Light and electron microscopic studies have identified elastotic changes in the extracellular component of pterygia 5 that resemble the actinic degenerative changes seen in chronic UV-exposed skin. Whether these degenerative changes are of primary importance or the development of pterygia is principally a consequence of altered cellular proliferation with associated tumorlike properties is unclear. 6 7  
Recently, we demonstrated expression of matrix metalloproteinases (MMPs) in resected pterygium specimens 8 9 and localized these enzymes at the advancing edges of the lesions. 10 In addition, we have presented in vitro data that suggest that proinflammatory cytokines (previously localized in pterygia 11 ) can modify the expression of these extracellular matrix denaturing enzymes. 8 These investigations imply that MMPs may play a significant role in tissue remodeling and invasion and in the dissolution of Bowman’s layer associated with pterygia. 
Although MMPs may be important effector molecules in the pathogenesis of pterygia, 8 9 10 the roles of cytokines and growth factors have yet to be established. Several studies have documented the expression of cytokines, such as tumor necrosis factor (TNF)-α, basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β, and platelet-derived growth factor (PDGF) in pterygia and cultured pterygium cells. 11 12 In addition, the localization of vascular endothelial growth factor (VEGF) in the pterygium epithelium and vascular endothelium (Di Girolamo N, Kumar RK, Coroneo MT, Wakefield D, unpublished observations, 2000), and the presence of intraepithelial capillaries in pterygia 13 suggest a role for angiogenic cytokines in this disease. 
IL-8 is a multifunctional cytokine with angiogenic, 14 neutrophil chemotactic, 15 and keratinocyte proliferative activity. 16 This cytokine has also been shown to induce the production of MMPs. 17 IL-8 is a product of activated monocytes and fibroblasts and of endothelial and epithelial cells. Similarly, IL-6 is a pleiotropic proinflammatory cytokine synthesized by various cells, such as fibroblasts, endothelial cells, and keratinocytes, in response to numerous cytokines including TNF-α and IL-1. Similar to IL-8, IL-6 can also induce the expression of MMPs. 18 19  
Consistent with the potential involvement of cytokines and angiogenic factors and the possible role of UV radiation in the development of pterygia, previous studies have shown that some of these mediators can be induced by UV radiation. Kennedy et al. 20 exposed human corneal fibroblasts to physiological doses of UVB and demonstrated significant expression of IL-1, -6, and -8 and TNF-α. Ansel et al. 21 demonstrated an upregulation of the same cytokines in corneal epithelium after exposure to UV radiation. Their data suggest that this induction is mediated by nuclear factor (NF)-κB. In similar experiments, expression of IL-6 mRNA was maximally enhanced 2 to 6 hours after UVB irradiation in human keratinocytes 22 and in UVA-exposed skin fibroblasts. 23  
The purposes of this study were to determine the expression and cellular source of IL-6 and -8 in pterygium tissue, compared with normal conjunctiva, cornea, and limbus, and to determine whether UVB irradiation modulates the expression of these cytokines in pterygium tissue ex vivo and in cultured pterygium epithelial cells (PECs). Our results establish a correlation between exposure to UV light and expression of cytokines that may offer some insight into the pathogenesis of pterygia. 
Materials and Methods
Ocular Tissue
For immunohistochemical analysis, excised primary pterygia (n = 10) and normal conjunctival tissue (n = 14) were obtained at surgery from the Prince of Wales Hospital, Sydney, Australia. Four of the conjunctival specimens, containing normal limbus, were excess (1–2 mm2) tissue after free conjunctival autografting. Tissue specimens were fixed in formalin and paraffin embedded according to routine procedures. Fresh primary pterygia (n = 8) were obtained for ex vivo organ culture experiments. Informed consent was obtained from each subject and the experimental protocol was approved by the University of New South Wales Ethics Committee and performed in accordance with the tenets of the World Medical Association’s Declaration of Helsinki. 
Immunohistochemical Analysis
Tissue blocks were serially sectioned (2–4 μm), placed on slides treated with 3-aminopropyltriethoxysilane (Sigma, Sydney, Australia), and processed for immunohistochemistry, as previously described. 8 9 10 24 Briefly, sections were deparaffinized in xylene, rehydrated, quenched for endogenous peroxidase with methanol/H2O2, and incubated with a 1:5 dilution of goat serum for 30 minutes. To facilitate detection of IL-8, antigen retrieval was performed by microwaving tissue sections twice for 3 minutes in 0.01 M citrate buffer (pH 6.0). Tissue sections were equilibrated in Tris-buffered saline (TBS) and then incubated with optimized dilutions of antibodies to mouse anti-human IL-6 (1:100), IL-8 (1:50; R & D Systems, Minneapolis, MN), human neutrophil elastase (1:200; ICN Biomedicals, Sydney, Australia), or an irrelevant mouse primary antibody as an isotype control (1:50; Clone DAK-G01; Dako, Carpinteria, CA) overnight at 4°C. Sections were extensively washed in TBS before the addition of a goat anti-mouse biotinylated secondary antibody for 30 minutes. Sections were again washed and incubated for 1 hour with horseradish peroxidase-conjugated streptavidin (Dako Corp.) and the immunoreactivity developed by adding 3-amino-9-ethylcarbazole (Sigma). Other control reactions included incubating the tissue without a primary antibody. 
Culture of PECs
Pure long-term cultures of PECs were established as previously described. 25 Briefly, pterygia were cut into several 2- to 3-mm2 segments and placed on tissue culture plastic as explants. Epithelial cell outgrowth from the explants began as early as 3 days in culture. Fibroblast contamination was minimized by removing the tissue when sufficient epithelial cell numbers surrounded each explant. Epithelial cells were passaged and the purity (>98%) established by flow cytometry using a pancytokeratin marker. 25  
UVB Irradiation of Cultured Cells
Human PECs were seeded at approximately 1 × 106 cells in 100-mm tissue culture dishes (Corning, Corning, NY) and grown in the presence of 10% FBS-Eagle’s minimum essential medium (EMEM). Once the cells reached semiconfluence, the medium was aspirated, and cells were washed three times with sterile PBS and left in serum-free medium for 16 hours, as previously described. 8 This medium was replaced with PBS (5 mL) and the monolayers irradiated with 0 to 100 mJ/cm2 UVB light (FL20SE bulbs; Philips, Sydney, Australia) as previously reported. 20 UVB light intensity was monitored and calibrated before each experiment with the aid of a radiometer-photometer (model IL1400A; International Light, Newburyport, MA). After each exposure, PECs were rinsed once with PBS and placed in 6 mL fresh serum-free medium. Some cells were treated with 10 ng/mL phorbol myristate acetate (PMA; Sigma) for 48 hours. Supernatants were collected at specific time points, cleared of cells and debris by centrifugation, and stored frozen in small aliquots at −70°C. 
UVB Irradiation of Pterygia
To determine the effect of UV light on the production of cytokines, fresh pterygia (n = 8) were surgically excised and cut symmetrically in half by the surgeon (MTC). Tissue was placed in sterile saline and transported immediately (<2 hours) to the laboratory on ice. One half of each pterygium was placed in a 24-well plate (Nunc, Roskilde, Denmark), covered with 250 μL sterile PBS, and irradiated with 40 mJ/cm2 UVB (as described earlier). The other half of each pterygium was exposed to ambient light (for the same length of time) in a laminar flow cabinet (Westinghouse, Sydney, Australia). All tissue specimens were subsequently washed in PBS and incubated for 72 hours in serum-free medium. 8 9 10 Supernatants were harvested, stored in small aliquots at −70°C, standardized for total protein (BSA Protein Assay Kit; Pierce, Rockford, IL), and appropriate dilutions of 0.5 μg total protein analyzed by ELISA for IL-6 and -8 production (see below). 
Enzyme Immunoassays
Human IL-1β, -6, and -8 (Immunotech, Marseilles, France) and human TNF-α (DuoSet; Genzyme Diagnostics, Cambridge, MA) were quantified with sandwich immunoassays. Cytokines in supernatants from control, PMA-treated, or UVB-irradiated PECs or UVB-exposed pterygia were captured on antibody-coated 96-well plates and detection performed precisely as directed by the manufacturer. The optical density of the reaction product was read at the appropriate wavelength with a microplate reader (Spectramax Plus; Molecular Devices, Sunnyvale, CA). 
