|Molecular Vision 2003;
Received 3 February 2003 | Accepted 26 March 2003 | Published 2 April 2003
Characterization and functionality of CXCR4 chemokine receptor and SDF-1 in human corneal fibroblasts
Tristan Bourcier,1,2 Tsouria Berbar,2 Sophie
Paquet,2 Nicolas Rondeau,1,2 Frédéric Thomas,1
Vincent Borderie,1 Laurent Laroche,1 William
Rostène,2 France Haour,2
1Cornea Bank, EFS AP-HP, EA 3123 Paris 6 University, Paris, France; 2INSERM U339, Saint-Antoine Hospital, Paris, France
Correspondence to: Alain Lombet, Ph.D., Laboratory of Remodelage Tissulaire et Fonctionnel: Signalisation et Physiopathologie, CNRS UMR 8078, Hôpital Marie Lannelongue, 133 Avenue de la Résistance, 92350 Le Plessis-Robinson, France; Phone: (33) 1 46315611; FAX: (33) 1 46304564; email: email@example.com
Purpose: The aim of this study was to investigate whether cultured human corneal fibroblasts express functional chemokine CXCR4 receptors on their cell surface and to determine the presence of its specific ligand, SDF-1 (CXCL12), in human corneal fibroblasts.
Methods: Human corneal fibroblast cultures were obtained using human donor corneas. CXCR4 receptors were characterized using binding studies and autoradiography with [125I]SDF-1. The functionality of CXCR4 receptors was assessed by intracellular calcium measurement using a dynamic imaging microscopy system. CXCR4 and SDF-1 mRNA were detected in human corneal fibroblasts using reverse transcriptase polymerase chain reaction (RT-PCR). The CXCR4 protein was detected by western blot analysis.
Results: [125I]SDF-1 specifically bound to cultured corneal fibroblasts with a KD value of 8.3±1.2 nM. The presence of CXCR4 was confirmed by autoradiography of the radioligand on slices of corneal stroma. SDF-1 induced a rapid and transient intracellular calcium increase in cultured corneal fibroblasts that was blocked by the specific antagonist bicyclam. Moreover, a 48 kDa protein was detected by western blot analysis of corneal fibroblast extracts, using a specific CXCR4 polyclonal antibody. RT-PCR showed the expression of both CXCR4 and SDF-1 mRNAs in human corneal fibroblasts.
Conclusions: These results indicate for the first time that cultured human corneal fibroblasts express the chemokine receptors CXCR4 and its ligand SDF-1. This latter might exert physiological effects on the cornea and could be involved in pathological conditions such as corneal angiogenesis.
The cornea is a transparent and avascular tissue that functions as the major refractive structure for the eye. It is also an essential anatomical and physiological barrier between the eye and the external environment. A wide variety of growth factors, cytokines, chemokines, and their receptors are synthesized by corneal epithelial cells, stromal keratocytes, and lachrymal glands and are found in tears . In addition to their maintenance functions in the healthy eye, these cells and molecules are involved in corneal wound healing and in inflammatory responses to various disorders such as traumatism, immunologic disease, or microbial infection .
Chemokines are 8 to 10 kDa proteins that are potent activators and chemoattractants for different leucocyte subpopulations and some non-hematopoietic cells (epithelial cells, fibroblasts, and endothelial cells, amongst others). More than 50 chemokines have been currently identified . The chemokine super-family is subdivided into four groups (CXC, CC, C3XC, and C) on the basis of the relative position of the two first cysteine residues in the mature protein .
Chemokines induce cell migration and activation by binding to specific G-protein coupled cell surface receptors, a group with 18 known members . Some chemokine receptors are also expressed on non-hematopoietic cells, including neurons, astrocytes, epithelial and endothelial cells. This suggests that the chemokine system has many other functions than leucocyte chemotaxis. Very little information is available on the expression and role of chemokines and their receptors in the cornea. Previous studies have suggested that corneal keratocytes may release chemokines and thereby contribute to the local accumulation of inflammatory cell in vernal keratoconjunctivitis [6-8]. Increased expression of chemokines of the CXC and CC families has been associated with corneal allograft rejection . Interleukin-8 may play a role in the development of subepithelial infiltrates in adenovirus keratitis  whereas MIP-2 is involved in leucocyte migration and tissue injury in HSV keratitis .
