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(Investigative Ophthalmology and Visual Science. 2006;47:2430-2437.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-1130
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UVB-Mediated Induction of Cytokines and Growth Factors in Pterygium Epithelial Cells Involves Cell Surface Receptors and Intracellular Signaling

Nick Di Girolamo,1 Denis Wakefield,1 and Minas T. Coroneo2

1From the Inflammatory Diseases Research Unit, Department of Pathology, School of Medical Sciences, University of New South Wales, Sydney, Australia; and the 2Department of Ophthalmology, Prince of Wales Hospital, Sydney, Australia.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Pterygium is a proliferative, inflammatory, and invasive ocular surface disease associated with excessive ultraviolet (UV) exposure. This investigation was conducted to identify UV activated signaling pathways in pterygium epithelial cells (PECs) that mediate cytokine and growth factor production and to determine whether these pathways are sensitive to blockade by anti-inflammatory agents such as retinoic acid (RA) and interferon (IFN)-{alpha}.

METHODS PECs were pretreated with or without inhibitors of the ERK1/2, JNK, and p38 (PD98059, SB202190, and SB203580, respectively) mitogen-activated protein kinases (MAPK) or with inhibitors of the tyrosine kinase activity of epidermal growth factor receptor (EGFR; PD153035) and platelet-derived growth factor (PDGF; AG1295); exposed to UVB (20 mJ/cm2); and then further treated with the same inhibitors. Media were harvested and analyzed by ELISA for interleukin (IL)-6, IL-8, and vascular endothelial growth factor (VEGF). Cytokine mRNA was assessed by reverse transcription–polymerase chain reaction (RT-PCR).

RESULTS Inhibitors of ERK1/2, JNK, and p38 MAPKs significantly abolished the UVB-mediated increase in IL-6, IL-8, and VEGF. PD153035 reduced IL-8, AG1295 repressed IL-6, and both inhibitors partially downregulated VEGF production in UV-exposed PECs. RA and IFN-{alpha} dose dependently abrogated IL-6 and IL-8 but had no effect on VEGF expression after UV exposure.

CONCLUSIONS The results have identified a stress-induced intracellular pathway and potential cell-surface transmitters that may be relevant to pterygium development. Moreover, two anti-inflammatory/antiangiogenic agents were identified that reduced cytokine production in the study model. Topical application of these drugs may benefit patients with pterygia, potentially reducing the necessity for surgical interven-tion.

Pterygium is an invasive and proliferative disease of the human ocular surface particularly prevalent in sun-exposed individuals.1 2 Histologic examinations have identified foci of connective tissue elastosis3 and regions of severely damaged Bowman’s membrane,4 suggesting either direct damage due to solar radiation or indirect damage due to excessive proteolytic activity.4 5 6 7 8 9 Pterygia are characterized by an inflammatory infiltrate, composed of neutrophils,5 mast cells,4 10 and lymphocytes.4 11 They can present with a prominent vascular reaction4 12 that is likely to be exacerbated by excessive cytokine13 and growth factor13 14 15 16 17 18 production involving resident cells, as well as infiltrating leukocytes.

Recently, our laboratory has identified several key effector molecules that are likely to participate in the inflammatory, proliferative, and remodeling phase of this disease. These include interleukin (IL)-619 and IL-8,19 heparin-binding epidermal growth factor-like growth factor (HB-EGF),20 vascular endothelial growth factor (VEGF),4 and several matrix metalloproteinases (MMPs).5 6 7 Relevant to the abundant epidemiologic evidence implicating ultraviolet (UV) exposure as a probable trigger for this disease,1 2 these molecules are induced by physiological doses of UVB radiation in ex vivo pterygium specimens19 as well as in cultured pterygium epithelial cells (PECs).19 21 Our studies have also demonstrated that the induction of collagenase-1 (MMP-1) by UVB is specifically mediated by the ERK1/2 mitogen activated protein kinase (MAPK) pathway and did not involve other MAPK pathways—namely, p38 or JNK.21 22 Moreover, we noted that the UVB signal was partially transmitted by the epidermal growth factor receptor (EGFR).22 This too is highly relevant, as this receptor22 23 and other growth factor receptors, such as platelet-derived growth factor receptor (PDGFR),24 have been identified in pterygia.