RNA Extraction and Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted (RNAgents Total RNA Extraction Kit; Promega, Sydney, Australia) from control and UVB-exposed PECs, as previously outlined. 8 24 Reverse transcription was performed according to the manufacturer’s instructions (Preamplification System for First-Strand cDNA Synthesis kit; Gibco BRL, Gaithersburg, MD), as previously described. 8 24 Aliquots (1 μL) of cDNA were amplified by PCR, in which 100 nM each of the forward and reverse gene-specific primers for IL-6, IL-8, and GAPDH was used (Table 1) . Initially, a 2-minute hot start at 95°C was performed to denature the double-stranded cDNA, followed by 26 to 32 cycles of PCR (each cycle: 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 30 seconds), and the reactions terminated with a 2-minute extension at 72°C. The cycle number was predetermined so that the products formed fell within the linear portion of the amplification curve. Products were visualized on a 1.2% agarose gel, precast with ethidium bromide. Semiquantitative assessment was performed on computer (Gel Doc 2000 and Quantity One software programs; Bio-Rad, Sydney, Australia). 
RNase Protection Assay
An RNase protection assay (RPA) was used to determine the mRNA expression of multiple cytokines in UV-stimulated PECs. The template for this assay included IL-6 but not IL-8. This assay was performed as previously described 26 with minor modifications as specified by the manufacturer. Briefly, the RNA samples (10 μg) were hybridized with [α32P]-labeled RNA probes (RiboQuant Human Template Set; BD Biosciences, Mountain View, CA) at 56°C for 12 to 16 hours. Unpaired RNA was degraded by treatment with RNase A and RNase T1 at 37°C for 30 minutes. The purified and protected fragments were denatured and electrophoresed in a standard 6% polyacrylamide, 7 M urea, 0.5% Tris-borate EDTA-buffered sequencing gel. Dried gels were placed on autoradiographic film (XAR; Eastman Kodak, Sydney, Australia) and exposed at −70°C. The identity and quantity of each mRNA species in the original RNA sample was estimated based on the signal intensity and comparison to the appropriately sized probe fragment bands. For quantitation, autoradiograms were scanned with a phosphorescence imager (Molecular Imager Systems GS-525; Bio-Rad), and the band intensity was assessed by computer (Multi-Analyst software; Bio-Rad) after standardization to the housekeeping gene GAPDH. 
Statistical Analysis
Cytokine concentrations were determined from the corresponding standard curve and expressed as the mean picograms per milliliter ± SD. The difference in cytokine levels between nonirradiated and UV-treated cells was assessed initially by one-way analysis of variance, followed by the Dunnett test for multiple comparison of treatment groups with the control group. Comparisons between treated and control PEC groups at different time points were made with an unpaired Student’s t-test. Comparisons between UV-irradiated and nonirradiated pterygia were made with a paired Student’s t-test. A commercial software package (Prism; GraphPad Software, San Diego, CA) was used for all data analysis and preparation of graphs. 
Results
Localization of IL-6 in Pterygia
The expression and distribution of IL-6 in diseased pterygium and control conjunctival, corneal, and limbal tissue was assessed. IL-6 protein was demonstrable in all pterygium specimens, predominantly associated with the superficial epithelium (Figs. 1A 1B 1C 1J) , but was absent in the basal pterygium epithelium (Figs. 1A 1B 1C 1J , arrows). Very faint staining for this cytokine was occasionally detected in some vascular endothelial cells (Fig. 1J , arrowheads). IL-6 reactivity in normal conjunctiva (Fig. 1D) , cornea (Fig. 1I) , and limbus (Fig. 1H) was minimal. The limbal epithelium was identified by the palisades of Vogt (Fig. 1G) . No goblet cells were present in the limbal epithelium. Cells in the basal limbal epithelium were similar to those of the cornea but were generally smaller and more closely packed (Fig. 1G) . No immunoreactivity developed when tissue specimens were incubated with an appropriate isotype control antibody (Fig. 1E) or when the primary antibody was omitted (data not shown). As a positive control, inflamed tonsillar tissue was assessed for IL-6 protein expression. Similar to the pattern observed in pterygia, there was moderate staining of the superficial mucosal epithelium for this cytokine (Fig. 1F) as well as reactivity in some vascular endothelial cells (micrographs not shown) and no staining was found in the basal epithelium. IL-6 protein was differentially expressed at the advancing edge of the pterygium. Although the basal pterygium epithelium displayed minimal-to-absent immunoreactivity and the superficial pterygium epithelium demonstrated moderate IL-6 staining, a group of differentiated, proliferating, and invading squamous epithelial cells displayed intense cellular staining for this cytokine at the invading edge (Fig. 1J)
Localization of IL-8 in Pterygia
Formalin-fixed, paraffin-embedded pterygia were serially sectioned and stained to determine the distribution of IL-8. Intense immunoreactivity for this cytokine was observed in pterygia, particularly in superficial epithelium (Figs. 2A 2G) and the vascular endothelium (Fig. 2B) , although not all the vascular endothelial cells were reactive (Figs. 2B 2G , arrowhead). A similar pattern of IL-8 staining was observed in a specimen of inflamed ocular tissue (derived from a donor conjunctival site, post pterygium surgery) that served as a positive control (Fig. 2F) . Occasionally, IL-8 was detected in resident stromal pterygium fibroblasts (micrographs not shown). IL-8 protein was also detected in the majority of normal human conjunctival (Fig. 2C) , limbal (Fig. 2D) , and corneal (Fig. 2G , inset) tissue specimens analyzed but the intensity and extent of staining for this cytokine were significantly less than in pterygia. Consistent with the potent neutrophil chemotactic activity exhibited by IL-8, 15 recruitment of neutrophils was demonstrated in most pterygium specimens. These cells were found either adherent to blood vessel walls (Fig. 2E) , extravasating from blood vessels 8 (micrographs not shown), or among pterygium epithelial cells (Fig. 2E) . Epithelial expression of IL-8 protein was most striking at the advancing edge of the pterygium, where a cluster of differentiated, proliferating, and invading squamous epithelial cells exhibited intense cell-associated staining for this cytokine (Fig. 2G) . In contrast, the basal columnar epithelium demonstrated minimal IL-8 reactivity (Fig. 2G)
Dose Effect of UV Light on Cytokine Production in PECs
Monolayers of semiconfluent PECs were irradiated with various doses of UVB, and IL-6 and -8 levels in the supernatants quantified by enzyme immunoassay. Exposure of PECs to UVB resulted in a dose-dependent induction of both IL-6 and -8 (Fig. 3) . IL-6 production was significantly increased at doses of 20 mJ/cm2 (2.2-fold induction) and 40 mJ/cm2 (4.6-fold induction), relative to control values. The production of IL-8 paralleled that of IL-6. This cytokine was significantly increased (7.5-fold) when exposed to 20 mJ/cm2 of UVB and peaked (71-fold) when PECs were exposed to 40 mJ/cm2 of UVB (Fig. 3) . IL-1β and TNF-α were not detected in the same conditioned media. Doses of UV ranging from 0 to 40 mJ/cm2 did not cause morphologic changes, nor was there a loss in cell viability as assessed by trypan blue exclusion. However, PECs exposed to greater than 40 mJ/cm2 UVB changed from a flattened, cuboidal appearance to spindly and irregular cells, some of which detached from their substratum, and there was a substantial reduction in viability. Treatment with the positive control stimulus PMA potently increased production of IL-6 (8.6-fold) to a level of 15,460 ± 181 pg/mL (P < 0.01) and significantly enhanced IL-8 levels to 12,894 ± 390 pg/mL (P < 0.01), which represented a 170-fold induction. 
Time-Course–Dependent Induction of Cytokines after UV Exposure
A dose of 20 mJ/cm2 of UVB irradiation that caused no change in cell morphology or viability was used to assess the time course of secretion of IL-6 and -8. Exposure of PECs to this dose resulted in a significant induction (5.9-fold) of IL-6 above constitutive levels at 24 hours after irradiation, with maximal production (twofold increase over baseline) achieved at 72 hours after exposure to UV light (Fig. 4A) . Similarly, IL-8 was rapidly and significantly induced (4.9-fold) at 24 hours, with a peak at 48 hours after exposure to UV irradiation, which represented a 4.7-fold increase above baseline levels (Fig. 4B)
