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(Investigative Ophthalmology and Visual Science. 2005;46:4224-4234.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0346
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Microarray Analysis of Cytokine and Chemokine Gene Expression after Prednisolone Treatment in Murine Experimental Autoimmune Uveoretinitis

Noriyasu Hashida,1 Nobuyuki Ohguro,1 Kei Nakai,1 Michiyo Kobashi-Hashida,1 Shin-ichi Hashimoto,2 Kouji Matsushima,2 and Yasuo Tano1

1From the Department of Ophthalmology, Osaka University Medical School, Osaka, Japan; and the 2Department of Molecular Preventive Medicine, School of Medicine, University of Tokyo, Tokyo, Japan.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. The purpose of this study was to investigate changes in gene expression of cytokines and chemokines and their receptors after systemic prednisolone treatment in experimental autoimmune uveoretinitis (EAU).

METHODS. EAU mice received one intravenous injection of 7.5 mg/kg prednisolone (PDS) sodium phosphate at the peak of inflammation. EAU mice treated with only solvent served as the control. Total RNA was extracted from the whole eyes 1, 2, and 3 days after treatment. Gene expression analysis was conducted with a cDNA microarray, which contains 117 individual transcripts encoding the genes of the cytokines and chemokines and their receptors (29 cytokines and 34 cytokine receptors; 33 chemokines and 21 chemokine receptors). Comparisons of expression between PDS-treated and placebo-treated EAU mice at each time point were performed. The genes were sorted into clusters based on expression profiles, to clarify the gene regulation pattern after treatment.

RESULTS. Forty-seven genes had a significant decrease in expression 1 day after treatment, 10 genes on day 2, and 46 genes on day 3. Ten genes were upregulated on day 1, but no gene was upregulated thereafter. Hierarchical cluster analysis of the microarray demonstrated that gene expression changes in EAU after treatment with PDS showed four patterns: flat, mountain, steep downhill, and less steep downhill.

CONCLUSIONS. The implications of the clusters after treatment with PDS remain unclear. The results showed that hierarchical cluster analysis based on comprehensive gene expression profiles may provide a powerful tool for identifying genes not previously associated with the therapeutic targets in ocular inflammation.

Experimental autoimmune uveoretinitis (EAU) is an organ-specific, T-cell (CD4+)-mediated disease that is induced in genetically susceptible murine strains by immunization with retinal proteins (e.g., interphotoreceptor retinoid-binding proteins [IRBPs] of tyrosinase family proteins) or by the adoptive transfer of T-cells specific for these antigens into naïve syngenetic recipients.1 2 3 4 5 EAU is characterized by granuloma formations in the retina, infiltration of polynuclear lymphocytes and macrophages into the retina, vasculitis, and destruction of photoreceptor cells.6 7 8 EAU serves as an animal model for ocular inflammatory diseases with a putative autoimmune etiology (e.g., ocular sarcoidosis, Behçet’s disease, or the Vogt-Koyanagi-Harada syndrome).4 7

Administration of systemic corticosteroids is a standard therapy for severe panuveitis. The pharmacologic effects of corticosteroids are based on several mechanisms. The accumulation of neutrophils and macrophages in inflamed tissue decreases after corticosteroid administration.9 Corticosteroids also modulate cytokine expression and induce apoptosis of T-cells in a dose-dependent manner.10 11 12 13 Recently, the production of interferon (IFN)-{gamma}, one of the best known Th1 cytokines, was reported to decrease after corticosteroid treatment in an EAU rat model.14

In previous studies, it was reported that domination of Th1-type immune responses plays an important role in the pathogenesis of EAU.15 16 17 18 The expression of several chemokines and cytokines was identified by quantitative polymerase chain reaction (PCR) and immunohistochemistry in an EAU mice model.17 The roles of some cytokines and chemokines—for example, interleukin (IL)-2, IFN-{gamma}, tumor necrosis factor (TNF)-{alpha}, chemokine (C-X-C motif) ligand 9 (CXCL9)/monokine induced by IFN-{gamma} (Mig), CXCL10/IP-10 (IFN-{gamma}-inducible protein of 10 kDa), and chemokine (C-X-C motif) receptor 3 (CXCR3)—have been reported in the development of EAU.19 20 21 In addition, the Th2-type cytokines IL-4 and IL-10 play a role in remission of the disease.20 22 23 However, the regulatory impact of corticosteroids on inflammatory genes in the EAU model is still a matter for speculation.

We examined comprehensive gene expression analysis of cytokines and chemokines and their receptors after corticosteroid treatment in an EAU murine model. The results provide new information about which inflammatory genes play key roles in the remission of EAU after corticosteroid treatment.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Mice
Ninety C57BL/6 mice were purchased from Japan SLC, Inc. (Shizuoka, Japan), housed in pathogen-free conditions in our animal facility at Osaka University, and used at 8 to 10 weeks of age. The animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Peptides and Adjuvants
Human IRBP peptides corresponding to IRBP1-20 (GPTHLFQPSLVLDMAKVLLD) was selected as an uveitogenic peptide, as described previously.8 24 The peptide was purchased from Gene Net Co., Ltd. (Fukuoka, Japan). Complete Freund’s adjuvant (CFA) and Mycobacterium tuberculosis H37Ra were purchased from Sigma-Aldrich (St. Louis, MO) and DIFCO (Detroit, MO), respectively. Bordetella pertussis inactive bacterial suspension was purchased from Wako Pure Chemical Industries (Tokyo, Japan). IRBP peptides were dissolved in phosphate-buffered saline at 5 mg/mL.