Given the results of our previous investigations, the present study was designed to explore whether a similar intracellular pathway and mode of signal transmission is responsible for cytokine production in a well-defined in vitro system. To assist us in elucidating the potential mechanism, we developed a working model (Fig. 1) . In brief, we hypothesize that UVB radiation triggers pterygium growth by activating (1) extracellular molecules (such as growth factor receptors), (2) intracellular signaling pathways (such as the MAPKs), and (3) nuclear transcription factors (such as AP-1) that ultimately result in enhanced production of effector proteins including cytokines, growth factors, and MMPs. In this study, experiments were designed to address several key questions specifically: What intracellular signaling pathways are responsible for the UVB-mediated induction of IL-6, IL-8, and VEGF? Are cell-surface receptors such as the EGFR and PDGFR capable of transmitting this signal? Are antiproliferative/antiangiogenic agents capable of suppressing cytokine expression in UVB-exposed pterygium-derived epithelial cells?


Figure 1
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  		FIGURE 1. Extracellular and intracellular molecules and pathways activated by UVB. A model for pterygium development. In this model, it is proposed that cytokine and/or growth factor receptors (C/GF-R) can be activated by two distinct pathways. The first is via ligand binding to a specific cell-surface receptor, a process termed "ligand-dependent" activation. Alternatively, these receptors can be activated directly by stress signals such as UV radiation, and this is termed "ligand-independent" activation. Irrespective of the pathway activated, receptor clustering, phosphorylation (P), and internalization are subsequent early events. Ras, a small G-protein transmits the signal from the cell membrane to the cytoplasm where one or more MAPK (JNK, ERK, p38) pathways may be activated. This signal reaches the nucleus where downstream transcription factors such as AP-1 (c-jun/c-fos) may be targeted to modulate the transcription of effector molecules such as cytokines, growth factors, and MMPs. Cytokines and MMPs may facilitate pterygium development, as these proteins are involved in survival, proliferation, and cell invasion. Antiproliferative and antiangiogenic compounds such as RA and IFN-{alpha} may selectively target certain levels of this cascade, resulting in suppression of effector molecules.

 
Surgical intervention25 accompanied by adjunct therapy with ß-irradiation26 or mitomycin-C (MMC)27 have been used to treat pterygia. However, follow-up studies have identified unacceptable recurrence rates with the use of these latter agents. Significantly better results have been observed with conjunctival autografts28 and amniotic membrane transplantation.29 30 In addition to understanding the molecular mechanisms that regulate pterygium development after UV stimulation, our research was also intended to determine whether antiproliferative/anti-inflammatory drugs such as retinoic acid (RA) and type I interferons (IFNs) could modify the expression of UV-inducible cytokines in our model system.

Type I IFNs comprise three subtypes (-{alpha}, -ß, and -{omega}), some of which have been used to treat other proliferative or invasive diseases such as cancer31 and rheumatoid arthritis.32 Although their mechanism of action remains poorly understood, this class of cytokines influences cell proliferation, differentiation, and apoptosis possibly by influencing the expression of the EGFR33 and the subsequent activation of intracellular signaling pathways, such as the MAPKs.34 IFNs also possess antiangiogenic activity31 thought to be via a mechanism that involves abrogation of endothelial cell proliferation and migration in vitro35 and suppression of neovascularization in vivo in the cornea.36 Like the IFNs, retinoids modulate immunologic and inflammatory responses by affecting cytokine and chemokine production.37 38 RA has been shown to influence cell growth and proliferation by suppressing several key inflammatory transcription factors such as nuclear factor (NF)-{kappa}B and activator protein (AP)-1.38 RA has also been used to treat various forms of human cancer39 and rheumatoid arthritis40 with mixed success. In the current investigation, we demonstrated the effectiveness of both RA and IFN-{alpha} at reducing the production of two potent proinflammatory cytokines that have been implicated in the pathogenesis of pterygia.19


