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(Investigative Ophthalmology and Visual Science. 2003;44:514-520.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0498
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The Effects of Proinflammatory Cytokines on Cytokine-Chemokine Gene Expression Profiles in the Human Corneal Endothelium

Hiroko Yamagami,1 Satoru Yamagami,2 Taeko Inoki,3 Shiro Amano,4 and Kazunori Miyata5

1From the Department of Ophthalmology, Omiya Medical Center, Jichi Medical School, Saitama, Japan; the 2Departments of Corneal Tissue Regeneration and 4Ophthalmology, Tokyo University Graduate School of Medicine, Tokyo, Japan; the 3Department of Ophthalmology, Jichi Medical School, Tochigi, Japan; and 4Miyata Eye Hospital, Miyakonojo, Miyazaki, Japan.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the effects of proinflammatory cytokines on differential gene expression profiles in the human corneal endothelium (HCE), by using a cDNA expression array.

METHODS. A human cDNA expression array technology was used to study the simultaneous expression of HCE incubated with interleukin (IL)-1{alpha} and tumor necrosis factor-(TNF)-{alpha}. Gene-specific semiquantitative reverse transcription–polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were performed to examine the gene and protein expression patterns revealed by the cDNA expression array, in the presence and absence of proinflammatory cytokines. Moreover, the expression of these genes was studied in ex vivo HCE of donor cornea by RT-PCR.

RESULTS. IL-1{alpha} and TNF-{alpha} upregulated the expression of 46 of 268 genes for cytokines, chemokines, and their receptors in stimulated HCE. The most upregulated genes in the cDNA expression array, those of monocyte chemotactic protein (MCP)-1 (CCL2), IL-8 (CXCL8), IL-6, and growth-related ß (GROß, CXCL2), were studied. Semiquantitative RT-PCR and ELISA analyses revealed the proinflammatory cytokine-mediated changes in the respective gene transcription and protein expression levels. The mRNAs were detected in ex vivo HCE of donor cornea stimulated with proinflammatory cytokines.

CONCLUSIONS. HCE can abundantly express cytokines and chemokines through the stimulation of proinflammatory cytokines. The detected genes, those of CCL2, CXCL8, IL-6, and CXCL2, in HCE could facilitate understanding of the inflammatory responses, including the production of keratic precipitates and the correlation between CE and an inflamed cornea or aqueous humor.

The corneal endothelium (CE) is the posterior surface of the stroma, comprises a single layer of flat hexagonal cells, and lays on Descemet’s membrane. It faces the aqueous humor as a part of the inner side of the anterior chamber of the eye and permits the passage of nutrients from the aqueous humor to the cornea. The CE constitutively expresses various genes to maintain stromal dehydration, corneal transparency, metabolic activity, and signal transduction.1

In corneal diseases such as herpetic keratitis, endotheliitis, and corneal allograft rejection, inflammatory cells infiltrate the cornea, including the CE. Moreover, inflammation of the iris and ciliary body breaks down the blood–aqueous barrier in uveitis and endophthalmitis, and thus inflammatory cells in the aqueous humor affect the CE.2 In inflammation of both the cornea and aqueous humor, clinicians often observe keratic precipitates (KPs) adhering to the CE and focal corneal stromal edema caused by endothelial decompensation, indicating that the CE is closely associated with anterior segment inflammation of the eye.3 The coordinated changes of gene transcription levels in the CE, however, remain unknown in inflammatory conditions.

Interleukin (IL)-1 and tumor necrosis factor (TNF)-{alpha} are mediators of inflammatory reactions and are representative proinflammatory cytokines. The cornea synthesizes IL-1 and TNF-{alpha} in an inflamed condition.4 5 6 7 These proinflammatory cytokines also mediate the immune response in uveitis,8 9 10 herpetic stromal keratitis,11 12 and corneal allograft rejection.13 14 Therefore, these cytokines are critical factors that promote inflammation in the anterior segment of the eye.

