Human Corneal Epithelial Cells Express Functional PAR-1 and PAR-2

Abstract

purpose. The objective of this study was to examine whether HCECs express functional proteinase-activated receptor (PAR)-1 and -2 and evaluate the effects of receptor activation on corneal epithelial cell proinflammatory cytokine production.

methods. Expression of PAR-1 and -2 mRNAs was determined by RT-PCR in cultured primary human corneal epithelial cells (HCECs) and the human corneal epithelial cell line HCE-T. Localization of PAR-1 and -2 in whole normal human corneas was determined by immunofluorescence with PAR-1 and -2 antibodies. The functional competence of PAR-1 and -2 in corneal epithelial cells was assessed by measuring the rapid induction of intracellular [Ca²⁺] in response to thrombin, trypsin, and specific receptor-activating peptides derived from the tethered ligands of the PAR receptors. HCE-T expression of cytokines (IL-6, IL-8, and TNFα) in response to activation of PAR-1 and -2 was measured by quantitative RT-PCR and ELISA.

results. Functional PAR-1 and -2 were expressed in both HCECs and HCE-T cells. Immunoreactivity for PAR-1 and -2 was detected in the outer epithelial layer of the cornea in whole human corneal sections. Activation of PAR-1 and -2 led to upregulation in HCE-T cells of both expression of mRNA and secretion of the proinflammatory cytokines IL-6, IL-8, and TNFα.

conclusions. The results show for the first time that functional PAR-1 and -2 are present in human cornea. Activation of these receptors results in the production of various corneal epithelial cell proinflammatory cytokines. These observations indicate that PAR-1 and -2 may play an important role in modulating corneal inflammatory and wound-healing responses. These receptors may be useful therapeutic targets in several corneal disease processes.

Proteinase-activated receptors (PARs) are members of a family of G-protein-coupled receptors that are activated by the cleavage of their N-terminal domain by serine proteinases. ^{1 2 3} The proteolytic cleavage of PAR exposes tethered ligand domains of the receptor that bind to and activate the cleaved receptor. Receptor activation can also be mimicked nonproteolytically by native peptides that correspond to the cleaved N-terminal amino acid sequence of the PAR. There are four known members of this receptor family of PARs: PAR-1, -3, and -4, which are activated by thrombin, and PAR-2, which is activated by trypsin or mast cell tryptase. ^{4 5 6}  

Recent studies indicate that PARs are important mediators of cellular responses to injury or infection and are widely distributed. PAR-1 is expressed in various tissues and cell lines, ³ including endothelial cells ⁴ and epithelial cells, such as keratinocytes ^{7 8} and lung epithelial cells. ⁹ Thrombin, which is generated after tissue injury as a part of the coagulation cascade, has numerous biological functions that are related to inflammation, tissue remodeling, and wound healing. Many of the biological actions of thrombin are mediated by PAR-1. PAR-1 agonists and serine proteases are capable of inducing increased vascular permeability, extravasation of plasma proteins, ¹⁰ stimulation of chemoattractants, ¹¹ and activation of T cells and production of cytokines. ¹² In addition, PAR-1 agonists are mitogenic for many cell types, including endothelial cells ¹³ and epidermal keratinocytes. ⁸ Similar to PAR-1, PAR-2 is expressed in a variety of cells, including keratinocytes, ^{7 14} endothelial cells, ¹⁵ and cells derived from other human tissues. ¹⁶ Therefore, PAR-2, similar to PAR-1, is an important regulator of tissue inflammation and repair. PAR-2 agonists increase adhesion, extravasation, and migration of leukocytes ¹⁷ and stimulate secretion of IL-8 by keratinocytes. ¹⁸ The effects of PAR-2 agonists on cellular growth appear to depend on the cell type. PAR-2 agonists also stimulate proliferation of endothelial cells, ¹³ whereas they inhibit keratinocyte growth and differentiation. ¹⁹  

Although the presence of PARs has been demonstrated in various tissues, little is known regarding the expression and function of these receptors in the human eye. Previous studies support the possibility that PARs may have a role in a variety of biological processes in the eye. These reports demonstrated that thrombin and trypsin are capable of inducing proliferation and migration of cultured rabbit lens epithelial cells, which are blocked by proteinase inhibitors. ²⁰ In addition, serine proteinase inhibitors have been found to inhibit the migration of corneal epithelial cells. ²¹ Moreover, thrombin and trypsin binding sites have been identified on cultured bovine corneal endothelial cells. It has been found that endothelial proteinase-bound membrane complexes are internalized and degraded after binding to these cells. ^{22 23 24} In this study, we examined the expression and function of PAR-1 and -2 receptors in human corneal cells and the possible biological implications of their activation. 