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
PEC Culture
All research protocols relating the use of human primary cells were approved by the University of New South Wales Human Ethics Committee and performed in accordance with the tenets of the World Medical Association’s Declaration of Helsinki. PECs from at least two donors were cultured from pterygium tissue obtained immediately after surgical resection. Pure long-term cultures of PECs were established as previously described.41 Briefly, pterygia were cut into several 2- to 3-mm2 segments and placed on tissue culture plates as explants in serum-containing medium. Epithelial cell outgrowth from the explants began as early as 3 days in culture. Fibroblast contamination was minimized by removing the tissue when a sufficient number of epithelial cells surrounded each explant. Epithelial cells were passaged, and the purity (>98%) established by flow cytometry using cytokeratin antibodies.41 All cell culture experiments were performed with cells from passages 5 to 10.

UVB Irradiation of Epithelial Cells
PECs were seeded at 1 x 106 cells in 100-mm tissue culture dishes (Corning-Costar, Corning, NY) or at a density of 1.5 x 105 cells per well in six-well plates (Nunc, Roskilde, Denmark) and grown in the presence of 10% fetal bovine serum/Eagle’s minimum essential medium (FBS/EMEM). Once cells reached semiconfluence, the medium was aspirated, and the cells were washed three times with sterile PBS and left in serum-free medium (EMEM without FBS) for 16 hours, as previously described.4 5 19 20 21 In some experiments, cells were preincubated with selective inhibitors of ERK1/2, JNK, and p38 MAPKs (Calbiochem, CA). These included 10 µM PD98059, 1 µM SB202190, and 1 µM SB203580, respectively. In addition, 1 µM hydrocortisone (Sigma-Aldrich, St. Louis, MO) was added to some cells as a broad-spectrum downregulator of cytokine production. To determine whether surface receptors were involved in transmitting the UVB signal, some cells were preincubated for 1 hour in 0 to 1.0 µM PD153035 (Calbiochem, La Jolla, CA), an inhibitor of the tyrosine kinase activity of the EGFR or with 0 to 1.0 µM AG1295 (Calbiochem) an inhibitor of the tyrosine kinase activity of the PDGFR. Whereas cells were pretreated in other experiments with RA (0–0.1 µM; Sigma-Aldrich) or IFN-{alpha} (0 to 15,000U; PBL Biomedical Laboratories, Piscataway, NJ). After the preincubation period, the medium containing the respective agents was replaced with PBS and the monolayers irradiated with 20 mJ/cm2 of UVB (ITL 20W/12 RS bulbs; Philips, Sydney, Australia), as previously reported.4 19 20 21 This amount of radiation equated to a 3-minute exposure and was an amount that did not affect cell morphology or viability. UVB light intensity was monitored and calibrated before each experiment with the aid of a radiometer (IL1400A; International Light, Newburyport, MA). After each exposure, cells were rinsed once with PBS and placed in fresh serum-free medium with the respective agents and incubated for a further 48 hours. Supernatants were collected, cleared of any cell debris by centrifugation, and stored frozen in small aliquots at –70°C. Compounds tested in these experiments were nontoxic at the doses used.

RNA Extraction and Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted (RNAgents Total RNA Extraction Kit; Promega, Sydney, Australia) after 24 hours as previously outlined4 5 19 20 21 from control (mock-irradiated), and UVB-exposed PEC that were pre- and posttreated with the agents described earlier. This time point was selected as it corresponded to the peak in cytokine and growth factor transcriptional activity.19 20 Reverse transcription was performed according to the manufacturer’s instructions (Preamplification system for first strand cDNA synthesis kit; Invitrogen-Gibco, Gaithersburg, MD), as previously described.5 7 19 20 21 22 An aliquot (1 µL) of cDNA was amplified by PCR with 100 nM each of the forward and reverse gene specific primers for IL-6,19 IL-8,19 VEGF (reverse 5'-TCG ATC GTT CTG TAT CAG TCT-3'; forward 5'-CCA TGA ACT TTC TGC TGT CTT-3'), and GAPDH.19 20 21 22 The VEGF primers used in the study were designed to amplify at least three of the four spliced variants including VEGF121, VEGF165, and VEGF189. 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 number of cycles was predetermined for each PCR product. PCR products were visualized on either 1.2% or 2% agarose gels that contained ethidium bromide. Semiquantitative assessment was performed after normalization to GAPDH (Gel Doc 2000; Bio-Rad, Sydney, Australia; and the Quantity One software program; Bio-Rad).