Recently, much attention has been paid to one particular member of the chemokine receptor family, named CXCR4. Current investigations show that CXCR4 is functionally expressed on several tissues and cell types , but has never been described in the cornea. Moreover, increasing evidence indicates that CXCR4 and its unique ligand stromal cell derived factor-1 (SDF-1 or CXCL12) play a key role in angiogenesis and could be involved in ocular neovascularization [12,13].
In this respect it was particularly important to investigate whether cultured human corneal fibroblasts express functional CXCR4 receptors and whether corneal fibroblasts are able to express SDF-1.
Human corneal fibroblast cultures
This study was carried out according to the tenets of the Helsinki Declaration. Primary human corneal fibroblast cultures were obtained using human donor corneas that were discarded before transplantation because of low endothelial cell counts. Stromal explants were obtained as previously described . Corneal fibroblasts were grown in culture medium that consisted of 1:1 mixture of TC199 and Ham F12 medium (Gibco Invitrogen, Cergy-Pontoise, France) with 10% fetal calf serum (Gibco), 20 μg/ml insulin (Sigma-Aldrich Chimie, Saint Quentin Fallavier, France), 20 μg/ml L-ascorbic acid (Sigma), 250 ng/ml heparin (Leo, Saint Quentin en Yvelines, France), 10 ng/ml FGF-1 (Sigma), 0.4 mg/ml chondroitin sulfate (Sigma), 2 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin. The medium was changed every 2 days. Cells were maintained at 37 °C in a water saturated atmosphere with 5% CO2.
Human recombinant SDF-1 was purchased from PeproTech (Tebu, Le Perrey en Yvelines, France). [125I]SDF-1 (Specific activity 2000 Ci/mmol) was obtained from Amersham Biosciences (Saclay, France). Bicyclam was a generous gift from N. Heveker and M. Alizon (Institut Cochin, Paris). RANTES, MCP-1, IL-8, MIP-1, IP-10, Eotaxin, TARC, and Fractalkine were purchased from R &D (Oxon, UK). Trasylol was obtained from Bayer Pharma (Puteaux, France).
Human corneal fibroblast binding assays
All experiments were carried out in triplicate using first, second and third passage human corneal fibroblasts obtained from different donors. The experiments were performed after a minimum of two days and the cells were cultured in culture medium containing FCS.
Binding studies were carried out according to Banisadr, et al. . Briefly, cells were grown in 24 well multiplates (5x104 cells/well). They were incubated with 10 pM [125I]SDF-1 (5x104 cpm/well) in the presence or absence of increasing concentrations of unlabeled SDF-1 or various chemokines, in 200 μl of binding buffer (MEM-1% BSA-20 mM Hepes-Tris, pH 7.4) at 20 °C for 2 h. Cells were then washed twice with cold phosphate buffer saline (PBS)-1% BSA and scraped in 1 ml NaOH (0.1 N). The cellular extract was then counted in a 1470 Wizard (Perkin Elmer Life Sciences SA, Orsay, France) gamma counter. IC50 values were determined using EBDA-LIGAND software .
Cornea section binding assays and autoradiography
Human donor corneas (from 55 to 74 year old donors) were obtained from the Cornea Bank, EFS AP-HP, Saint-Antoine Hospital, Paris, France. Autoradiography was performed on whole human corneas on which the epithelium and endothelium had been removed prior to autoradiography following SDF-1 binding. Transversal cornea sections (20 μm thick) were cut on a cryostat (model CM 3050, Leica Microsystèmes SA, Rueil-Malmaison, France) at -18 °C, mounted flat onto Superfrost-plus slides (Menzel-Glaser, Madison, WI), and kept at -20 °C until used. The slide mounted sections were dried with a cold air stream and treated as previously described . Binding experiments were achieved in 300 μl of MEM medium containing 33 pM (1.5x105 cpm/ml) [125I]SDF-1, 1% BSA, 250 KIU/ml Trasylol, and Tris-HCl (0.25 M), pH 7.4 (binding buffer) for 18 h at 4 °C. Additional sections were incubated in the presence of 20 nM unlabeled SDF-1 for determination of non-specific binding. Following incubations, slides were washed three times (10 min each) at 4 °C in 50 mM ice cold Tris-HCl buffer, pH 7.4, dipped quickly in ice cold distilled water and dried with a cold air stream. Sections were placed in X-ray cassettes, and exposed to a Phosphorscreen for 6 h. The Phosphorscreen was then scanned using the Phosphor Imager STORM (Molecular Dynamics, Sunnyvale, CA).