Induction of IL-6 and -8 mRNAs in UVB-Irradiated PECs
Total RNA was extracted from PECs and analyzed by RT-PCR to examine the effect of 20 mJ/cm2 UVB irradiation on expression of cytokine mRNA 24 hours after irradiation. IL-6 mRNA was constitutively expressed in untreated PECs, but was induced 2.2-fold after exposure to UVB (Fig. 5A) . Similarly, IL-8 mRNA was increased by 18-fold after UVB irradiation (Fig. 5B) . The expression of the house-keeping gene GAPDH remained relatively unaltered after UV treatment (Fig. 5C)
Multicytokine Gene mRNA Analysis
Having localized both IL-6 and -8 in pterygia and demonstrated the UVB-mediated induction of both cytokines in cultured PECs, a multicytokine RPA was performed to determine whether UVB radiation could influence the expression of other relevant cytokines. Of the eight detectable cytokines, IL-6 was the only mRNA species observed, a result that corroborated the RT-PCR (Fig. 5A) . After standardizing the RNA loading and signal intensity to GAPDH, IL-6 was constitutively expressed in nonstimulated cells (Fig. 6 , lane 1), but was enhanced approximately 1.8-fold in UVB-exposed PECs (Fig. 6 , lane 2). 
Induction of IL-6 and -8 in UVB-Irradiated Pterygia
IL-6 (Fig. 7A) and -8 (Fig. 7B) proteins were significantly (P < 0.05) enhanced (1.5-fold and 1.7-fold, respectively) in UVB-exposed pterygia compared with the corresponding nonirradiated tissue specimens. High levels of both cytokines were noted in nonirradiated pterygia, and significant variations in cytokine concentration were observed between pterygium samples, which may reflect differences in pterygium development or disease activity at the time of surgery. 
Discussion
In the present study, we examined the expression of two potent proinflammatory cytokines in pterygium specimens, as well as the effect of UVB irradiation on cytokine expression by both pterygium tissue and by epithelial cells in culture. We demonstrated abundant immunoreactivity of IL-8 in pterygium epithelium and in the vascular endothelium. Similarly, abundant expression of IL-6 was identified in the pterygium epithelium. A striking feature of the pattern of expression of these two cytokines was that they appeared to be markedly upregulated at the advancing or invading edge of the pterygium, with moderate expression elsewhere in the superficial epithelium and little expression in the basal epithelium (Figs. 1 2) . The specific roles of IL-6 and -8 in pterygia are uncertain, but it is tempting to speculate that IL-8 may be responsible for the accumulation of leukocytes, formation of new blood vessels, and proliferation of PECs, because previous studies have documented its potent angiogenic, 14 neutrophil chemotactic, 15 and keratinocyte proliferative 16 activities. Similar to IL-8, the pluripotent cytokine IL-6 has been assigned various roles including angiogenesis, in that it has been shown to induce VEGF 27 ; mitogenic and tumor cell growth factor activity 28 ; metastatic activity, in that its overexpression has been shown to correlate with cancer 29 30 ; and antiapoptotic activity. 30 The data presented in the current investigation suggest that IL-6 and -8 may act in concert to promote inflammation, cellular proliferation, and angiogenesis in pterygia, because minimal expression of both cytokines was observed in normal conjunctiva and cornea. Although pterygia are thought to arise at the limbus, significantly less staining was revealed for both IL-6 (Fig. 1H) and -8 (Fig. 2D) in the superior limbus, suggesting that limbal tissue in this region is relatively quiescent. 
Although the pathogenesis of pterygia is still poorly understood, epidemiologic evidence suggests that environmental stress may have a role. Of the potential agents, UV irradiation has received the greatest attention. 2 3 31 In the present study, we observed the induction of IL-6 and -8 mRNA and protein in UVB-irradiated PECs (Figs. 3 4 5 6) and in UVB-exposed pterygia (Fig. 7) . It is well established that ocular surface epithelial cells produce these cytokines either constitutively or in response to a stimulus. 14 32 Corroborating data have also been presented by other investigators, who have shown that UVB-exposed human skin keratinocytes produce TNF-α and IL-8, which correlates with increased expression of E-selectin and accumulation of neutrophils. 33 Other studies in UVB-irradiated and implanted human cutaneous melanomas have demonstrated increased expression of IL-8 that correlates with angiogenesis, tumorigenicity, and metastatic ability, possibly because of enhanced expression of MMPs. 34 In addition, abundant epidermal expression of bFGF and VEGF has been noted in UVB-exposed mouse skin. 35 Similar in vitro investigations have shown increased production of IL-1, -6, and -8 and TNF-α in cultured human corneal fibroblasts, 20 and corneal epithelium 21 after UVB irradiation. de Vos et al. 22 irradiated human keratinocytes with exposures similar in duration and dose to those used in the present study and found that IL-6 mRNA was enhanced in a time- and dose-dependent manner. Other investigators have linked UVB irradiation to the production of the immunosuppressive cytokine IL-10 from human keratinocytes. 36 37 Although most epithelial cell–derived cytokines seem to be induced after UVB exposure, perhaps as a consequence of enhancing mRNA stability, 22 the same cannot be said for IL-7. The downregulated expression of this cytokine is thought to be mediated by the enhancement of a transcription repressor. 38  
Despite detecting immunoreactivity for both IL-6 and -8 in the superficial cells of resected pterygia, significantly elevated amounts were detected in a group of proliferating and migrating epithelial cells at the advancing edge. This pattern of staining was identified when tissue specimens were oriented so that Bowman’s layer and the advancing edge could be distinguished (Figs. 1J 2G) . Although this was a surprising result, cytokines in the superficial-to-intermediate layers of the pterygium epithelium may diffuse to the more basal epithelium and signal those cells for the induction of other gene products such as the MMPs. 
The results of the present study, together with those presented by other investigators has led us to develop a hypothetical model of how pterygia form (Fig. 8) . We propose that UV light could be the initial trigger that activates epithelial cells at or near the limbus to produce cytokines such as IL-6 and -8. These multifunctional proteins set up a cascade of events that include inflammation, 15 proliferation, 16 28 angiogenesis, 14 27 and antiapoptosis. 30 In other models, these cytokines are able to induce the expression of MMPs 17 18 19 and their tissue inhibitors (TIMPs), 18 19 making it likely that they would also indirectly affect the rate of tissue remodeling, such as destruction of Bowman’s membrane and the invasion of pterygium. The abundant expression of IL-8 and the obvious increased leukocyte infiltration is consistent with its chemotactic activity and suggests that the accumulation of neutrophils in pterygia may be due in part to the expression of this cytokine. It is also clear from other studies that apoptosis may be directly related to UV exposure. It is well established that UV is mutagenic to the p53 tumor-suppressor gene, 39 and abnormal p53 expression has been reported in pterygia. 40 41 Although p53 has recently received considerable attention in pterygia, an alternative mechanism of apoptosis could be through the induction of IL-6 30 or the TIMPs 42 43 (Fig. 8)
In a recent investigation, Wang et al. 44 demonstrated the potential role of UV in the abnormal elastin accumulation in pterygia. They demonstrated several point mutations in the 3′ untranslated region of the elastin gene in UV-irradiated normal conjunctival fibroblasts that were absent from untreated conjunctival fibroblasts, but were identified in nonexposed pterygium fibroblasts. Thus, it is likely that elastosis in pterygia may be due to UV-mediated damage, 5 because a similar histologic pattern has pterygium demonstrated in solar-exposed skin. 45 Induction of proteolytic enzymes by proinflammatory cytokines may also result in pterygium elastosis, as has been observed in human skin. 23 46  
We conclude that the two multifunctional cytokines IL-6 and -8 are expressed in pterygia and that their production may be significantly enhanced by UVB radiation. Minimizing UV light exposure may be the best approach to preventing development of these lesions. 
 
