|Molecular Vision 2005;
Received 2 October 2004 | Accepted 23 February 2005 | Published 10 March 2005
Chemokine receptor gene expression in giant papillae of atopic keratoconjunctivitis
Nobuyuki Ebihara,2 Seiichi Yokoo,1 Shiro Amano3
Departments of 1Corneal Tissue Regeneration and 3Ophthalmology, Tokyo University Graduate School of Medicine, Tokyo, Japan; 2Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan
Correspondence to: Satoru Yamagami, MD, PhD, Department of Corneal Tissue Regeneration, Tokyo University Graduate School of Medicine, Bunkyo-ku, Tokyo, 113-8655, Japan; Phone: +81-3-5800-8660; FAX: +81-3-3817-0798; email: firstname.lastname@example.org
Purpose: Major chemokine receptors in the giant papillae of atopic keratoconjunctivitis (AKC) have yet to be determined. We evaluated chemokine receptor genes and their ligand expressions in upper tarsal conjunctival giant papillae of AKC with atopic dermatitis and/or asthma.
Methods: CC, CXC, and CX3C chemokine receptor (R) gene expression levels in giant papillae of five clinically active AKC patients and in three age matched non-allergic control conjunctiva were measured with a multi-probe ribonuclease protection assay (RPA) system. The ligands of abundant chemokine receptors in the giant papillae were examined by immunohistochemistry or reverse transcription-polymerase chain reaction. Interleukin (IL)-4 and IL-13 gene expression levels were measured with RPA. Ligand expression in cultured human conjunctival fibroblasts was examined by reverse transcription-polymerase chain reaction.
Results: High CXCR4 and CCR4 gene expression levels were detected in the giant papillae of all (CXCR4) and four out of five (CCR4) patients. As a CCR4-ligand, thymus and activation regulated cytokine (TARC/CCL17) rather than macrophage derived chemokine (MDC/CCL22), was predominant immunohistochemicaly in the giant papillae. Giant papillae with high CCR4 gene expression levels showed high IL-4 and IL-13 expression. Cultured human conjunctival fibroblasts express stromal cell derived factor-1 (SDF-1/CXCL12) in vitro.
Conclusions: CXCR4 and CCR4 are the major chemokine receptor genes expressed in the giant papillae of AKC with atopic dermatitis and/or asthma. Our findings suggest a role for CXCR4 and CCR4 in the formation of giant papillae.
Atopic keratoconjunctivitis (AKC) is characterized by chronic allergic inflammation of the conjunctiva accompanied by ocular itching and conjunctival hyperemia. Histopathologically, cells that infiltrate the conjunctiva are eosinophils, T-and B-lymphocytes, mast cells, macrophages, basophils, plasma cells, and dendritic cells . Vernal keratoconjunctivitis (VKC) and AKC patients have many elevated giant papillae, a gelatinous conjunctival elevation in the superior tarsal conjunctiva . Clinically, the giant papillae and chemical mediators often produce corneal epithelial lesions subsequent to the conjunctival allergic reaction.
Chemokines are low molecular weight proteins critical to immune and inflammatory responses by their regulating leukocyte trafficking in a multistep process [3,4]. Chemokines are divided into families based on the relative positions of their cysteine residues . The CXC (α) chemokine family has the first two cysteines separated by one amino acid residue. In the CC (β) chemokine group, the first two cysteine residues are adjacent to each other. Two other subfamilies, the C (XCL1, lymphotactin) and CX3C (CX3CL1, fractalkine) families, have only single members [5,6]. Chemokines form a complex functional network locally and systemically in a variety of inflammatory, infectious, and immune diseases [4,7]. In allergic diseases that T helper 2 cytokine are predominant, CCR3 and CCR4 are candidate chemokine receptors relevant to pathophysiology. In allergic conjunctivitis, CXCR3 is reported to be the predominant chemokine receptor in limbal form VKC . Eotaxin and its receptor, CCR3, were detected in tear and mucus of AKC and VKC patients [9-11]. Major chemokine receptors in the giant papillae of AKC have yet to be determined by systemic comparison.
We evaluated CC, CXC, and CX3C chemokine receptor mRNA expression in the giant papillae of severe AKC patients. CXCR4 and CCR4 were the major chemokine receptors in the giant papillae, suggesting that they may play a role in the formation of tarsal conjunctival giant papillae.