Cell extraction and immunoblotting
Cultured corneal fibroblasts were harvested, centrifuged (2,000 g, 7 min) and the pellet lysed using 0.6 ml of 1% deoxycholic acid at pH 11.3 . After denaturation in boiling water for 10 min, lysates were clarified by centrifugation (11,000 g, 20 min) and the supernatants were aliquoted and frozen. Protein concentration of the lysates were quantified using the bicinchoninic acid protein assay according to the manufacturer's instructions (Pierce, Interchim, France). For western blot analysis, 10-20 μg of cell lysate was boiled for 5 min in a denaturing sample buffer according to Laemmli  and electrophoresed on a 10% SDS-polyacrylamide gel before transfer to PVDF membrane Hybond-P (Amersham). After blocking the membrane with 5% non-fat milk in NaCl (150 mM), Tris-Cl (25 mM), 0.1% Tween 20, pH 8.1 for 30 min, antibody binding reactions were performed in the same buffer supplemented with 1% non-fat milk at 4 °C. Overnight incubations were carried out using primary rabbit polyclonal antibody against human CXCR4 (1:2,000; ABR, Interbiotech, Montluçon, France). Secondary goat anti-rabbit antibody coupled to horseradish peroxidase (1:20,000; Sigma Aldrich) was incubated at room temperature for 1 h. The reactivity was evaluated using the blotting western detection reagents ECL+PLUS (Amersham), visualized with Hyperfilm/ECL (Amersham) and quantified using a PhosphorImager and the Image Quant software (Molecular Dynamics). Relative levels of protein were normalized against standard actin protein, revealed using a rabbit polyclonal antibody against the C-terminal actin fragment (1:4,000; Sigma Aldrich).
Intracellular calcium measurements
The intracellular calcium concentration was measured in response to activation by CXCR4 and other chemokine receptors using a dynamic imaging microscopy system QuantiCell 700 (VisiTech international Ltd., UK) with 15-20 corneal fibroblasts per field as described earlier [15,19]. Cells were grown on glass coverslips (22 mm) for 4 days. They were then loaded with 4 mM Fura-2 AM (Molecular Probes, Interbiotech, France) in PBS supplemented with 1.3 mM CaCl2, 0.8 mM MgCl2 and 20 mM Hepes-Tris pH 7.4 at 37 °C for 1 h. After washing with this buffer, the cells were treated with either SDF-1 (10 nM), or with RANTES, MCP-1, IL-8, MIP-1, IP-10, Eotaxin, TARC, or Fractalkine (all at 100 nM). Fluorescence images were recorded every 2 s and intracellular calcium concentration was calculated from the ratio of the fluorescence intensities at 340 and 380 nm on a pixel basis.
Reverse transcription polymerase chain reaction (RT-PCR) analysis for CXCR4 and SDF-1
Total RNA extraction was performed on corneal fibroblasts taken in primary cultures, and on the first and second subcultures from different stromal explants. Total RNA was extracted by RNA Now using methods described by Chomczynski and Sacchi . Total RNA recovery was then measured by spectrophotometric absorbance at 260 nm.
Total RNA (5 μg) was reverse transcribed in a 30 μl reaction mixture containing 20 mM Tris-Cl pH 8.3, 50 mM KCl, 5 mM MgCl2, 10 mM DTT, 1 mM concentration of each dNTP, 0.2 pmoles oligo (dT)15, 1 U/ml RNAsin (Promega Corporation, Madison, WI) and 200 U of Moloney Murine Leukemia Virus Reverse Transcriptase (Gibco, USA) at 37 °C for 1 h. The PCR amplification was performed on 1:5 (vol/vol) of the RT reaction in a mixture containing 16 mM Tris-Cl pH 8.3, 40 mM KCl, 1.5 mM MgCl2, 0.2 mM dXTP, 10% DMSO, 0.2 mM concentration of each dNTP, 25 pmoles of the sense primer, 25 pmoles of the antisens primer, and 1 U of Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT) in a final volume of 50 μl. The PCR primers used were S-CXCR4: (5'-TTCTACCCCAATGACTTGTG-3') and AS-CXCR4: (5'-ATGTAGTAAGGCAGCCAACA-3') for CXCR4 , S-SDF-1: (5'-ATGAACGCCAAGGTCGTGGTC-3') and AS-SDF-1: (5'-TGGCTGTTGTGCTTACTTGTTT-3') for SDF-1 . S14 was chosen as the housekeeping gene using the following PCR primers, S-S14: (5'-GGCAGACCGAGATGAATCCTCA-3') and AS-S14: (5'-CAGGTCCAGGGGTCTTGGTCC-3') . PCR products for CXCR4, SDF-1 and S14 were 206, 305, and 144 bp, respectively.