In this study, we determined the effects of proinflammatory cytokines on differential gene expression profiles in the human CE (HCE) observed with a cDNA expression array. cDNA array technology allows the quantification of the simultaneous expression of many genes. This in turn has the obvious advantage of allowing the analysis of multiple clones and large-scale comparison of multiple nucleic acid sequences with a single hybridization. Our findings reveal important candidate genes in the HCE for understanding of inflammatory responses in the anterior segment of the eye.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Cytokine Treatment
Research involving human donor corneas was conducted according to the provisions of the Declaration of Helsinki. Primary HCE cells from normal donor corneas were cultured as described elsewhere.15 Fourth-passage HCE cells were transferred to 5 mL RPMI 1640 containing 1% fetal bovine serum in 35-mm culture dishes and then treated for 12 hours with 20 ng/mL each of human IL-1{alpha} and TNF-{alpha} (R&D Systems, Minneapolis, MN; IL-1{alpha}/TNF-{alpha} group) or the vehicle only (control group). Cells were used immediately after incubation with cytokines or the vehicle for isolation of RNA for gene array analysis or RT-PCR. The supernatants were stored at -70°C while awaiting use for enzyme-linked immunosorbent assay (ELISA).

Human Cytokine-Chemokine Receptor Gene Array
A human cDNA expression array (Atlas; Clontech, Palo Alto, CA), in which 268 known cytokine-chemokine receptor genes are represented, was used in these experiments. (A complete list of the 268 genes included in this human array is provided by Clontech at http://www.clontech.com). Total RNA was isolated with an RNA extraction reagent (RNA Stat 60; Tel-Test Inc.), according to the manufacturer’s instructions. After DNase treatment, an {alpha}-32P-labeled cDNA probe was synthesized from the total RNA according to the manufacturer’s protocol. Each cDNA probe was purified (NucleoSpin Extraction Spin column; Clontech). Incorporation of the label was assessed by scintillation counting. Equal counts per minute of the cDNA probe from the IL-1{alpha}/TNF-{alpha} or control group were hybridized in a hybridization solution (Express Hyb; Clontech) with the cytokine-chemokine receptor array membranes overnight at 68°C with continuous agitation. The arrays were washed in solution 1 (2x SSC, 1% SDS) and then in solution 2 (0.1x SSC, 0.5% SDS) at 68°C. The array membranes were exposed to x-ray film (BioMax MS; Eastman Kodak, Rochester, NY) at -70°C. The two sets of autoradiographic results were analyzed and compared on computer (Mac BAS 2000 ver. 2.4 software; Fujifilm, Tokyo, Japan). The average intensity differences among genes upregulated or downregulated in two hybridizations were calculated.

RNA Preparation and RT-PCR
Total RNA was isolated from cultured HCE and peeled off HCE from donor corneas (RNA Stat 60 reagent; Tel-Test), according to the manufacturer’s instructions. Water was used as a negative control. After DNase treatment, first-strand cDNA was synthesized with a reverse-transcription system (Promega Corp., Tokyo, Japan). cDNA was constructed from the total RNA. The PCR reaction mixtures comprised 1% cDNA, 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTPs, 20 pmol oligonucleotides, and 2.5 U Taq polymerase (AmpliTaq Gold; Applied Biosystems, Foster City, CA) in a 50-µL reaction volume. After incubation at 95°C for 9 minutes, amplification was performed at 94°C for 30 seconds and then at 60°C for 30 seconds. (Gene Amp PCR System 2400; Applied Biosystems, Foster City, CA). Samples were separated in a 2% agarose gel, and the products were visualized with ethidium bromide. An optical scanner was used to determine the density of the gel bands of the PCR products and to standardize them in comparison with those for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The linear amplified curve of the PCR product of each sample was examined at four-cycle intervals. Within the linear range of amplification, four sets of PCR products were prepared under appropriate cycling conditions, and the band densities were compared between the IL-1{alpha}/TNF-{alpha} and control groups.