Enzyme Immunoassays
Human IL-6, IL-8, and VEGF (Quantikine; R&D Systems, Minneapolis, MN) were quantified using sandwich immunoassays. The VEGF ELISA used in this study specifically detected VEGF165, one of the soluble variants of this growth factor. Cytokines in supernatants from control (mock irradiated), or UVB-exposed PECs that were incubated with specified agents, were captured on antibody-coated 96-well plates and detected precisely as directed by the manufacturer. The optical density of the reaction product was read at the appropriate wavelength using a microplate reader (Spectramax Plus; Molecular Devices, Sunnyvale, CA).

Statistical Analysis
Cytokine levels in the culture supernatants were expressed as mean ± SD. Difference in cytokine level between control and UV-treated cells was assessed by one-way analysis of variance, followed by the Newman-Keuls test for multiple comparisons of treatment groups with the control group. A commercial software package (Prism; GraphPad Software, San Diego, CA) was used for analysis.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Mediation of Induction of IL-6, IL-8, and VEGF
To determine whether cytokine and growth factor induction after UVB exposure is mediated by a MAPK pathway, PECs were pretreated, irradiated, and posttreated with specific intracellular pathway inhibitors. Initially, we noted a significant upregulation of IL-8, IL-6, and VEGF protein production (Fig. 2) after UVB exposure. The addition of specific MAPK inhibitors (SB202190, SB203580, and PD98059) abrogated IL-8, IL-6, and VEGF production, respectively, after irradiation (Fig. 2) . These agents inhibited IL-8 (Fig. 2A) and IL-6 (Fig. 2B) , whereas VEGF production was only partially, but still significantly, reduced. Hydrocortisone suppressed the production of all three proteins to near control levels, whereas the diluent control (DMSO) had no significant effect on cytokine production (Fig. 2) .


Figure 2
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  		FIGURE 2. Abrogation of cytokine production with selective MAPK inhibitors. PECs were pre- and then posttreated with selective MAPK inhibitors after UVB (20 mJ/cm2) irradiation. Supernatants were harvested 48 hours later and analyzed by ELISA. Some cells were irradiated and incubated with the diluent control (DMSO), whereas other cells were mock irradiated (Control). Data represent the mean ± SD of results in triplicate experiments. *P < 0.01.

 
Similar experiments were conducted to determine whether selective MAPK inhibitors were effective at the level of gene transcription. Indeed, when PECs were exposed to the same level of UVB radiation, we observed a 5.5-, 1.8-, and 1.5-fold increase in IL-8, IL-6, and VEGF gene expression, respectively (Figs. 3A 3B, 3C) . Furthermore, the addition of the MAPK inhibitors (SB202190, SB203580, and PD98059) significantly suppressed IL-8 (Fig. 3A) and IL-6 (Fig. 3B) , whereas they only moderately inhibited VEGF (Fig. 3C) mRNA expression. The results of this analysis closely resembled the cytokine protein production data (Fig. 2) .


Figure 3
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  		FIGURE 3. Inhibition of cytokine gene transcription by MAPK inhibitors. PECs were cultured under control conditions (lane 1: mock irradiated) or exposed to 20 mJ/cm2 of UVB (lanes 2–7) before pre- and posttreatment with specific MAPK inhibitors, including SB202190 (SB02), SB203580 (SB03), and PD98059 (PD98) or with hydrocortisone (HYC). Total RNA was extracted after 24 hours and analyzed by RT-PCR. No products were formed in reactions that contained template that had not been reverse transcribed (lane 7) or lacked primer pairs or a cDNA template (data not shown). The values above each band indicate the x-fold increase compared with nonirradiated cells after standardizing to GAPDH expression. Similar results were obtained with PECS from another donor.