Table 1.
 
Primer Pairs Used for PCR Analysis
Table 1.
 
Primer Pairs Used for PCR Analysis
Interleukin-6 45 F 5′-GTACCCCCAGGAGAAGATTC-3′
R 5′-CAAACTGCATAGCCACTTTC-3′
Interleukin-8 45 F 5′-GCTTTCTGATGGAAGAGAGC-3′
R 5′-GGCACAGTGGAACAAGGACT-3′
GAPDH 8 F 5′-TGATGACATCAAGAAGGTGGTGAA G-3′
R 5′-TCCTTGGAGGCCATGTGGGCCAT-3′
Figure 1.
 
Expression of IL-6 in pterygia. Pterygia (AC, E, J), normal conjunctiva (D), an inflamed tonsil (F), normal limbus (G, H), and normal cornea (I) were sectioned and stained for IL-6 (AD, F, HJ) or incubated with an appropriate isotype control antibody (E). Positive immunoreactivity was seen as cell-associated red staining, with hematoxylin counterstaining. (J, arrowheads) Faint IL-6 reactivity in some blood vessels; (AC, arrows) absent IL-6 reactivity in the basal epithelium. A similar pattern of staining was observed with other pterygium, conjunctival, and limbal tissue specimens. (A, B, C, J) Tissue sections derived from different patients. To identify normal limbus, some tissue sections were stained with hematoxylin and eosin (G). Original magnification: (AF, HJ) ×500; (G) ×250.
Figure 1.
 
Expression of IL-6 in pterygia. Pterygia (AC, E, J), normal conjunctiva (D), an inflamed tonsil (F), normal limbus (G, H), and normal cornea (I) were sectioned and stained for IL-6 (AD, F, HJ) or incubated with an appropriate isotype control antibody (E). Positive immunoreactivity was seen as cell-associated red staining, with hematoxylin counterstaining. (J, arrowheads) Faint IL-6 reactivity in some blood vessels; (AC, arrows) absent IL-6 reactivity in the basal epithelium. A similar pattern of staining was observed with other pterygium, conjunctival, and limbal tissue specimens. (A, B, C, J) Tissue sections derived from different patients. To identify normal limbus, some tissue sections were stained with hematoxylin and eosin (G). Original magnification: (AF, HJ) ×500; (G) ×250.
Figure 2.
 
Expression of IL-8 in pterygia. Pterygia (A, B, E, G), normal conjunctiva (C), normal limbus (D), normal central cornea (G, inset), and a nonspecific inflammatory ocular lesion (F) were sectioned and analyzed immunohistochemically to determine the expression of IL-8 (AD, F, G). Some sections were incubated with a neutrophil elastase monoclonal antibody (E) or a relevant isotype control antibody (D, inset). Note the numerous intravascular neutrophils often observed in pterygium specimens (E). A similar pattern of immunostaining was observed with other pterygium, conjunctival, and limbal tissue specimens. (A, B, E, G) Tissue sections derived from different patients. (E) Numbers correspond to intravascular (1, 2), interstitial (3–6), and intraepithelial (7) neutrophils. (B, G, arrowheads) Absent IL-8 reactivity in some blood vessels. Original magnification: (AD, F, G) ×500; (E) ×640.
Figure 2.
 