All the experiments followed the guidelines of the Declaration of Helsinki. The Institutional Review Board of the University of Tokyo School of Medicine approved the study. Giant papillae were surgically resected in cases where patients suffered from severe corneal damage and profound itching in spite of steroid or non-steroid treatment, and five patients with giant papillae of AKC were studied. After obtaining informed consent of the patients or persons with parental authority, giant papillae were resected for treatment, as described elsewhere , of five patients (Table 1). Normal conjunctivae were obtained during strabismus surgery from three non-allergic normal control patients (12, 16, and 32 years old) after informed consent was obtained. Papillae from each patient were homogenized separately in Isogen® (Nippon Gene, Tokyo, Japan) then quick frozen in OCT compound (Miles, Elkhart, IN) with liquid nitrogen. Normal conjunctivae used for total RNA extraction were processed as a group because of the small amount of total RNA available per sample. All samples were stored at -70 °C until further examination.
RNA preparation and ribonuclease protection assay
After centrifugation to remove cellular debris, total RNA was isolated with Isogen® according to the manufacturer's instructions, and the RNA pellet was resuspended in nuclease free water. Detection and quantification of chemokine receptor mRNAs were carried out with a multiprobe ribonuclease protection assay (RPA) system (BD PharMingen, San Diego, CA) as described previously . Briefly, a mixture of [α-32P] UTP labeled antisense riboprobes was generated from the chemokine receptor template set hCR-5, hCR-6, and hCK-1 (BD PharMingen). Total RNA (5 μg) was used in each sample. Total RNA was hybridized overnight at 56 °C with 300 pg of the 32P-anti-sense riboprobe mixture. After ethanol purification of the nuclease protected RNA fragments, samples were separated on 5% polyacrylamide sequencing gels, and the gels dried and subjected to autoradiography. Protected bands were observed after exposure of the gels to x-ray film. Specific bands were identified by their distinctive migration patterns as compared to patterns of the undigested probes. Densitometric analysis (NIH Image, version 1.63) was used for quantitation. Bands were normalized to the liposome 32 (L32) housekeeping gene. Two sets of results on separate experiments were analyzed, and averaged density was shown as the results of densitometric analysis.
The antibody (Ab) used for immunostaining was mouse anti-human thymus and activation regulated cytokine (TARC/CCL17) Ab (54026.11; R&D Systems, Minneapolis, MN), goat anti-CCR4 polyclonal antibody (C-20; Santa Cruz biotechnology, Inc., Santa Cruz, CA), mouse anti-human macrophage derived chemokine (MDC/CCL22) Ab (57226.11; R&D Systems), anti-CXCR4 Ab (12G5; R&D Systems), and anti-stromal cell derived factor (SDF)-1/CXCL12 Ab (79018.111; R&D Systems). Non-immunized mouse IgG and goat serum were negative controls. Frozen specimens were cut into 7 μm sections with a cryostat, air dried, fixed in cold acetone 10 min, then washed with phosphate buffered saline. The sections were blocked with 3% bovine serum albumin. Primary Ab was added, and the slides kept for 30 min at room temperature. After three washes in phosphate buffered saline, they were incubated for 30 min with biotinylated goat anti-mouse Ab (Dako Cytomations, Carpinteria, CA) and exposed to horseradish peroxidase labeled streptavidin for 20 min. Fluorescein isothiocyanate conjugated bovine anti-goat Ab (Santa Cruz) was used for CCR4 Ab. The sections were incubated for 2 min in 3,3-diaminobenzidine, then stained with Mayer's hematoxylin for 10 s. The specimens for CCR4 staining were analyzed by a fluorescent microscope (model BH2-RFL-T3 and BX50; Olympus, Tokyo, Japan).
The resected giant papillae were fixed in 10% formalin and then stained with hematoxylin and eosin (HE) for light microscopy.
First-strand cDNA was synthesized with a Reverse Transcription System® (Promega Corporation, Tokyo, Japan). cDNA was constructed from the total RNA. PCR amplification was done in a Gene Amp PCR System 2400® (Applied Biosystems, Foster City, CA) with Ampli Taq Gold® (Roche Molecular Systems, Branchburg, NJ) in 50 μl of the reaction mixture. The primer sequences for stromal cell derived factor-1 (SDF-1/CXCL12), TARC/CCL17, and glyceraldehyde phosphate dehydrogenase (GAPDH) are shown in Table 2. The PCR primer pairs was selected to discriminate between cDNA and genomic DNA by using primers specific for different exons. After incubation at 95 °C for 9 min, amplification was performed at 94 °C for 30 s, then at 60 °C for 30 s. Samples were separated in a 2% agarose gel, and the products made visible with ethidium bromide. An optical scanner was used to determine the densities of the gel bands of the PCR products and to normalize them to those of GAPDH. The linear amplification curve of the PCR product of each sample was examined after every fourth cycle (for example, band densities of 24 cycles, 28 cycles, and 32 cycles were examined). Four sets of PCR products were prepared under appropriate cycling conditions within the linear range of amplification, and the band densities of the AKC and control conjunctivae compared.