Methods

Culture of Human Corneal Epithelial Cells

The human corneal epithelial cell line 10.014 pRSV-T (HCE-T) was kindly provided by Sherry Ward at the Gillette Medical Evaluation Laboratories (Gaithersburg, MD). HCE-T cells were cultured on a surface coated with a bovine fibronectin and collagen mix (FNC; BRFF, Ijamsville, MD) using a serum-free keratinocyte growth medium kit (KGM; Clonetics, San Diego, CA) supplemented with 100 U/mL penicillin, 250 μg/mL amphotericin B, and 10 μg/mL streptomycin (Life Technologies, Gaithersburg, MD) at 37°C in a humidified atmosphere containing 5% CO₂. 

Normal HCECs (cell line HCEC-3) were obtained from Cascade Biologics, Inc. (Portland, OR) and grown in medium (EpiLife: Sigma-Aldrich, St. Louis, MO) supplemented with human corneal growth supplement and 100 U/mL penicillin G, 100 μg/mL streptomycin sulfate, and 0.25 μg/mL amphotericin B (Cascade Biologics, Inc.) at 37°C in a humidified atmosphere containing 5% CO₂. 

Reagents

Human thrombin was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Bovine trypsin (TPCK-treated) was obtained from Worthington Biochemicals, Inc. (Lakewood, NJ). Peptides corresponding to the tethered ligand domains of PAR-2 and -1 were synthesized on solid phase and purified by high-pressure liquid chromatography. ²⁵ The following peptides were prepared: SLIGRL-NH₂ (tethered ligand of rat PAR-2), LRGILS-NH₂ (reverse of rat PAR-2 tethered ligand), and TFLLR-NH₂ (selective agonist of PAR-1). Recombinant human TNFα was purchased from R&D Systems (Minneapolis, MN), and phorbol 12-myristate 13-acetate (PMA) was from Sigma (St. Louis, MO). 

Determination of PAR-1 and -2 mRNA Expression in HCECs

HCEC-3 and HCE-T cells were grown to 70% to 80% confluence as described earlier. Total RNA was isolated from the cells with extraction reagent (Tri Reagent; Sigma-Aldrich) according to the manufacturer’s instructions. First-strand cDNA was synthesized by reverse transcription of the RNA by an avian myeloblastosis virus (AMV) reverse transcriptase transcription system (Promega, Madison, WI), according to the manufacturer’s protocol. PCR was performed with 40 cycles, as previously described. ²⁶ The oligonucleotide primers used to amplify PAR-1 and -2 were based on published sequences of these genes. ^{27 28} PAR-1 primers (forward, 5′-CACCGGAGTGTTTGTAGTCA-3′; reverse, 5′-TAACTGCTGGGATCGGAACT-3′) ²⁷ were chosen to amplify an 864-bp fragment. Conditions were as follows: denaturation for 5 minutes; 40 cycles of 94°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute; and elongation at 72°C for 7 minutes. PAR-2 primers (forward, 5′-GTTGATGGCACATCCCACGTC-3′; reverse, 5′-GTACAGGGCATAGACATGGC-3′) ²⁸ were chosen to amplify a product of the expected size of 865 bp. Conditions were as follows: denaturation for 5 minutes; 40 cycles at 94°C for 1 minute, 64°C for 1 minute, and 72°C for 1 minute; and elongation at 72°C for 7 minutes. PCR products were analyzed by electrophoresis on a 1.5% agarose gel with ethidium bromide. 