Expression of IL-8 in pterygia. Pterygia (A, B, E, G), normal conjunctiva (C), normal limbus (D), normal central cornea (G, inset), and a nonspecific inflammatory ocular lesion (F) were sectioned and analyzed immunohistochemically to determine the expression of IL-8 (AD, F, G). Some sections were incubated with a neutrophil elastase monoclonal antibody (E) or a relevant isotype control antibody (D, inset). Note the numerous intravascular neutrophils often observed in pterygium specimens (E). A similar pattern of immunostaining was observed with other pterygium, conjunctival, and limbal tissue specimens. (A, B, E, G) Tissue sections derived from different patients. (E) Numbers correspond to intravascular (1, 2), interstitial (3–6), and intraepithelial (7) neutrophils. (B, G, arrowheads) Absent IL-8 reactivity in some blood vessels. Original magnification: (AD, F, G) ×500; (E) ×640.
Figure 3.
 
Dose-dependent induction of IL-6 and -8 in UV-irradiated PECs. Conditioned medium from control and UVB-irradiated PECs was analyzed by enzyme immunoassay to determine cytokine levels. UVB-irradiation induced both IL-6 and -8 in a dose-dependent manner. Data points are the mean results from triplicate samples. SE bars were usually smaller than the symbol. Both cytokines were significantly (P < 0.01) induced at 20 and 40 mJ/cm2 of UVB when compared with untreated cells. Similar results were obtained with two other PEC lines.
Figure 3.
 
Dose-dependent induction of IL-6 and -8 in UV-irradiated PECs. Conditioned medium from control and UVB-irradiated PECs was analyzed by enzyme immunoassay to determine cytokine levels. UVB-irradiation induced both IL-6 and -8 in a dose-dependent manner. Data points are the mean results from triplicate samples. SE bars were usually smaller than the symbol. Both cytokines were significantly (P < 0.01) induced at 20 and 40 mJ/cm2 of UVB when compared with untreated cells. Similar results were obtained with two other PEC lines.
Figure 4.
 
Time-course–dependent induction of cytokines in UV-irradiated PECs. Semiconfluent PECs were exposed to UVB irradiation and the cell supernatants collected and analyzed by enzyme immunoassay to determine the production of IL-6 (A) and -8 (B). This treatment caused a time-course–dependent induction of both cytokines. Data points are mean results from triplicate samples ± SE. Both cytokines were significantly (P < 0.01) enhanced at each of the time points of harvest when compared with untreated cells. Similar results were obtained with two other PEC lines.
Figure 4.
 
Time-course–dependent induction of cytokines in UV-irradiated PECs. Semiconfluent PECs were exposed to UVB irradiation and the cell supernatants collected and analyzed by enzyme immunoassay to determine the production of IL-6 (A) and -8 (B). This treatment caused a time-course–dependent induction of both cytokines. Data points are mean results from triplicate samples ± SE. Both cytokines were significantly (P < 0.01) enhanced at each of the time points of harvest when compared with untreated cells. Similar results were obtained with two other PEC lines.
Figure 5.
 
Cytokine mRNA expression in UVB-irradiated PECs. An equal amount of RNA was reverse transcribed from unstimulated (lane 1) or UVB-irradiated (lanes 2-4) PECs. When no reverse transcriptase enzyme (lane 3) and no gene-specific primers (lane 4) were included, PCR products did not form. Otherwise, products at the expected size were amplified for IL-6 (A), IL-8 (B), and GAPDH (C). A 100-bp ladder was run in an adjacent lane (not shown). The same results were obtained in at least three separate experiments.
Figure 5.
 
Cytokine mRNA expression in UVB-irradiated PECs. An equal amount of RNA was reverse transcribed from unstimulated (lane 1) or UVB-irradiated (lanes 2-4) PECs. When no reverse transcriptase enzyme (lane 3) and no gene-specific primers (lane 4) were included, PCR products did not form. Otherwise, products at the expected size were amplified for IL-6 (A), IL-8 (B), and GAPDH (C). A 100-bp ladder was run in an adjacent lane (not shown). The same results were obtained in at least three separate experiments.
Figure 6.
 
Expression of multiple cytokine mRNAs. Equal amounts of total RNA from control (lane 1) and 20 mJ/cm2–irradiated PECs (lane 2) were analyzed by RPA to determine the expression of multiple cytokines. To identify the protected bands, the full-length probes (without-RNase treatment) were also applied (lane 3). Of the eight possible cytokines, only IL-6 mRNA was detected (arrow).
Figure 6.
 
Expression of multiple cytokine mRNAs. Equal amounts of total RNA from control (lane 1) and 20 mJ/cm2–irradiated PECs (lane 2) were analyzed by RPA to determine the expression of multiple cytokines. To identify the protected bands, the full-length probes (without-RNase treatment) were also applied (lane 3). Of the eight possible cytokines, only IL-6 mRNA was detected (arrow).
Figure 7.
 
Cytokine protein production in UVB-irradiated pterygia. Fresh surgically excised pterygia (n = 8) were cut into halves (represented by pairs of symbols). Paired specimens were either UV irradiated or treated under control conditions and the supernatants analyzed by ELISA for production of IL-6 (A) or -8 (B). Both cytokines were significantly (P < 0.05) elevated in UVB-exposed pterygia when compared with the paired nonirradiated specimens.
Figure 7.
 
Cytokine protein production in UVB-irradiated pterygia. Fresh surgically excised pterygia (n = 8) were cut into halves (represented by pairs of symbols). Paired specimens were either UV irradiated or treated under control conditions and the supernatants analyzed by ELISA for production of IL-6 (A) or -8 (B). Both cytokines were significantly (P < 0.05) elevated in UVB-exposed pterygia when compared with the paired nonirradiated specimens.
Figure 8.
 
A model of pterygium development. In this model, it is proposed that UV irradiation is one environmental factor that activates cells at the limbus to produce cytokines such as IL-6 and -8 and proteolytic enzymes such as MMPs. Both classes of proteins may be responsible (directly or indirectly) for initiating a series of concurrent events, such as increased cellular proliferation and migration, angiogenesis, inflammation, apoptosis, and tissue invasion and degradation, that may lead to formation of pterygium.
Figure 8.
 