Cell culture and cytokine treatment
Normal conjunctival tenon capsules containing fibroblasts, obtained from non-allergic normal patients during strabismus surgery, were used for primary culture of human conjunctival fibroblasts (HCFs). These fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). Third passage conjunctival fibroblasts (4x105) were transferred to 0.5 ml serum free DMEM in 24 well plates and kept for 3 days, then treated for 24 h. Supernatants from each well were stored separately at -70 °C until used in the enzyme linked immunosorbent assay (ELISA). SDF-1/CXCL12 and TARC/CCL17 concentrations in the culture supernatants were measured with an ELISA kit according to the manufacturer's protocol (R&D Systems). HCFs remaining in the wells were exposed to trypsin-ethylene diamine tetraacetic acid, and the average number present was determined with a hematocytometer. Protein concentration in the culture medium was normalized as nanograms of protein per 106 cells. Plates were read with a Microplate Reader (Molecular Bioscience Group, Hercules, CA) at the optical densities of 450 nm for measurement and 550 nm for reference.
One way analysis of variance (ANOVA) and Fisher's protected least significant difference was used to compare the band densities in the RT-PCR. The α level was set at 0.05. The analysis was done with the Stat View statistical software package (Statview, version 5; Abacus Concepts, Berkeley, CA).
CC, CXC, and CX3C chemokine receptor gene expression in upper tarsal conjunctiva
Figure 1 shows the representative RPA autoradiographs (Figure 1A,B) and the amounts of chemokine receptor mRNA normalized to housekeeping gene L32. Averaged density of two sets of results was shown in Figure 1C,D. The CCR1, CCR2, and CCR4 genes are barely detectable in normal conjunctivae. The AKC, but not the normal, conjunctivae show appreciable CCR3 gene expression. Overexpression of CCR4 gene is present in four of five upper tarsal giant papillae as compared with the normal control conjunctivae. In contrast, patient 5 does not show a significant amount of CCR4 gene expression. In CXC and CX3C chemokine receptor, the CXCR1, CXCR2, CXCR4, and CX3CR genes are detected in the normal conjunctivae. There are no appreciable differences in CXCR1, CXCR3, or CX3CR1 between the normal and allergic conjunctivae. CXCR2 gene expression level in normal conjunctivae is higher than those in giant papillae of AKC patients, suggesting a comparatively high expression of neutrophil related chemokines/chemokine receptors in the normal conjunctivae [14-16]. The AKC, but not the normal, conjunctivae show appreciable CXCR5 gene expression. A significant high amount of CXCR4 gene is present in all five conjunctivae as compared with the normal control conjunctivae.
Immunohistochemical study of CCR4 ligands, TARC/CCL17 and MDC/CCL22 in conjunctivae
TARC/CCL17 and MDC/CCL22 expression were examined immunohistochemically in three AKC conjunctivae (patients 1, 2, and 4) and three normal ones. No positive staining was detected in the conjunctival epithelium of giant papillae. In the AKC conjunctivae, many TARC/CCL17 positive stainings are present in the subepithelial area, in the representative photograph of TARC/CCL17 positive stains in Figure 2A (Patient 1). In contrast, MDC/CCL22 positive staining was present, but not as much as compared with the number of TARC/CCL17 positive staining cells (Figure 2B). No positive staining was detected in the normal conjunctivae with anti-TARC/CCL17 and MDC/CCL22 Abs (data not shown). The AKC conjunctivae were not stained with non-immunized control IgG (Figure 2C). Similar findings were detected in patients 1 and 4 (data not shown).