The amplification profile was composed of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, and extension at 72 °C for 30 s (CXCR4) or 1 min (SDF-1). The 30 cycles (22 cycles for S14) were preceded by denaturation at 95 °C for 5 min and immediately followed by a final extension at 72 °C for 10 min. The amplification was performed in a DNA thermal cycler (Perkin-Elmer Cetus, Gene Amp PCR system 9700, Norwalk, CT). PCR samples (20 μl) were electrophoresed on 1% agarose gels in 90 mM Tris borate/2 mM ethylenediaminetetraacetic acid (EDTA) buffer. Gels were stained with ethidium bromide and photographed under an UV lamp (Polaroid 665 film).
A negative control was routinely used for all assays to confirm the absence of contamination. For these controls, RNA was omitted from the RT reaction mixture and the reverse transcription was carried out as described above. Human neuroblastoma cells SH-SY5Y expressing CXCR4 receptors were used as a positive control .
As shown in Figure 1, [125I]SDF-1 binds to its receptors with high affinity. IC50 values were 8.3±1.2 nM. Results represent the mean from a representative experiment performed in triplicate and normalized to 100% specific binding. The binding of SDF-1 was highly specific since various chemokines (RANTES, MCP-1, Figure 1) were unable to compete for the binding of [125I]SDF-1, up to 0.1 μM. Other chemokines such as IL-8, MIP-1, TARC, Fractalkine and Eotaxin tested at the same concentration were also unable to compete for the binding of [125I]SDF-1 (data not shown). Only IP-10 competed for 20% of SDF-1 binding at 0.1 μM.
Figure 2 demonstrates the autoradiographic localization of [125I]SDF-1 binding sites in human cornea sections. [125I]SDF-1 binding sites were highly present at the level of the corneal stroma (Figure 2A). As shown in Figure 2B, [125I]SDF-1 binding was specific since it was abolished in the presence of 20 nM unlabeled SDF-1.
The functionality of CXCR4 was assessed using calcium imaging to visualize the transduction pathway as shown in Figure 3. Various chemokines were tested in comparison to SDF-1 for their ability to induce intracellular calcium transients. When corneal fibroblasts were triggered with MIP-1 (Figure 3A), RANTES (Figure 3B), IL-8 (Figure 3C), MCP-1, TARC, Fractalkine, Eotaxin or IP-10 (data not shown), we did not observe any effect on intracellular calcium levels, even at 100 nM. Conversely, when SDF-1 was added after application of the various chemokines, a rapid and transient increase in the intracellular calcium concentration was observed after a few s. The addition of 1 μM of bicyclam (a non-peptidic CXCR4 antagonist , Figure 3D) before stimulation with SDF-1 totally inhibited the calcium response. Bicyclam alone had no effect.
The presence of SDF-1 and CXCR4 was also assessed by detection of specific protein and mRNA. A 48 kDa protein revealed by specific polyclonal antibody against human CXCR4 was detected by western blot analysis (Figure 4). When actin was used as a control, antibodies against actin showed a second band of 42 kDa (Figure 4).
Expression of SDF-1, CXCR4, and S14 (a housekeeping gene) mRNAs were detected in cultured human corneal fibroblasts. For SDF-1, CXCR4 and S14, a unique PCR product was detected with ethidium bromide after gel electrophoresis (Figure 5). The sizes of these bands were respectively 305, 206, and 144 bp and were consistent with the expected fragment size as determined from the human SDF-1, CXCR4 and S14 cDNA [21,22].
Chemokines are thought to be involved in many pathological conditions including corneal inflammation and wound healing . Until now, no characterization of chemokine receptors has been reported in human cornea. The gene of the IL-8 receptor (CXCR1/CXCR2) was the only gene shown to be upregulated in keratocytes following stimulation with pro-inflammatory cytokines . We thus investigated by functional analysis a wide range of chemokine receptors to test their presence in corneal fibroblasts.
We have thus demonstrated the presence of CXCR4 using a radioactive tracer in corneal tissue as well as in cultured corneal fibroblasts. The sensitivity of this technique allowed for a clear characterization of CXCR4 in spite of the relatively low level of mRNA after RT-PCR amplification. Western blotting also confirmed the expression of the CXCR4 protein in these cells. The low level of CXCR4 mRNA relative to high amounts of CXCR4 protein could be due to a low stability of the expressed mRNA. Conversely, the CXCR4 protein might possess good stability against proteolysis.