Immunohistochemical Detection of PAR-1 and -2

Human corneas were obtained from the Department of Ophthalmology, Emory University (Atlanta, GA) in preservative (Optisol; Chiron Vision, Irvine, CA), fixed in Zamboni fixative for 24 hours at room temperature, and embedded in paraffin. Tissue sections were prepared for immunohistochemistry as previously described. ²⁹ Briefly, the sections were preincubated with PBS containing 0.5% Triton X-100, 1% normal goat serum, and 0.01% thimerosal. The sections were incubated with primary antibodies overnight at 4°C. Antibodies used were mouse anti-human PAR-1 antibody TR31–2 (Cor Therapeutics, South San Francisco, CA) at a 1:500 to 1:1000 dilution and the polyclonal rabbit anti-rat PAR-2 antibody B5 (raised against the NH₂-terminal of rat PAR-2, ³⁰GPNSKGRSLIGRLDT⁴⁶P; Molly D. Hollenberg, University of Calgary, Calgary, Alberta, Canada) at a 1:500 to 1:800 dilution. The sections were washed three times in PBS and incubated with a goat anti-rabbit and anti-mouse IgG conjugated to Texas red or fluorescein (Jackson ImmunoResearch, Inc., West Grove, PA) at a dilution of 1:200 for 2 hours at room temperature. Tissue sections were washed three times in PBS, postfixed with 4% paraformaldehyde, washed again, and mounted (Prolong; Molecular Probes, Eugene, OR). The immunolocalization of PAR-1 and -2 was observed and photographed by light microscopy (Olympus Optical Co., Ltd., Tokyo, Japan). 

Measurement of Intracellular [Ca²⁺]

HCEC-3 and HCE-T cells were cultured on 35-mm glass-bottomed microwell dishes (MatTek Corp., Ashland, MA) and starved overnight. Cells were incubated in Hanks’ balanced salt solution (HBSS) containing 3 μM fura-2/AM (Molecular Probes) for 2 hours at room temperature. Cells were washed twice with HBSS and kept in HBSS for an additional 45 minutes at 37°C. After the cells were washed once with HBSS, 1 mL HBSS was added to the culture dishes. 

The imaging system consisted of a microscope (model TMS-F; Nikon, Tokyo, Japan) with phase objectives (Fluor; Nikon) and a CCD camera (model 4915; Cohu, Irvine, CA). Analysis of video signals and system filter changer control were performed by computer (InCyt Im2 software; Intracellular Imaging Inc., Cincinnati, OH). All experiments were performed at room temperature with excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Ca²⁺ standard curves were obtained using fura-2 pentapotassium salt (Molecular Probes). 

Quantitative RT-PCR

HCE-T cells were grown to 70% to 80% confluence on 100-mm culture dishes (BD Bioscience, Bedford, MA) in culture medium. After overnight starvation, the cells were incubated with trypsin (10 nM), thrombin (10 nM), TNFα (10 ng/mL), and PMA (50 ng/mL) in medium. Medium alone was used as the negative control. After 3 and 6 hours of incubation, total RNA was isolated, and cDNA was generated as described earlier. 

Quantitative RT-PCR was performed using a fluorescent green PCR master mix (SYBR Green; Applied Biosystems, Foster City, CA) and the following primer sequences: human IL-6 primer sequences: sense, 5′-TGG CTG CAG GAC ATG ACA ACT-3′, and antisense, 5′-ACA ATC TGA GGT GCC CAT GCT-3′ ³⁰ ; oligonucleotide sequences to amplify human IL-8: sense, 5′-GCA GTT TTG CCA AGG AGT GTC-3′, and antisense, 5′-TTT CTG TGT TGG CGC AGT GTG-3′ ³¹ ; human TNFα primer sequences: sense, 5′-GCC CAT GTT GTA GCA AAC CCT-3′, and antisense, 5′-TCG GCA AAG TCG AGA TAG TCG-3′. ³² and human 18S rRNA primer sequences: sense, 5′-CGG CTA CAT CCA AGG AA-3′, and antisense, 5′-GCT GGA ATT ACC GCG GCT-3′. ³³  

Amplification was performed with a sequence detection system (Gene Amp 5700; Applied Biosystems, Foster City, CA) using a standard amplification program (1 cycle at 50°C for 2 minutes, 1 cycle at 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute). Direct detection of PCR products was quantified by measuring the increase in fluorescence caused by the binding of the green fluorescent dye to double-stranded DNA, and the specificity of the PCR products was verified by melting-curve analysis. Using a standard curve of serial dilutions of an untreated cDNA sample, a relative quantitation of the respective target cDNAs expressed in multiples of differences was performed. All quantities were normalized to an endogenous control (18S rRNA) to account for variability in the initial concentration and quality of total RNA and in the conversion efficiency of the reverse transcription reaction. 