ELISA of Supernatants of Cultured HCE, with or without IL-1{alpha} and TNF-{alpha} Stimulation
Passaged HCE cells were treated in 5 mL RPMI 1640 containing 1% fetal bovine serum, and 20 ng/mL each of human IL-1{alpha} and TNF-{alpha}, or the vehicle only in 35-mm culture dishes for 12, 24, and 48 hours. The supernatants were harvested and stored at -70°C until used for ELISA. The protein concentrations of monocyte chemotactic protein 1 (MCP-1, CCL2), IL-8 (CXCL8), and IL-6 in the culture supernatant were measured with an ELISA kit according to the manufacturer’s protocol (Biosource International, Camarillo, CA). The GROß (MIP-2{alpha}, CXCL2) concentration in the supernatant was measured by direct ELISA. Recombinant CXCL2 (Santa Cruz Biotechnology, Santa Cruz, CA) and diluted serum were incubated at 37°C overnight in a 96-well coating plate. Polyclonal goat anti-human CXCL2 (0.5 µg/mL; Santa Cruz Biotechnology) was applied for 1 hour as a detection antibody after blocking was performed with 3% bovine serum albumin at 37°C for 30 minutes. Biotin-conjugated anti-goat IgG antibody (1 µg/mL; Sigma-Aldrich, Tokyo, Japan) was reacted for 1 hour at room temperature. The plates were read with a microplate reader (Molecular Bioscience Group, Hercules, CA) at an optical density of 450 nm.

Proinflammatory Cytokine Treatment of Ex Vivo HCE of Donor Corneas
An eye bank cornea with the CE side up was placed on the upside-down cap of a 15 mL tube, and the epithelial cell side was immersed in preservative (Optisol GS; Chiron Vision, Irvine, CA). The HCE side was treated with the preservative, which contained both 20 ng/mL human IL-{alpha} and 20 ng/mL TNF-{alpha} at 37°C for 12 hours. After this treatment, the HCE was peeled off and used for mRNA extraction, DNase treatment, and subsequent RT-PCR. Water was used as a negative control.

Statistical Analysis
The Mann-Whitney test was used to compare the band densities on RT-PCR and the protein concentrations on ELISA. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
cDNA Expression Array
A series of experiments was performed twice with RNA isolated from two separate cultures of either normal or IL-1{alpha}/TNF-{alpha}–treated HCE (four hybridizations of four cDNA array membranes). The left-side duplicated lane of these membranes contains nine housekeeping controls, three negative controls, and four calibration markers from the top. No signals were observed for the negative control spots, indicating that the hybridization was highly specific (Fig. 1) . Forty-six of the tested cytokine, chemokine and receptor genes were upregulated, and 26 were downregulated in the IL-1{alpha}/TNF-{alpha}–treated HCE. The average positive and negative intensity differences greater than 7 (in arbitrary units) between the cytokine-treated and vehicle-treated cell–probed arrays are listed in Table 1 . The genes that appeared to be markedly upregulated were those of MCP-1 (CCL2), IL-8 (CXCL8), IL-6, GROß (MIP-2{alpha}, CXCL2), interferon regulatory factor-1, and IL-1ß. The most downregulated genes were those of CC chemokine receptor 2 and connective tissue growth factor. Of these genes, we further studied those of CCL2, CXCL8, IL-6, and CXCL2, which showed the highest numbers of intensity differences on densitometry analysis.



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  		FIGURE 1. Representative cytokine-chemokine gene arrays hybridized with labeled RNA probes isolated from cultured HCE exposed to the vehicle, as a control (A), and 20 ng/mL each of IL-1{alpha} and TNF-{alpha} (B) for 12 hours. No signals were visible for the negative control spots. The markedly upregulated genes were MCP-1 (CCL2; thick filled arrow), IL-8 (CXCL8; single thin arrow), IL-6 (double thin arrows), and GROß (MIP-2{alpha}, CXCL2; thick open arrow). Data are representative of two independent experiments.

 

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  		TABLE 1. Genes Upregulated or Downregulated on IL-1{alpha} and TNF-{alpha} Treatment

 
Semiquantitative RT-PCR of Cultured HCE
To confirm the results of gene array analysis, semiquantitative RT-PCR was performed. The oligonucleotide primers used are listed in Table 2 . GAPDH was detected on RT-PCR in the IL-1{alpha}/TNF-{alpha} and control groups. There was no significant difference in GAPDH between the two groups (Fig. 2) . CCL2, CXCL8, IL-6, and CXCL2 mRNAs were detected in the cultured HCE in both groups. After confirmation of linear amplification of the PCR products, the transcription levels of the genes were compared. Figure 2 shows that the gene transcription levels of CCL2, CXCL8, IL-6, and CXCL2 in the IL-1{alpha}/TNF-{alpha} group were significantly higher than those in the control group, consistent with the results of cDNA array analysis.