Chemokine receptors, like other members of the family of G-protein coupled receptors, are functionally linked to phospholipases through G proteins. Receptor activation leads to a cascade of cellular activation, including the generation of inositol triphosphate, the release of intracellular calcium, and the activation of protein kinase C. The intracellular calcium measurement experiments presented here demonstrate that CXCR4 receptors expressed in corneal fibroblasts are functional. In contrast, other chemokines tested in the same system did not induce intracellular calcium transients. Taken together, these results suggest that CXCR4 might be the most important chemokine receptor for corneal fibroblast functions.
On the other hand, a number of chemokines can be expressed or secreted by corneal keratocytes [6-11] but to our knowledge SDF-1, the only ligand reported so far for CXCR4 , has never been reported in these cells. RT-PCR analysis demonstrated not only the expression of CXCR4 receptors but also of its ligand SDF-1 in human corneal fibroblasts. Therefore this chemokine/receptor system appears to be of particular interest for physiological and pathological functions in the cornea. Although the present results show that corneal fibroblasts are capable of expressing SDF-1 under certain culture conditions (when transformed into corneal fibroblasts, for instance), further investigations are required to determine in vivo or ex vivo if normal quiescent or activated keratocytes similarly express SDF-1.
Current investigations show that CXCR4 is expressed on a multitude of tissues and cell types, including different leucocyte subsets, hematopoietic progenitor cells, endothelial cells [27-30], epithelial cells [21,30,31], and human microglia and neurons . We can thus hypothesize that CXCR4/SDF-1 might be involved in the recruitment of inflammatory or vascular endothelial cells to sites of corneal injury.
The contribution of CXCR4 and SDF-1 to angiogenesis is currently a focus of intensive investigation. In the eye, Crane, et al., have shown SDF-1 and CXCR4 expression by retinal pigment epithelial (RPE) cells . The authors postulated that CXCR4/SDF-1 interactions might modulate the effects of chronic inflammation and subretinal neovascularization at the RPE site of the blood-retina barrier. Certain members of the CXC chemokine family can induce bovine capillary endothelial cell migration in vitro and corneal angiogenesis in vivo . The CXC chemokine subfamily includes interleukin (IL)-8, GRO, interferon inducible protein (IP)-10, MIG, PF4, and SDF-1 among others. Interestingly, the presence of an amino terminal proximal Glu-Leu-Arg (ELR) motif correlates with angiogenic chemokines, whereas CXC chemokines lacking this motif, such as PF4 and IP-10, are reported to be angiostatic . Although it lacks the ELR-motif, SDF-1 appears to stimulate both endothelial cell migration and angiogenesis [12,35]. In vivo, subcutaneous injection of SDF-1 into mice induced the formation of local small blood vessels, and SDF-1 stimulated endothelial vessel development in a rat aortic ring sprouting assay . SDF-1 also induced an angiogenic activity as shown in the model of the rabbit corneal pocket . Vascular endothelial cells are able to bind and respond to a number of CXC chemokines [34,36]. It has been shown that stimulation of human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (FGF-2) increased the levels of CXCR4 expression on HUVECs .
Taken together these data provide evidence that SDF-1 could act as a potent chemoattractant for endothelial cells and, like other CXC chemokines, participate in angiogenesis, presumably via CXCR4. The expression of this receptor might be regulated by growth factors or pro-inflammatory cytokines (such as TNF-α and IL-1α). We can thus hypothesize that SDF-1 produced by activated keratocytes might trigger the influx of inflammatory and vascular endothelial cells into the stroma and participate in the development of corneal neovascularization. In clear corneal tissue, the proangiogenic effect of SDF-1 could be controlled by an antiangiogenic compound such as Pigment Epithelium Derived Factor (PEDF) whose presence has been recently reported in human cornea . On the other hand, CXCR4 expressed on corneal fibroblasts could be involved in the control of cell proliferation, viability, or differentiation .
In conclusion, we have characterized the expression of CXCR4 in human corneal fibroblasts. These findings provide new insight into the activities of chemokine receptors on non-hematopoietic cells and indicate that these molecules have a more widespread cellular expression and perhaps a wider biological role than previously envisaged. Thus, SDF-1 might exert physiological effects on the cornea and could be involved in the control of corneal angiogenesis.
We thank Warwick Fifield for skilful reading of this manuscript. Supported by a grant from INSERM, Association Claude Bernard and MESR
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