Determination of Secreted and Cell-Associated Cytokines by ELISA

HCE-T cells were grown to 70% to 80% confluence on 100-mm culture dishes (BD Bioscience) in culture medium. After overnight starvation, the cells were incubated with trypsin (10 nM), SLIGRL-NH₂ (100 μM), LRGILS-NH₂ (100 μM), thrombin (10 nM), TFLLR-NH₂ (100 μM), TNFα (10 ng/mL), and PMA (50 ng/mL) in medium. Medium alone was used for negative control for 24 hours. Cell supernatants were collected and stored at −80°C. Cell lysates were prepared by incubating cells with 1% igepal (CA-630; Sigma, St. Louis, MO) in Dulbecco’s phosphate-buffered salt solution (DPBSS) on ice for 30 minutes. After centrifugation (2440g for 15 minutes), clarified cell lysates were stored at −80°C until used. Both cell lysates and tissue culture supernatants were tested for human IL-6 (R&D Systems), IL-8 (Endogen, Woburn, MA), and TNFα (R&D Systems) using commercially available ELISA kits, as previously described. ³⁴  

Statistical Analysis

Statistical differences were determined on computer by unpaired t-test with the Welch correction (Prism 2 software; GraphPad, San Diego, CA). P < 0.05 was considered to be significant. 

Results

Corneal Epithelial Cell Expression of PAR-1 and -2 mRNAs

We examined expression of PAR-1 and -2 mRNAs in primary HCECs by RT-PCR followed by partial DNA sequencing of the PCR products. As indicated in Figure 1A , PAR-1 and -2 mRNAs were constitutively expressed in primary HCECs. The PAR-1 and -2 mRNAs were also detected in the HCE-T cell line (Fig. 1B) . The identity of amplified gene products in HCECs and HCE-T cells was confirmed by nucleotide sequencing, as shown in Figures 1C and 1D . These nucleotide sequences were identical with the GenBank (http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD) sequences reported for PAR-1 and -2. ^{27 28} This observation indicates that both primary HCECs and a HCE-T cells expressed PAR-1 and -2 mRNAs. 

Immunolocalization of PAR-1 and -2 in Whole Human Cornea

We localized PAR-1 and -2 protein in whole human cornea by immunohistochemistry. Prominent immunoreactive staining for PAR-1 was detected in the outer cell layers of the corneal epithelium, whereas the corneal endothelium did not stain with the PAR-1 antibody (Fig. 2A) . Similar staining patterns were observed for PAR-2. Immunoreactivity was also clearly visible in the outer corneal epithelial cell layers (Fig. 2C) . These findings localize PAR-1 and -2 to the apical surface of the most superficial corneal epithelial cells. 

Functional Activity of PAR-1 and -2 in HCECs

The functional competence of corneal epithelial cell PAR-1 and -2 was determined by measuring intracellular [Ca²⁺] responses after exposure to specific PAR-1 and -2 agonists. As indicated in Figure 3 , 10 nM thrombin, which activates PAR-1, induced a rapid increase in intracellular [Ca²⁺] levels in primary HCECs. A peptide corresponding to the receptor-activating tethered ligand of human PAR-1 (100 μM TFLLRN-NH₂) similarly increased Ca²⁺ levels in these cells (Fig. 3B) . Similarly, exposure of HCECs to 10 nM trypsin, which activates PAR-2, stimulated a rapid increase in intracellular [Ca²⁺] levels (Fig. 3C) . A peptide corresponding to the receptor-activating tethered ligand of PAR-2 (100 μM SLIGRL-NH₂) induced an increase in HCEC intracellular [Ca²⁺] levels similar to that induced by trypsin (Fig. 3D) . Additionally, as indicated in Figure 4 , activation of PAR-1 and -2 also resulted in a rapid induction of intracellular [Ca²⁺] levels in the HCE-T cell line. Taken together, these results demonstrate the functionality of PAR-1 and -2 receptors in HCECs. 