IL-4 and IL-13 gene expression in giant papillae
To examine the correlation between helper T2 (Th2) cytokine and CCR4 gene expression, IL-4 and IL-13 gene expression was measured with the RPA method. Figure 3 shows a representative autoradiograph and the averaged amounts of chemokine receptor mRNA of two sets of results normalized to housekeeping gene L32. IL-4 gene expression was upregulated in high CCR4 gene giant papillae (patients 1-4) as compared with the normal conjunctivae, whereas IL-4 gene expression level was low in giant papillae of patient 5 with no CCR4 gene expression. A high amount of IL-13 gene expression as compared with the normal conjunctivae was detected in high CCR4 gene expressing giant papillae (patients 1-4), but not in the giant papillae of patient 5.
HE staining in giant papillae of AKC patients 1 and 5
HE staining in patient 1 shows infiltrations of mononuclear cells and eosinophils are predominant (Figure 4A). Focal fibroblast-like cell infiltrating areas are also detected in giant papillae of patient 1. Similar findings are observed in patients 2 and 3 (data not shown). Marked fibroblast-like cells are infiltrating in giant papillae of patient 5 (Figure 4B). Some fibroblast-like cells are present under normal conjunctival epithelium (Figure 4C).
SDF-1/CXCL12 gene expression in conjunctivae
The CXCR4 ligand's SDF-1/CXCL12 gene expression level was measured by semi-quantitative RT-PCR, because commercially available antibody did not work immunohistochemically. The gene was present in both normal and patient conjunctivae. A representative photograph of 4 sets of data and the average density within the linear range of amplification obtained by NIH Image are shown in Figure 5. Significantly higher amounts of SDF-1/CXCL12 are present in all the conjunctivae from the patients as compared to the normal conjunctivae (p<0.01).
TARC/CCL17 and SDF-1/CXCL12 expression in cultured human conjunctival fibroblasts
To examine the possibility that normal HCFs in addition to infiltrating cells expresses the TARC/CCL17 and SDF-1/CXCL12, we tested whether cultured HCFs can express TARC/CCL17 and SDF-1/CXCL12. The TARC/CCL17 (38 cycles) and SDF-1/CXCL12 (30 cycles) genes were expressed in cultured HCFs (Figure 6A). Then cultured HCFs were stimulated with recombinant IFN-γ, IL-4, IL-13, TNF-α, or the vehicle (control group). As shown in Figure 6B, cultured HCFs produced SDF-1/CXCL12 in the serum free condition (control group). In comparison with the control samples, a significantly large amount of SDF-1/CXCL12 was produced in HCFs stimulated with recombinant IFN-γ, but not with IL-4, IL-13, or TNF-α. The high SDF-1/CXCL12 concentration produced with IFN-γ was decreased in the presence of the same amount of recombinant TNF-α. In ELISA, TARC/CCL17 expression was undetectable in HCFs stimulated with recombinant IFN-γ, IL-4, IL-13, TNF-α, or vehicle (data not shown).
We used a multiprobe RPA system to quantify a panel of CC, CXC, and CX3C chemokine receptor mRNA from individual samples (five samples five results) of the total RNA of giant papillae from five AKC patients. This RPA method allows the comparative analysis of different mRNA species in an mRNA sample. All five patients had corneal damage and giant papillae with atopic dermatitis, asthma, or both. Although the patient numbers were not satisfactory for comprehensive analysis, identification of common abundant chemokine receptor genes can become a clue to reveal the pathogenesis of giant papillae formation in allergic conjunctival diseases.
Generally, Th2 cytokines and some chemoattractants play a role in the pathogenesis of allergic inflammation. The effects of Th2 cytokines, such as IL-4, IL-5, IL-9, and IL-13, reflect the pathophysiological manifestations of allergy and asthma. Moreover, both Th2 cells and the effector cells (basophils, mast cells, and eosinophils) express chemoattractant receptors such as CCR3, CCR4, and CCR8 . Therefore, interactions of eotaxin(s), eotaxin/CCL11, RANTES/CCL5, and MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, and MCP-4/CCL13 with CCR3 are responsible for the recruitment of basophils, eosinophils, and mast cells, whereas interactions of CCR4 with MDC/CCL22 or TARC/CCL17, and CCR8 with I-309/CCL1 have a role in the allergen induced recruitment of Th2 cells in the target tissues of allergic inflammation [18,19]. Our findings that an interaction of CCR4 with TARC/CCL17 rather than MDC/CCL22 among possible interactions of chemokine-chemokine receptors is predominant suggests a critical role of CCR4 with TARC/CCL17 in Th2 cytokine and effector cell recruitment in giant papillae of allergic conjunctivitis with atopic dermatitis and asthma.