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  		TABLE 2. Oligonucleotide Primers for RT-PCR

 


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  		FIGURE 2. Semiquantitative gene transcription levels in the control and IL-1{alpha}/TNF-{alpha} groups. The linear amplified curve of the PCR product of each sample was examined at four-cycle intervals. PCR products were prepared under appropriate cycling conditions, and the band densities were compared. There was no significant difference in GAPDH between the groups. The gene transcription levels of CCL2, CXCL8, IL-6, and CXCL2 in the IL-1{alpha}/TNF-{alpha} group were significantly higher than those in the control group. The data represent the mean ± SD of four sets of PCR products. *P < 0.05.

 
Cytokine and Chemokine Protein Production
A HCE culture supernatant was harvested separately after 12, 24, or 48 hours and then subjected to ELISA. Figure 3 shows the CCL2, CXCL8, IL-6, and CXCL2 production results in the control and IL-1{alpha}/TNF-{alpha} groups. CCL2, CXCL8, and IL-6 production in the IL-1{alpha}/TNF-{alpha} group was significantly higher than that in the control group at 12, 24, and 48 hours, consistent with the results of cDNA array and RT-PCR analyses. The culture supernatants of HCE cells in the IL-1{alpha}/TNF-{alpha} group contained a significantly higher amount of CXCL2 than those in the control group at 24 and 48 hours. Production of CXCL2 in the control group was undetectable at 12 hours. This cytokine and chemokine production in the IL-1{alpha}/TNF-{alpha} group increased in a time-dependent manner.



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  		FIGURE 3. Protein concentrations in cultured HCE supernatants. Passaged HCE cells were treated in 5 mL RPMI 1640 containing 1% fetal bovine serum, and 20 ng/mL human IL-1{alpha} and TNF-{alpha}, or vehicle only, in 35-mm culture dishes for 12, 24, and 48 hours. ELISA was performed to determine the protein concentrations in the culture supernatants. The protein concentrations of CCL2, CXCL2, IL-6, and CXCL2 in the IL-1{alpha}/TNF-{alpha} group were significantly higher than those in the control group. CXCL2 production in the control group was undetectable at 12 hours. The results are the mean ± SD of quadruplicate determinations in a representative experiment. ** P < 0.05, * P < 0.02.

 
RT-PCR Analysis of Ex Vivo HCE
RT-PCR was performed to detect mRNA expression of CCL2, CXCL8, IL-6, and CXCL2 in ex vivo HCE of donor corneas stimulated with IL-1{alpha}/TNF-{alpha}. Two donor corneas were examined separately. GAPDH was detected in both cases, but not in a negative control sample. CCL2, CXCL8, IL-6, and CXCL2 mRNAs were all detected in ex vivo HCE of donor corneas under appropriate cycling conditions (Fig. 4) .



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  		FIGURE 4. mRNA expression in ex vivo donor HCE on proinflammatory cytokines stimulation. RT-PCR was performed to detect mRNA expression in ex vivo human corneal endothelium (HCE) of donor corneas stimulated with IL-1{alpha}/TNF-{alpha}. Two donor corneas were examined separately. Water was used as a negative control. GAPDH was detected in both cases, but not in a negative control sample. CCL2, CXCL8, IL-6, and CXCL2 mRNAs were all detected in ex vivo HCE under appropriate cycling conditions. Lanes 1 and 2: donor HCE samples 1 and 2; lane 3: negative control sample.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We investigated the effects of proinflammatory cytokines on the differential gene expression profiles in cultured HCE by means of cDNA array analysis. Our data showed that the highly upregulated genes were those of CCL2 (MCP-1), CXCL8 (IL-8), IL-6, and CXCL2 (GROß) with stimulation by proinflammatory cytokines, whereas the numbers and levels of downregulated genes were, respectively, fewer and lower than those of upregulated ones. The upregulated gene expression levels were confirmed by the respective semiquantitative RT-PCR and ELISA analyses. Moreover, the upregulated mRNAs were detected on RT-PCR in ex vivo donor HCE stimulated with proinflammatory cytokines. These results indicate that the results of gene expression array analysis are in good agreement as to the gene transcription and protein expression levels and that cultured HCE can reflect the cytokine gene expression profile of an ex vivo donor HCE in an inflamed condition.