A model of pterygium development. In this model, it is proposed that UV irradiation is one environmental factor that activates cells at the limbus to produce cytokines such as IL-6 and -8 and proteolytic enzymes such as MMPs. Both classes of proteins may be responsible (directly or indirectly) for initiating a series of concurrent events, such as increased cellular proliferation and migration, angiogenesis, inflammation, apoptosis, and tissue invasion and degradation, that may lead to formation of pterygium.
The authors thank Sandy Beynon for technical assistance with the RNase protection assay. 
Dushku N, Reid TW. Immunohistochemical evidence that human pterygia originate from an invasion of vimentin-expressing altered limbal epithelial basal cells. Curr Eye Res. 1994;13:473–481. [CrossRef] [PubMed]
Coroneo MT. Pterygium as an early indicator of ultraviolet insolation: a hypothesis. Br J Ophthalmol. 1993;77:734–739. [CrossRef] [PubMed]
Coroneo MT, Di Girolamo N, Wakefield D. The pathogenesis of pterygia. Curr Opin Ophthalmol. 1999;10:282–288. [CrossRef] [PubMed]
Maloof AJ, Ho A, Coroneo MT. Influence of corneal shape on limbal light focusing. Invest Ophthalmol Vis Sci. 1994;35:2592–2598. [PubMed]
Cameron ME. Histology of pterygium: an electron microscopic study. Br J Ophthalmol. 1983;67:604–608. [CrossRef] [PubMed]
Tan DTH, Liu Y-P, Sun L. Flow cytometry measurements of DNA content in primary and recurrent pterygia. Invest Ophthalmol Vis Sci. 2000;41:1684–1686. [PubMed]
Spandidos DA, Sourvinos G, Kiaris H, Tsamparlakis J. Microsatellite instability and loss of heterozygosity in human pterygia. Br J Ophthalmol. 1997;81:493–496. [CrossRef] [PubMed]
Di Girolamo N, McCluskey PJ, Lloyd A, Coroneo MT, Wakefield D. Expression of MMPs and TIMPs in human pterygia and cultured pterygium epithelial cells. Invest Ophthalmol Vis Sci. 2000;41:671–679. [PubMed]
Di Girolamo N, Coroneo MT, Wakefield D. Active matrilysin (MMP-7) in human pterygia: potential role in angiogenesis. Invest Ophthalmol Vis Sci. 2001;42:1963–1968. [PubMed]
Di Girolamo N, Wakefield D, Coroneo MT. Differential expression of matrix metalloproteinases and their tissue inhibitors at the advancing pterygium head. Invest Ophthalmol Vis Sci. 2000;41:4142–4149. [PubMed]
Kria L, Ohira A, Amemiya T. Immunohistochemical localization of basic fibroblast growth factor, platelet derived growth factor, transforming growth factor-β and tumor necrosis factor-α in the pterygium. Acta Histochem. 1996;98:195–201. [CrossRef] [PubMed]
Kria L, Ohira A, Amemiya T. Growth factors in cultured pterygium fibroblasts: immunohistochemical and ELISA analysis. Graefes Arch Clin Exp Ophthalmol. 1998;236:702–708. [CrossRef] [PubMed]
Seifert P, Sekundo W. Capillaries in the epithelium of pterygium. Br J Ophthalmol. 1998;82:77–81. [CrossRef] [PubMed]
Strieter RM, Kunkel SL, Elner VM, et al. Interleukin-8: a corneal factor that induces neovascularization. Am J Pathol. 1992;141:1279–1284. [PubMed]
Mo J-S, Matsukawa A, Ohkawara S, Yoshinaga M. CXC chemokine GRO is essential for neutrophil infiltration in LPS-induced uveitis in rabbits. Exp Eye Res. 2000;70:221–226. [CrossRef] [PubMed]
Tuschil A, Lam C, Haslberger A, Lindley I. Interleukin-8 stimulates calcium transients and promotes epithelial cell proliferation. J Invest Dermatol. 1992;99:294–300. [CrossRef] [PubMed]
Luca M, Huang S, Gershenwald JE, Singh RK, Reich R, Bareli M. Expression of interleukin-8 by human melanoma cells up-regulates MMP-2 activity and increases tumor growth and metastasis. Am J Pathol. 1997;151:1105–1113. [PubMed]
Franchimont N, Rydziel S, Delany AM, Canalis E. Interleukin-6 and its soluble receptor cause a marked induction of collagenase 3 expression in rat osteoblast cultures. J Biol Chem. 1997;272:12144–12150. [CrossRef] [PubMed]
Ito A, Itoh Y, Sasaguri Y, Morimatsu M, Mori Y. Effects of interleukin-6 on the metabolism of connective tissue components in rheumatoid synovial fibroblasts. Arthritis Rheum. 1992;35:1197–1201. [CrossRef] [PubMed]
Kennedy M, Kim KH, Harten B, et al. Ultraviolet irradiation induces the production of multiple cytokines by human corneal cells. Invest Ophthalmol Vis Sci. 1997;38:2483–2491. [PubMed]
Ansel JC, Abraham TA, Zivony AS, Edelhauser HF, Armstrong CA, Song PI. UV induces human corneal epithelial cell NF-κB activation and results in the production of proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα [ARVO Abstract]. Invest Ophthalmol Vis Sci. 2001;42(4)S575.Abstract nr 3087
de Vos S, Brach M, Budnik A, Grewe M, Herrmann F, Krutmann J. Post-transcriptional regulation of interleukin-6 gene expression in human keratinocytes by ultraviolet B radiation. J Invest Dermatol. 1994;103:92–96. [CrossRef] [PubMed]
Wlaschek M, Bolsen K, Herrmann G, et al. UVA-induced autocrine stimulation of fibroblast-derived-collagenase by IL-6: a possible mechanism in dermal photodamage. J Invest Dermatol. 1993;101:164–168. [CrossRef] [PubMed]
Di Girolamo N, Tedla N, Lloyd A, Wakefield D. Expression of matrix metalloproteinases by human plasma cells and B lymphocytes. Eur J Immunol. 1998;28:1773–1784. [CrossRef] [PubMed]
Di Girolamo N, Tedla N, Kumar RK, et al. Culture and characterisation of epithelial cells from human pterygia. Br J Ophthalmol. 1999;83:1077–1082. [CrossRef] [PubMed]
Stalder AK, Campbell IL. Simultaneous analysis of multiple cytokine receptor mRNAs by RNase protection assay in LPS-induced endotoxemia. Lymphokine Cytokine Res. 1994;13:107–112. [PubMed]
Cohen T, Nahari D, Cerem LW, Neufeld G, Levi B-Z. Interleukin 6 induces the expression of vascular endothelial growth factor. J Biol Chem. 1996;271:736–741. [CrossRef] [PubMed]
Goswami S, Gupta A, Sharma SK. Interleukin-6-mediated autocrine growth promotion in human glioblastoma multiforme cell line U87MG. J Neurochem. 1998;71:1837–1845. [PubMed]
Rolhion C, Penault-Llorca F, Kemeny J-L, et al. Interleukin-6 overexpression as a marker of malignancy in human gliomas. J Neurosurg. 2001;94:97–101. [CrossRef]
Jee SH, Shen SC, Chiu HC, Tsai WL, Kuo ML. Overexpression of interleukin-6 in human basal cell carcinoma cell lines increases anti-apoptotic activity and tumorigenic potency. Oncogene. 2001;20:198–208. [CrossRef] [PubMed]
Threlfall TJ, English DR. Sun exposure and pterygium of the eye: a dose-response curve. Am J Ophthalmol. 1999;128:280–287. [CrossRef] [PubMed]
Sotozono C, He JC, Matsumoto Y, Kita M, Imanishi J, Kinoshita S. Cytokine expression in the alkali-burned cornea. Curr Eye Res. 1997;16:670–676. [CrossRef] [PubMed]
Strickland I, Rhodes LE, Flanagan BF, Friedmann PS. TNF-α and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: correlation with neutrophil accumulation and E-selectin expression. J Invest Dermatol. 1997;108:763–768. [CrossRef] [PubMed]
Singh RK, Gutman M, Reich R, Bareli M. Ultraviolet B irradiation promotes tumorigenic and metastatic properties in primary cutaneous melanoma via induction of interleukin 8. Cancer Res. 1995;55:3669–3674. [PubMed]
Bielenberg DR, Bucana CD, Sanchez R, Donawho CK, Kripke ML, Fidler IJ. Molecular regulation of UVB-induced cutaneous angiogenesis. J Invest Dermatol. 1998;111:864–872. [CrossRef] [PubMed]
Enk CD, Sredni D, Blauvelt A, Katz SI. Induction of IL-10 gene expression in human keratinocytes by UVB exposure in vivo and in vitro. J Immunol. 1995;154:4851–4856. [PubMed]
Grewe M, Gyufko K, Krutmann J. Interleukin-10 production by cultured human keratinocytes: regulation by ultraviolet B and ultraviolet A1 radiation. J Invest Dermatol. 1995;104:3–6. [CrossRef] [PubMed]
Aragane Y, Schwarz A, Luger TA, Arizumi K, Takashima A, Schwarz T. Ultraviolet light suppresses IFN-γ-induced IL-7 gene expression in murine keratinocytes by interfering with IFN regulatory factors. J Immunol. 1997;158:5393–5399. [PubMed]
Brash DE, Rudolph J, Simon J, et al. A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc Natl Acad Sci USA. 1991;88:10124–10128. [CrossRef] [PubMed]
Tan DTH, Tang WY, Liu YP, Goh H-S, Smith DR. Apoptosis and apoptosis related gene expression in normal conjunctiva and pterygium. Br J Ophthalmol. 2000;84:212–216. [CrossRef] [PubMed]
Dushku N, Reid TW. P53 expression in altered limbal basal cells of pingueculae, pterygia, and limbal tumors. Curr Eye Res. 1997;16:1179–1192. [CrossRef] [PubMed]
Li GY, Fridman K, Kim HRC. Tissue inhibitor of metalloproteinase-1 inhibits apoptosis of human breast epithelial cells. Cancer Res. 1999;54:6267–6275.
Tummalapalli CM, Heath BJ, Tyagi SC. Tissue inhibitor of metalloproteinase-4 instigates apoptosis in transformed cardiac fibroblasts. J Cell Biochem. 2001;80:512–521. [CrossRef] [PubMed]
Wang I-J, Hu F-R, Chen P-J, Lin C-T. Mechanism of abnormal elastin gene expression in the pinguecular part of pterygia. Am J Pathol. 2000;157:1269–1276. [CrossRef] [PubMed]
Bernstein EF, Chen YQ, Kopp JB, et al. Long-term sun exposure alters the collagen of the papillary dermis. J Am Acad Dermatol. 1996;34:209–218. [CrossRef] [PubMed]
Fisher GJ, Wang ZQ, Datta SC, Varani J, Kang S, Voorhees JJ. Pathophysiology of premature skin aging induced by ultraviolet light. N Engl J Med. 1997;337:1419–1428. [CrossRef] [PubMed]
Hieda O, Sotozono C, Kinoshita S. Expression of matrix metalloproteinases and cytokines in cultured human scleral fibroblasts [ARVO Abstract]. Invest Ophthalmol Vis Sci. 2000;41(4)S697.Abstract nr 3714
Figure 1.
 