Giant papillae of a patient (patient 5) did not show overexpression of CCR4 gene and Th2 cytokines, IL-4 and IL-13 genes. As shown in HE staining (Figure 4B), fibroblast-like cell infiltrations were predominant in giant papillae, whereas numerous eosinophilic and mononuclear cells were mainly infiltrating in the giant papillae of the other patients as shown in representative HE staining. Systemically, patient 5 also has asthma and severe atopic dermatitis including facial skin and clinical finding of giant papillae was similar to those of other patients, suggesting a systemic Th2 cytokine dominant condition. In this patient, resection of giant papillae was performed on the chronic stage after the proliferating stage. Although Th2 cytokine should affect formation of giant papillae, it may depend on the chronic or acute stage or the difference of giant papillae formation etiology.
SDF-1/CXCL12 is a member of a family of chemokines and initially was characterized as a growth stimulating factor for B cell precursors [20,21]. It is a potent chemotactic factor for T and pre-B lymphocytes [22,23], plasma cells , and dendritic cells (DCs)  and is a co-stimulatory factor for CD4+ T-cell activation  and affects T-cell rolling and tight adhesion to activated endothelial cells . Moreover, SDF-1/CXCL12 and its specific receptor, CXCR4, are involved in tumorgenesis [28-30] and angiogenesis [31,32]. As a regulator of local inflammation in the rheumatoid arthritis synovium, CXCR4 and SD-1/CXCL12 accumulate CD4+ T cells within the inflamed synovium . These findings suggest that SDF-1/CXCL12 may recruit a large number of mature and immature CXCR4 positive leukocytes to the giant papillae and stimulate neovascularization in the giant papillae of tarsal conjunctivae. Moreover, the enlargement of papillae in tarsal conjunctivae is due to the combination of conjunctival thickening, subepithelial fibrosis, mucous metaplasia, neovascularization, and scarring . This tissue remodeling is involved in overproduction of the extracellular matrix promoted by T-helper 2 type cytokines and growth factors [34-36]. Although our data revealed that resident conjunctival fibroblasts express SDF-1/CXCL12, there is a possibility that the other infiltrating cells in giant papillae also express SDF-1/CXCL12. Massive induced CXCR4 positive inflammatory cell infiltration may enhance the formation of giant papillae by supplying an extracellular matrix, cytokines, and neovasculariztion.
CXCR4 is also expressed on eosinophils [37,38] and mast cells [39,40], critical components in allergic conjunctivitis . CXCR4 and SDF-1/CXCL12 are considered to be essential factors of allergic airway disease in the mouse [42,43] and humans . Human mast cells migrate across the vascular endothelium to tissues in response to SDF-1/CXCL12 . Neutralization of CXCR4 and SDF-1/CXCL12 reduced lung eosinophilia and airway hyper-responsiveness in the mouse model of allergic airway disease [42,43]. Although we did not directly identify which cells express CXCR4 in this study, the blockade of CXCR4 and SDF-1/CXCL12 interaction may offer candidate treatment for tarsal conjunctival giant papillae. Because corneal damage seen in allergic conjunctival disease is associated with the eosinophil infiltration of the conjunctiva and the release of various eosinophilic proteins , mast cells have critical roles in the pathogenesis of allergic conjunctivitis . The findings that eotaxin and its receptor CCR3 expression have been detected in tear and mucous of AKC patients [8-11] are not contradictory to our data because our findings show comparatively predominant expression of CCR4 and CXCR4 genes in the giant papillae.
It is important to explain a limitation of this study. We showed comparative data of chemokine receptor gene expression levels, but not protein expression of chemokine receptor because commercially available antibody did not work well for immunohistochemical studies. To our knowledge, protein expressions of IL-4 and IL-13 have not been detected yet in the giant papillae of chronic allergy diseases, although Th2 cytokines are considered to be associated with formation of giant papillae. Further definitive protein level studies such as western blotting method may be required in the analysis of chemokines and cytokines.
In summary, CCR4 and CXCR4 are the major chemokine receptor genes of giant papillae in tarsal conjunctivae and TARC/CCL17, rather than MDC/CCL22, is a predominant CCR4 ligand. Cultured HCFs express SDF-1/CXCL12. Our findings point to a role for CXCR4 and CCR4 in the pathogenesis of allergic conjunctival giant papillae.
This study was supported by grants in aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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