Upregulation of Corneal Epithelial IL-6, IL-8, and TNFα mRNAs by Activation of PAR-1 and -2

Real-time quantitative RT-PCR was used to determine whether thrombin and trypsin were capable of inducing an increase in corneal epithelial cell expression of proinflammatory cytokine mRNAs. As summarized in Table 1 , stimulation of HCE-T cells with either trypsin or thrombin for 3 hours increased expression levels of IL-6, IL-8, and TNFα mRNA 7.5-to 13-fold compared with untreated cells, thus demonstrating the capability of both thrombin and trypsin to upregulate the expression in HCE-T cells of the mRNAs of these proinflammatory cytokines. 

Induction of Proinflammatory Cytokine Secretion by Activation of PAR-1 and -2

We determined the effect of activation of PAR-1 and -2 on secretion of proinflammatory cytokines by corneal epithelial cells. Cell lysates and supernatants from the HCE-T cell line were collected after incubation with thrombin or trypsin for 24 hours, and cytokine levels were measured by ELISA. To further determine the specificity of this response, we also assessed the ability of the human PAR-1-activating peptide TFLLRN-NH₂ and the PAR-2-activating peptide SLIGRL-NH₂ to induce the secretion of these cytokines (Figures 5 6 7) . Our results indicate that there was a significant increase in production of IL-6 in HCE-T cells treated with 10 nM thrombin (PAR-1 activator) and 10 nM trypsin (PAR-2 activator) compared with untreated cells (Fig. 5) . Cells treated with TNFα served as a positive control. Similarly, treatment of the HCE-T cells with 100 mM TFLLRN-NH₂ (PAR-1 activator) or 100 mM SLIGRL-NH₂ (PAR-2 activator) also resulted in a significant increase in production of IL-6 protein in supernatants (Fig. 5A) and cell lysates (Fig. 5B) . 

The effect of activation of PAR-1 and -2 on HCE-T cell secretion of IL-8 was also examined. Significantly increased secretion of IL-8 was detected in supernatants after treatment with thrombin or with the PAR-1-activating peptide TFLLRN-NH₂ and with trypsin or the PAR-2-activating peptide SLIGRL-NH₂ (Fig. 6A) _. TNFα again served as a positive control for these studies. For further confirmation of the specificity of the PAR-2 response, HCE-T cells were treated with the reverse sequence of the PAR-2 tethered ligand peptide LRGILS-NH₂, and there was no increase in IL-8 secretion after treatment with this inactive PAR-2 receptor ligand (Fig. 6B) . 

Supernatants from HCE-T cells also contained significantly enhanced levels of TNFα after treatment with thrombin, the PAR-1-activating peptide TFLLRN-NH₂, trypsin, or the PAR-2-activating peptide SLIGRL-NH₂ (Fig. 7) . Cells were treated with PMA as a positive control in this experiment. Thus, our studies demonstrated that the specific activation of PAR-1 and -2 resulted in increased production of IL-6, IL-8, and TNFα by HCECs. 

Discussion

PAR-1 and -2 represent a novel family of proteinase-activated G protein-coupled receptors, which are characterized by autoactivation by a tethered peptide ligand. ³⁵  

PAR-1 and -2 have been detected in a variety of cell lines and tissues, ^{3 14 16} but little is known about the localization of PAR-1 and -2 in ocular tissues. To date, only bovine corneal endothelial cells have been reported to possess binding sites for thrombin and trypsin. ^{22 24} In this report PAR-1 and -2 mRNAs were expressed in primary HCECs and in the HCE-T cell line. Our studies also immunolocalized PAR-1 and -2 to the apical surface of the superficial corneal epithelial cells. Furthermore, the functionality of corneal epithelial cell PAR-1 and -2 was demonstrated by intracellular [Ca²⁺] mobilization in response to thrombin, TFLLR-NH₂ (a selective agonist of PAR-1), trypsin, and SLIGRL-NH₂ (a tethered ligand of rat PAR-2). An increase of intracellular [Ca²⁺] after activation of PAR-1 and -2 has also been demonstrated in human keratinocytes, fibroblasts, and guinea pig myenteric neurons. ^{7 14 25 36}  