Expression of IL-6 in pterygia. Pterygia (AC, E, J), normal conjunctiva (D), an inflamed tonsil (F), normal limbus (G, H), and normal cornea (I) were sectioned and stained for IL-6 (AD, F, HJ) or incubated with an appropriate isotype control antibody (E). Positive immunoreactivity was seen as cell-associated red staining, with hematoxylin counterstaining. (J, arrowheads) Faint IL-6 reactivity in some blood vessels; (AC, arrows) absent IL-6 reactivity in the basal epithelium. A similar pattern of staining was observed with other pterygium, conjunctival, and limbal tissue specimens. (A, B, C, J) Tissue sections derived from different patients. To identify normal limbus, some tissue sections were stained with hematoxylin and eosin (G). Original magnification: (AF, HJ) ×500; (G) ×250.
Figure 1.
 
Expression of IL-6 in pterygia. Pterygia (AC, E, J), normal conjunctiva (D), an inflamed tonsil (F), normal limbus (G, H), and normal cornea (I) were sectioned and stained for IL-6 (AD, F, HJ) or incubated with an appropriate isotype control antibody (E). Positive immunoreactivity was seen as cell-associated red staining, with hematoxylin counterstaining. (J, arrowheads) Faint IL-6 reactivity in some blood vessels; (AC, arrows) absent IL-6 reactivity in the basal epithelium. A similar pattern of staining was observed with other pterygium, conjunctival, and limbal tissue specimens. (A, B, C, J) Tissue sections derived from different patients. To identify normal limbus, some tissue sections were stained with hematoxylin and eosin (G). Original magnification: (AF, HJ) ×500; (G) ×250.
Figure 2.
 
Expression of IL-8 in pterygia. Pterygia (A, B, E, G), normal conjunctiva (C), normal limbus (D), normal central cornea (G, inset), and a nonspecific inflammatory ocular lesion (F) were sectioned and analyzed immunohistochemically to determine the expression of IL-8 (AD, F, G). Some sections were incubated with a neutrophil elastase monoclonal antibody (E) or a relevant isotype control antibody (D, inset). Note the numerous intravascular neutrophils often observed in pterygium specimens (E). A similar pattern of immunostaining was observed with other pterygium, conjunctival, and limbal tissue specimens. (A, B, E, G) Tissue sections derived from different patients. (E) Numbers correspond to intravascular (1, 2), interstitial (3–6), and intraepithelial (7) neutrophils. (B, G, arrowheads) Absent IL-8 reactivity in some blood vessels. Original magnification: (AD, F, G) ×500; (E) ×640.
Figure 2.
 