 
Cell-Surface Receptors and Transmission of the UVB Signal
Next, we investigated whether cell-surface receptors (previously localized in pterygia22 23 24 ) might function as transmitters of the UVB signal. In a recent investigation, the EGFR was found to be partially responsible for transmitting the signal that resulted in the induction of collagenase-1 (MMP-1).22 We demonstrated, using the same experimental model, that EGFR, but not PDGFR, was responsible for relaying the UVB signal that resulted in increased IL-8 production (Fig. 4A) . The addition of 0.1 µM PD153035 significantly reduced IL-8 levels from 166.6 ± 22.5 to 74.4 ± 23.6 pg/mL (P < 0.01) whereas 0.1 µM AG1295 had a negligible effect (174.4 ± 60.3 pg/mL). In contrast to IL-8 (Fig. 4A) , the UVB-mediated induction of IL-6 predominantly involved signal transmission through the PDGFR (Fig. 4B) as the PDGFR inhibitor (AG1295; 0.1 µM final) significantly reduced IL-6 levels from 356.2 ± 32.1 to 217 ± 42 pg/mL (P < 0.01). At a final concentration of 0.1 µM, PD153035 (109.1 ± 1.5 pg/mL) and AG1295 (132.7 ± 8.0 pg/mL) moderately suppressed (P < 0.01) VEGF production compared with UVB alone (166.5 ± 11.0 pg/mL), thus implicating both receptors in signal transmission (Fig. 4C) . Similar results were obtained when PD153035 and AG1295 were used at a final concentration of 1.0 µM (Fig. 4) .


Figure 4
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  		FIGURE 4. Signal transmission through growth factor receptors. PEC were pre- and then posttreated with either a selective inhibitor of the EGFR (PD153035) or an inhibitor of the PDGFR (AG1295) after UVB (20 mJ/cm2) irradiation. Supernatants were harvested 48 hours later and analyzed by ELISA. Data represent the mean ± SD of results in triplicate experiments. *P < 0.01.

 
Effect of RA and IFN-{alpha} on IL-6 and IL-8
PEC were pretreated, then irradiated, and finally posttreated with either RA or IFN-{alpha} (Fig. 5) . Both drugs dose dependently inhibited IL-8 (Figs. 5A 5B) and IL-6 (Figs. 5C 5D) protein production, but had no significant inhibitory effect on the UVB-mediated induction of VEGF at the concentrations used (Figs. 5E 5F) . Likewise, when cytokine mRNA expression was determined after treatment of UVB-irradiated PECs with either RA or IFN-{alpha}, we noted significant suppression of both IL-8 (Fig. 6A) and IL-6 (Fig. 6B) , but not of VEGF (Fig. 6C) , a result that corroborated the protein secretion data (Fig. 5) .


Figure 5
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  		FIGURE 5. Downregulation of IL-6 and IL-8 protein by RA and IFN-{alpha}. PECs were pre- and then posttreated with RA (A, C, E) or IFN-{alpha} (B, D, F) after UVB (20 mJ/cm2) irradiation. Supernatants were harvested 48 hours later and analyzed by ELISA. Some cells were mock irradiated (Control) or incubated with the diluent control (DMSO) without exposure. A similar pattern of inhibition was observed with at least one other diseased epithelial cell line. Data represent the mean ± SD of results in triplicate experiments. *P < 0.01.