PAR-1 mediates platelet aggregation, ³⁷ increased vascular permeability, extravasation of plasma proteins, ¹⁰ stimulation of chemoattractants, ¹¹ and activation of T cells. ¹² PAR-1- activates events associated with inflammation, tissue remodeling, and wound healing and also stimulates the production of proinflammatory cytokines in various cell lines. ^{38 39 40} Our studies indicate that thrombin was effective in stimulating expression of IL-6, IL-8, and TNFα mRNAs in HCECs. In addition, ELISA studies demonstrated that protein levels of these cytokines were significantly increased after activation of PAR-1. In these experiments, thrombin induced higher protein levels in comparison with the PAR-1-activating peptide, suggesting that activation of PAR-1 may not only account for all thrombin-induced effects. Because it is known that PAR-3 and -4 also mediate the effects of thrombin, ^{5 41} it is possible that activation of PAR-3 and -4 contributes to the thrombin-induced upregulation of these proinflammatory cytokines in HCE-T cells. This possibility is further supported by the finding that PAR-3 is expressed by HCE-T cells (Lang et al., unpublished observation, 2002). 

Like PAR-1, PAR-2 may be an important mediator in inflammation and cell growth. PAR-2 activation increases leukocyte adhesion, extravasation, and migration ¹⁷ and stimulates secretion of IL-6 and -8 by keratinocytes. ^{18 42} In the present study, trypsin also induced increased expression of the mRNAs of the proinflammatory cytokines IL-6, IL-8, and TNFα by HCE-T cells. Moreover, the activation of PAR-2 by trypsin and the PAR-2-activating peptide also resulted in increased production of these cytokines in HCE-T cells. The reduced cytokine production in HCE-T cells in response to the PAR-2-activating peptide in comparison with trypsin may indicate the involvement of other receptors such as PAR-4, which can also be activated by trypsin. ⁶ Our data demonstrating the induction of the proinflammatory cytokines IL-6, IL-8, and TNFα in HCECs by activation of PAR-1 and -2 strongly support the capacity of these receptors to participate in corneal inflammatory responses. 

The corneal epithelium provides a barrier between the external environment and the underlying structures of the eye and is frequently injured through physical or chemical insult, necessitating subsequent wound repair. The wound repair process may involve the local release of serine proteases such as thrombin and trypsin—for example, Jegorowa et al. ⁴³ found increased activity of trypsin-like proteases in corneal burns. Our results indicate that activation of corneal epithelial PAR-1 and -2 increased the production of IL-8, which has been shown to induce corneal neovascularization, ⁴⁴ and suggest that PAR-1 and -2 may also be involved in corneal inflammation and wound healing. In addition, PAR-1 and -2 agonists are mitogenic for many cell types, including endothelial cells. ¹³ Serine proteinase inhibitors, which are present in the cornea ⁴⁵ and other parts of the eye, ^{46 47} have been found to inhibit the migration of corneal epithelial cells. ²¹ Further studies demonstrating binding sites for thrombin and trypsin in cultured bovine corneal endothelial cells ^{22 23 24} also indicate that these serine proteases seem to play a role in inflammation and wound healing in the cornea. 

Although trypsin appears to be the main activator of PAR-2 in many systems, trypsin-like serine protease ⁴⁸ and mast cell-tryptase, which has been shown to activate PAR-2 in several cell types, ^{4 14} are present in human tears and may be activators of PAR-2 in the cornea. PAR-2 may serve as a modulator in the inflammatory cascade in allergic eye diseases, where increased mast cell densities in the conjunctiva and significant increase in tear tryptase levels have been found. ^{49 50}  

Our findings suggest that corneal PARs may represent a novel cellular activation pathway that could be particularly important in the regulation of inflammation and wound healing in the cornea. The clinical relevance of our findings is still to be determined, but further evaluation of these receptors could lead to new strategies to modulate inflammatory responses in the cornea. 

 Supported by National Institutes of Health Grants EY09218 (JCA), EY12304 (RML) and a grant from the Dermatology Foundation (PIS).

 Submitted for publication April 9, 2002; revised July 15, 2002; accepted August 9, 2002.

 Commercial relationships policy: N.

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Figure 1.

 

Detection of PAR-1 and -2 mRNAs by RT-PCR in cultures of primary HCEC-3 (A) and HCE-T (B) cells. Reverse transcribed mRNA was amplified by PCR with primers specific for human PAR-1 (864 bp) and -2 (865 bp). The amplified gene products were partially sequenced by automated DNA sequencer. The nucleotide sequence of PAR-1 and -2 are presented in (C) and (D), respectively.