Expression of IL-8 in pterygia. Pterygia (A, B, E, G), normal conjunctiva (C), normal limbus (D), normal central cornea (G, inset), and a nonspecific inflammatory ocular lesion (F) were sectioned and analyzed immunohistochemically to determine the expression of IL-8 (AD, F, G). Some sections were incubated with a neutrophil elastase monoclonal antibody (E) or a relevant isotype control antibody (D, inset). Note the numerous intravascular neutrophils often observed in pterygium specimens (E). A similar pattern of immunostaining was observed with other pterygium, conjunctival, and limbal tissue specimens. (A, B, E, G) Tissue sections derived from different patients. (E) Numbers correspond to intravascular (1, 2), interstitial (3–6), and intraepithelial (7) neutrophils. (B, G, arrowheads) Absent IL-8 reactivity in some blood vessels. Original magnification: (AD, F, G) ×500; (E) ×640.
Figure 3.
 
Dose-dependent induction of IL-6 and -8 in UV-irradiated PECs. Conditioned medium from control and UVB-irradiated PECs was analyzed by enzyme immunoassay to determine cytokine levels. UVB-irradiation induced both IL-6 and -8 in a dose-dependent manner. Data points are the mean results from triplicate samples. SE bars were usually smaller than the symbol. Both cytokines were significantly (P < 0.01) induced at 20 and 40 mJ/cm2 of UVB when compared with untreated cells. Similar results were obtained with two other PEC lines.
Figure 3.
 
Dose-dependent induction of IL-6 and -8 in UV-irradiated PECs. Conditioned medium from control and UVB-irradiated PECs was analyzed by enzyme immunoassay to determine cytokine levels. UVB-irradiation induced both IL-6 and -8 in a dose-dependent manner. Data points are the mean results from triplicate samples. SE bars were usually smaller than the symbol. Both cytokines were significantly (P < 0.01) induced at 20 and 40 mJ/cm2 of UVB when compared with untreated cells. Similar results were obtained with two other PEC lines.
Figure 4.
 
Time-course–dependent induction of cytokines in UV-irradiated PECs. Semiconfluent PECs were exposed to UVB irradiation and the cell supernatants collected and analyzed by enzyme immunoassay to determine the production of IL-6 (A) and -8 (B). This treatment caused a time-course–dependent induction of both cytokines. Data points are mean results from triplicate samples ± SE. Both cytokines were significantly (P < 0.01) enhanced at each of the time points of harvest when compared with untreated cells. Similar results were obtained with two other PEC lines.
Figure 4.
 
Time-course–dependent induction of cytokines in UV-irradiated PECs. Semiconfluent PECs were exposed to UVB irradiation and the cell supernatants collected and analyzed by enzyme immunoassay to determine the production of IL-6 (A) and -8 (B). This treatment caused a time-course–dependent induction of both cytokines. Data points are mean results from triplicate samples ± SE. Both cytokines were significantly (P < 0.01) enhanced at each of the time points of harvest when compared with untreated cells. Similar results were obtained with two other PEC lines.
Figure 5.
 
Cytokine mRNA expression in UVB-irradiated PECs. An equal amount of RNA was reverse transcribed from unstimulated (lane 1) or UVB-irradiated (lanes 2-4) PECs. When no reverse transcriptase enzyme (lane 3) and no gene-specific primers (lane 4) were included, PCR products did not form. Otherwise, products at the expected size were amplified for IL-6 (A), IL-8 (B), and GAPDH (C). A 100-bp ladder was run in an adjacent lane (not shown). The same results were obtained in at least three separate experiments.
Figure 5.
 
Cytokine mRNA expression in UVB-irradiated PECs. An equal amount of RNA was reverse transcribed from unstimulated (lane 1) or UVB-irradiated (lanes 2-4) PECs. When no reverse transcriptase enzyme (lane 3) and no gene-specific primers (lane 4) were included, PCR products did not form. Otherwise, products at the expected size were amplified for IL-6 (A), IL-8 (B), and GAPDH (C). A 100-bp ladder was run in an adjacent lane (not shown). The same results were obtained in at least three separate experiments.
Figure 6.
 
Expression of multiple cytokine mRNAs. Equal amounts of total RNA from control (lane 1) and 20 mJ/cm2–irradiated PECs (lane 2) were analyzed by RPA to determine the expression of multiple cytokines. To identify the protected bands, the full-length probes (without-RNase treatment) were also applied (lane 3). Of the eight possible cytokines, only IL-6 mRNA was detected (arrow).
Figure 6.
 
Expression of multiple cytokine mRNAs. Equal amounts of total RNA from control (lane 1) and 20 mJ/cm2–irradiated PECs (lane 2) were analyzed by RPA to determine the expression of multiple cytokines. To identify the protected bands, the full-length probes (without-RNase treatment) were also applied (lane 3). Of the eight possible cytokines, only IL-6 mRNA was detected (arrow).
Figure 7.
 
Cytokine protein production in UVB-irradiated pterygia. Fresh surgically excised pterygia (n = 8) were cut into halves (represented by pairs of symbols). Paired specimens were either UV irradiated or treated under control conditions and the supernatants analyzed by ELISA for production of IL-6 (A) or -8 (B). Both cytokines were significantly (P < 0.05) elevated in UVB-exposed pterygia when compared with the paired nonirradiated specimens.
Figure 7.
 
Cytokine protein production in UVB-irradiated pterygia. Fresh surgically excised pterygia (n = 8) were cut into halves (represented by pairs of symbols). Paired specimens were either UV irradiated or treated under control conditions and the supernatants analyzed by ELISA for production of IL-6 (A) or -8 (B). Both cytokines were significantly (P < 0.05) elevated in UVB-exposed pterygia when compared with the paired nonirradiated specimens.
Figure 8.
 
A model of pterygium development. In this model, it is proposed that UV irradiation is one environmental factor that activates cells at the limbus to produce cytokines such as IL-6 and -8 and proteolytic enzymes such as MMPs. Both classes of proteins may be responsible (directly or indirectly) for initiating a series of concurrent events, such as increased cellular proliferation and migration, angiogenesis, inflammation, apoptosis, and tissue invasion and degradation, that may lead to formation of pterygium.
Figure 8.
 
A model of pterygium development. In this model, it is proposed that UV irradiation is one environmental factor that activates cells at the limbus to produce cytokines such as IL-6 and -8 and proteolytic enzymes such as MMPs. Both classes of proteins may be responsible (directly or indirectly) for initiating a series of concurrent events, such as increased cellular proliferation and migration, angiogenesis, inflammation, apoptosis, and tissue invasion and degradation, that may lead to formation of pterygium.
Table 1.
 
Primer Pairs Used for PCR Analysis
Table 1.
 
Primer Pairs Used for PCR Analysis
Interleukin-6 45 F 5′-GTACCCCCAGGAGAAGATTC-3′
R 5′-CAAACTGCATAGCCACTTTC-3′
Interleukin-8 45 F 5′-GCTTTCTGATGGAAGAGAGC-3′
R 5′-GGCACAGTGGAACAAGGACT-3′
GAPDH 8 F 5′-TGATGACATCAAGAAGGTGGTGAA G-3′
R 5′-TCCTTGGAGGCCATGTGGGCCAT-3′
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×