 

Figure 6
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  		FIGURE 6. Suppression of IL-6 and IL-8 mRNA by RA and IFN-{alpha}. PECs were pre- and then posttreated with either RA (0.1 µM) or IFN-{alpha} (15,000 U) after UVB (20 mJ/cm2) irradiation. Total RNA was isolated 24 hours later and analyzed by RT-PCR. The values shown above each band indicate the x-fold increase compared with nonirradiated cells after standardizing to GAPDH expression. Similar results were obtained with PECs from another donor.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Pterygia have predominantly been described in humans, with only one report of a pterygium-like lesion in animals.42 This species-specific disease may be attributable to the unique anatomic features of the human eye.43 44 Currently, surgical excision with adjunctive therapy25 26 27 and tissue grafting28 29 30 are the major forms of disease management but have been associated with recurrences and ocular complications. Given that there are no animal models for pterygia, we have developed an in vitro system using epithelial cells derived from human pterygia. We simulated environmental UV exposure in a controlled laboratory setting and demonstrated that a single physiological dose of this agent increased the production of several inflammatory and angiogenic mediators (Figs. 2 3 4 5 6) .19 20

Enhanced production of IL-6,45 IL-8,46 and VEGF47 has been documented in UVB-irradiated human skin keratinocytes. Given that the skin is the largest and most exposed organ of the body, this comes as no surprise and is thought to be a tissue response to injury. The ocular surface is subjected to similar environmental stress and cytokines such as IL-1, -6, and -8 and TNF-{alpha} have been shown to be induced in UV exposed corneal epithelial cells48 and corneas in vivo.49

Multiple UV exposure to the skin triggers downstream events resulting in cell differentiation, proliferation, invasion, inflammation, and angiogenesis.46 50 Our model is not sufficiently robust to accommodate several exposures. Furthermore, a monolayer of epithelial cells cultured under serum-minimized conditions does not accurately represent the complex cell-to-cell and cell–matrix interaction of an intact tissue. Therefore, we cannot comment on the potential epithelial influence on the stromal component, but we postulate that cell-to-cell cross-talk is an integral part of pterygium development.4

Recently, our research has focused on mapping the likely molecules and pathways that support our working hypothesis (Fig. 1) . In addition to demonstrating the UVB-mediated enhanced expression of key proinflammatory cytokines (IL-6 and IL-8) and growth factors (VEGF and HB-EGF), we have identified several MAPK pathways that are activated in response to this stress signal (Fig. 1) . Furthermore, we have identified two stress sensitive cell-surface receptors (EGFR and PDGFR) as potential signal transmitters, as specific tyrosine kinase inhibitors of these receptors suppressed cytokine production after UVB exposure. Although the mechanism of action is not entirely clear (Fig. 1) , RA and IFN-{alpha} significantly abrogated both IL-6 and IL-8 mRNA expression and protein production in our model (Figs. 5 6) .

Receptor signaling is a complex process and can be mediated through direct binding of ligand51 (ligand-dependent) or indirectly by physical stress such as osmosis52 and UV radiation52 (ligand-independent; Fig. 1 ). Although several EGFR4 13 19 22 and PDGFR13 ligands have been identified in pterygium, we previously proposed that these receptors are activated through a ligand-independent pathway.22 Despite this circumstantial evidence, it is tempting to speculate that the UVB-mediated cytokine–growth factor induction observed in the present study may have been caused by a secondary mediator such as another cytokine (e.g., TNF-{alpha} or IL-1). We have excluded this possibility, as several proinflammatory cytokines including TNF-{alpha} and IL-1 were not induced by UVB in our model (data not shown). Furthermore, in a previous investigation, we attempted to measure both IL-1 and TNF-{alpha} in supernatants derived from UVB irradiated PECs using commercial ELISAs.19 However, neither cytokine was detectable, nor were mRNA transcripts identified by an RNase protection assay, further confirming the irrelevance of these cytokines in our assay system. These results closely resemble those of other investigators who demonstrated that the UV-mediated induction of VEGF in epithelial cells is totally independent of TNF-{alpha}.53 Likewise, Blaudschun et al.54 demonstrated that the UVB-mediated induction of VEGF in skin keratinocytes is dependent on phosphorylation of the EGFR and not due to ligand binding. Irrespective of the mode of activation, EGFR signaling has been reported to facilitate cell proliferation, apoptosis, angiogenesis, and metastasis.55 For this reason, tyrosine kinase inhibitors of such receptors have been used to treat patients with advanced malignancies55 and may be a nonsurgical strategy worth considering for pterygia.