Figure 2.

 

Immunolocalization of PAR-1 and -2 in whole human cornea. Immunohistochemistry was performed on human corneal sections by using monoclonal mouse anti-human PAR-1 antibody followed by a goat anti-mouse IgG labeled with Texas red as a secondary antibody (A). For PAR-2 staining, the sections were incubated with a polyclonal rabbit anti-rat PAR-2 antibody followed by a goat anti-rabbit IgG labeled with fluorescein (C). The epithelium and the stroma are shown. For negative controls for PAR-1 (B) and -2 (D) the primary antibody was omitted. Magnification, ×400.

Figure 3.

 

Induction of intracellular [Ca²⁺] in HCECs by activation of PAR-1 and -2. Cultured HCEC-3 cells were loaded with fura-2 and stimulated with thrombin (A), the human PAR-1 peptide agonist TFLLRN-NH₂ (PAR-1-AP) (B), trypsin (C), and the murine PAR-2 peptide agonist SLIGRL-NH₂ (PAR-2-AP) (D). Each line is a trace from a single cell. Results are representative of those obtained in three such experiments.

Figure 4.

 

Induction of intracellular [Ca²⁺] in HCE-T by activation of PAR-1 and -2. Cultured HCE-T cells were loaded with fura-2 and exposed to thrombin (A), the human PAR-1 peptide agonist TFLLRN-NH₂ (PAR-1-AP) (B), trypsin (C), and the murine PAR-2 peptide agonist SLIGRL-NH₂ (PAR-2-AP) (D). Each line is a trace from a single cell. Results are representative of those obtained in three such experiments.

Table 1.

 

Induction of Corneal Epithelial Cell Expression of IL-6, IL-8, and TNFα mRNA by Thrombin and Trypsin

	IL-6	IL-8	TNFα
Thrombin (10 nM)	11.3	13.0	7.5
Trypsin (10 nM)	8.0	7.5	8.6

Figure 5.

 

Induction of corneal epithelial cell secretion of IL-6 by activation of PAR-1 and -2. IL-6 protein levels were determined by ELISA in supernatants (A) and cell lysates (B) from HCE-T cells, either untreated or stimulated with thrombin, the human PAR-1 peptide agonist TFLLRN-NH₂ (PAR-1-AP), trypsin, or the murine PAR-2 peptide agonist SLIGRL-NH₂ (PAR-2-AP) for 24 hours. TNFα served as the positive control. Negative control (Co) was from cultured cells with normal medium. Data represent mean ± SEM of triplicates of a representative experiment. *P < 0.05 compared with untreated cells.

Figure 6.

 

Induction of corneal epithelial cell secretion of IL-8 by activation of PAR-1 and -2. IL-8 protein levels were determined by ELISA in supernatants from HCE-T cells, either untreated or stimulated with thrombin, the human PAR-1 peptide agonist TFLLRN-NH₂ (PAR-1-AP), trypsin, or the murine PAR-2 peptide agonist SLIGRL-NH₂ (PAR-2-AP) for 24 hours. TNFα served as the positive control. (A). Exposure to the reverse sequence of the tethered ligand peptide LRGILS-NH₂ had no effect on the secretion of IL-8 protein (B). Negative control (Co) was from cultured cells with normal medium. Data represent mean ± SEM of triplicates of a representative experiment. *P < 0.05 compared with untreated cells.

Figure 7.

 

Induction of corneal epithelial cell secretion of TNFα by activation of PAR-1 and -2. TNFα protein levels were determined by ELISA in supernatants from HCE-T cells, either untreated or stimulated with thrombin, the human PAR-1 peptide agonist TFLLRN-NH₂ (PAR-1-AP), trypsin, or the murine PAR-2 peptide agonist SLIGRL-NH₂ (PAR-2-AP) for 24 hours. PMA served as the positive control. Negative control (Co) is from cultured cells with normal medium. Data represent mean ± SEM of triplicates of a representative experiment. *P < 0.05 compared with untreated cells.

The authors thank Dale Geroski and Henry Edelhauser (Department of Ophthalmology, Emory University) in intracellular calcium studies and providing human corneas.