The most upregulated and downregulated genes in HCE were, respectively, those of CCL2 and CCR2 in this cDNA array. CCL2 attracts CCR2, expressing activated and memory T lymphocytes, monocytes, and macrophages.16 CCR2 expression has been reported to be downregulated by proinflammatory cytokines and CCL2 itself.17 CCL2 is synthesized in cultured human keratocytes, but not in the corneal epithelium, with IL-1{alpha} and TNF-{alpha} stimulation.18 19 In an in vivo animal model, CCL2 protein was detected in keratocytes after corneal epithelial injury.19 Our data indicate that not only corneal keratocytes but also CE can express CCL2 in the presence of inflammation.

Both CXCL8 (IL-8) and CXCL2 (GROß) are chemoattractants for neutrophils.20 CXCL8 mRNA and protein expression have been detected in HCE in vivo,21 consistent with our results. Among corneal cells, not only CE, but also keratocytes and the epithelium can express CXCL8 in vivo22 23 24 25 and in vitro.26 CXCL1 (GRO{alpha}) and KC (mouse homologue of CXCL1, -2, and -3) are also expressed in cultured human keratocytes and the epithelium after stimulation with proinflammatory cytokines27 and in a mouse corneal infection model,28 respectively. CXCR2 is a common receptor for CXCL1, -2, 33 (GRO{gamma}), and -8. Neutrophil recruitment to the cornea in CXCR2-knockout mice has been found to be impaired compared with in wild-type mice.27 These observations and CXCL2/CXCL8 expression in CE indicate that all corneal cells can express chemoattractants for neutrophils, and that CXCR2 and its ligands play a critical role in neutrophil recruitment in the cornea. This may be because the corneal cells in avascular tissues require the swift induction of an innate immune response to infection by chemoattractants for neutrophils.

IL-6 is a pleiotropic cytokine with functions that include stimulation of Ig secretion, acute-phase protein synthesis, and platelet production.29 IL-6 synthesis can be induced by other cytokines, especially IL-1 and TNF-{alpha}. In fact, IL-6, which is detectable in the ocular fluid of patients and rodents with anterior uveitis,30 31 32 has been implicated as a major mediator of uveitis, in that it antagonizes immunosuppressive transforming growth factor (TGF)-ß in the aqueous humor.33 34 Conversely, IL-6 inhibits the production of IL-1 and TNF-{alpha}35 and stimulates the secretion of anti-inflammatory corticosteroids.36 Our data suggest that not only the iris and ciliary body, but also HCE can produce IL-6 and participate in the contradictory reactions of promoting and suppressing inflammation in the aqueous humor. IL-6 is essential for ocular reactivation of herpes simplex virus type 1 (HSV-1) in mice,37 and HSV-1 is a possible cause of corneal endotheliitis.38 39 40 41 These findings suggest that expression of IL-6 in HCE after stimulation with cytokines may promote corneal endotheliitis through the reactivation of virus specific to CE.

KPs are common findings and an important marker for evaluating intraocular inflammation. They are mainly composed of monocytes, neutrophils, macrophages, and lymphocytes.3 42 43 The upregulated chemokines CXCL2, CXCL8, and CCL2 are potent chemoattractants for these cells. In the vascular endothelium, CXCL8 and CCL2 firmly attach monocytes to the endothelium with the assistance of two cell-adhesion molecules, vascular cell adhesion molecule-1, and E-selectin.44 These two cell adhesion molecules can be expressed in the CE of inflamed eyes in rats43 and in rejected allografts in humans.45 These findings suggest that KPs are actively rather than passively formed on CE in inflamed eyes through expression of chemokines and cell adhesion molecules.

In summary, we investigated the cytokine and chemokine expression in HCE on stimulation by proinflammatory cytokines, using cDNA array technology. The genes detected in HCE—CCL2, CXCL8, IL-6, and CXCL2—could facilitate understanding of the inflammatory responses including the production of KPs and the correlation between CE and an inflamed cornea or aqueous humor.


   Footnotes
 
Supported in part by Grant-in-Aid for Scientific Research 14657451 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Submitted for publication May 24, 2002; revised August 5, 2002; accepted August 14, 2002.

Commercial relationships policy: N.

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: Satoru Yamagami, Department of Corneal Tissue Regeneration, Tokyo University Graduate School of Medicine, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan; syamagami-tky{at}umin.ac.jp.


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 Materials and Methods
 Results
 Discussion
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