Once initiated, receptor phosphorylation proceeds within minutes, with subsequent activation of intracellular signaling pathways.56 In the current investigation, we focused our attention on the MAPK pathway for several reasons. First, we have documented the exclusive involvement of the ERK1/2 in the UVB-mediated induction of MMP-1 in PECs.21 22 Second, JNK, p38, and ERK1/2 are all activated, although to varying degrees, in UVB exposed skin keratinocytes.56 57 Third, activation of one or more of these pathways amplifies nuclear transcription factor expression (c-jun and c-fos) resulting in cytokine production in skin57 and ocular surface22 epithelial cells. Finally, MAPKs have overlapping as well as contrasting effects relating to apoptosis and survival58 and these processes have been a topic of debate in pterygia.59 60

In the current investigation we noted that RA and IFN-{alpha} inhibited IL-6 and IL-8 (but not VEGF) in UV-irradiated epithelial cells. This finding is highly relevant, as these cytokines have been identified in pterygium specimens,19 and, given their mitogenic, angiogenic, and proinflammatory activity,61 62 63 selective repression of these effector molecules may have therapeutic application. Several investigators have successfully used topical IFN-{alpha}64 65 and RA66 to treat primary as well as recurrent corneal/conjunctival neoplasia without side effects. Features of corneal neoplasia resemble pterygia. These include a strong association with excessive UV exposure67 ; a slow, progressive invasion of "altered" epithelial cells over the ocular-surface; and high recurrence rates, depending on the surgical approach. Given these striking similarities, clinical trials using topical IFN and/or RA should be considered. Indeed, a recent report has demonstrated the successful treatment of recurrent pterygium with topical IFN-{alpha}.68

Although the mechanism by which RA and IFN-{alpha} act is incompletely understood, it is likely both agents target intracellular signaling pathways. IFN-{alpha} can induce proliferative as well as antiproliferative effects in a cell-type–specific manner via a mechanism dependent on MAPK activation. For example, tumor cells displaying dramatic proliferative response to IFN-{alpha}, expressed substantial levels of phosphorylated (p)-ERK1/2. In contrast, tumor cells that display typical growth arrest in response to IFN-{alpha}, have little or no active ERK1/2.69 Furthermore, IFN-{alpha} at a concentration comparable to that used in the present study (1000 U/mL), selectively increases pERK1/2, without an appreciable effect on p38 and JNK.34 It is highly likely that the effects evoked by RA on decreasing IL-6 and IL-8 (Fig. 6) is via activation of similar signaling pathways (Fig. 1) . Like IFN-{alpha}, RA increased pERK1/2 in a cell-type and time-dependent manner70 and suppressed the growth factor (EGF)-mediated increase in epithelial cell proliferation by specifically inhibiting the activation of ERK1/2 without altering p38 or JNK.71 Of note, combination therapy with RA plus IFN-{alpha} synergistically retards the growth and neovascularization of tumor cells,31 and this may be a future strategy worth considering in our culture model. Finally, IFN-{alpha} not only influences MAPKs, but it is tempting to speculate that cell-surface receptors such as the EGFR33 may also be a target in our model (Fig. 1) .

Improved understanding of the immunopathogenesis of pterygia and the relevant molecular pathways that are activated in this common ocular-surface disorder may lead to new avenues of treatment that may have relevance to other ocular diseases.


   Footnotes
 
Supported by Grant 350919 National Health and Medical Research Council of Australia.

Submitted for publication August 24, 2005; revised December 5, 2005, and January 18, 2006; accepted March 13, 2006.

Disclosure: N. Di Girolamo, None; D. Wakefield, None; M.T. Coroneo, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Nick Di Girolamo, Inflammatory Diseases Research Unit, Department of Pathology, School of Medical Sciences, The University of New South Wales, Sydney, 2052, Australia; n.digirolamo{at}unsw.edu.au.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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