Induction and Clinical Assessment of EAU
C57BL/6 mice were inoculated subcutaneously in the right hind footpad, right thigh, and base of the tail with 100 µg IRBP peptide in emulsion with CFA supplemented with 6.0 mg/mL M. tuberculosis (1:1, vol/vol). Inactivated suspension of B. pertussis (1010 organisms/mouse) in a volume of 100 µL was injected intraperitoneally as additional adjuvants. Beginning 10 days after immunization, slit lamp examinations were performed daily. A peak inflammatory response was observed on day 16 after the IRBP peptide injection. The EAU clinical scores were graded from 0 to 4, as previously described.24 25 We analyzed mice with grade 2 EAU throughout the study.

Administration of Prednisolone Sodium Phosphate
The EAU mice at the peak of inflammation were treated with one intravenous injection of 7.5 mg/kg of body weight prednisolone (PDS) sodium phosphate (LKT Laboratories, Inc., St. Paul, MN) dissolved in sterile 0.9% NaCl solution.26 27 This dose is similar to that used clinically to treat uveitis, such as occurs in the Vogt-Koyanagi-Harada syndrome.28

Tissue Sampling from Mice
For the examination of gene expression in EAU, 10 mice were killed, and the eyeballs were collected on day 16. Mice injected with only adjuvant served as control animals. For the analysis of the effect of PDS, 10 mice were killed, and the eyeballs were collected on days 1, 2, and 3 after administration of PDS. EAU mice that received only sterile 0.9% NaCl solutions served as the control and the eyeballs were collected on days 1, 2, and 3 after administration of solvent.

Total RNA Isolation
The eyes of 10 mice were pooled as one sample at each time point, to minimize variation among the animals.29 30 The globes were removed from each mouse and prepared immediately. The pooled eyes were homogenized (model HG30 homogenizer; Hitachi Koki Co., Ltd., Tokyo, Japan) in RNA extraction reagent (TRIzol; Invitrogen Inc., Carlsbad, CA). Total RNA was isolated according to the manufacturer’s instructions. The cetyltrimethylammonium bromide method was used to remove polysaccharide contamination.29 31 Any DNA contamination was removed by incubation with DNase I. Final products yielded a 260/280-nm ratio of 1.9 to 2.0 and a 230/260-nm ratio less than 0.5. The quality was checked via gel electrophoresis by visualization of intact 28S and 18S ribosomal RNA bands. The quantity was determined based on 260-nm absorbance.

DNA Chips
A description of mouse cytokine and chemokine chips (Kakengeneqs Co., Ltd., Chiba, Japan) is available at http://www.geocities.jp/nohashida2005/genes.pdf. Individual transcripts (n = 117) were included on the DNA chip that contained most cytokines and chemokines and their receptors (29 cytokines, 34 cytokine receptors; 33 chemokines, 21 chemokine receptors). Two housekeeping genes (GAPDH and ß-actin) and three unrelated murine reagents (tobacco chloroplast DNA, part of a cloning vector, and a solvent that suspends the PCR products) were spotted onto the chip as internal positive and negative controls, respectively. This cDNA chip covers almost all genes of the cytokines and chemokines and their receptors.29

cDNA Preparation and Array Hybridization
Total RNA samples (100 µg) were converted to double-strand cDNA by using a custom kit (LabelStar Array kit; Qiagen, Valencia, CA) and labeled with cyanine 3 (Cy3)-conjugated dUTP. Reference total RNA was labeled with cyanine 5 (Cy5)-conjugated dUTP (PerkinElmer, Boston, MA), according to the manufacturer’s protocol. Each RNA sample was labeled in triplicate with Cy3 and Cy5. The labeled cDNAs were mixed and hybridized simultaneously to the cDNA microarray chip. Expression patterns in the EAU mice were compared with those of control mice. Expression patterns after PDS treatment in the treated mice were compared with those in the day-matched control mice. Array hybridization was performed according to the manufacturer’s instructions (Kakengeneqs Co., Ltd.), except that the hybridization and washing temperature was 65°C.

Microarray Quantification
The fluorescent images of hybridized microarrays were primarily obtained with an array scanner (model 428; Affymetrix, Santa Clara, CA), with independent laser excitation at 532- and 635-nm wavelengths for the Cy3 and -5 labels, respectively. Each chip was scanned twice. Raw fluorescence intensity data were used to calculate signal intensities of the spots (DNASIS Array; Hitachi Software Engineering Co., Ltd., Tokyo, Japan). The software provides absolute analysis of the data from each chip, including normalization of identified signal intensities, background calculation, and comparative analysis between any two chips. Data were normalized by the median of the intensity of the housekeeping gene. Triplicate data for one RNA sample were averaged for each gene. Transcripts not found in two of three experiments in either the control and experimental groups were not analyzed further. Normalized and averaged fluorescence ratios of genes from the experimental mice were compared with those from the control animals. Data were used to calculate the percentage increase or decrease of change. A twofold change in gene expression was used as the cutoff. Three independent experiments were performed to compare the experimental and control data.

Quantitative RT-PCR
We performed quantitative RT-PCR of the selected genes to validate the microarray data. Commercially available primers and probe sets of the genes were prepared for the analysis. IL21r, TNFa, and CXCL1 were selected for the experiment involving developing EAU, and CXCL4, CXCL5, CXCR6, and IL16 were selected for that after corticosteroid treatment. Two genes (GAPDH, 18s rRNA) were selected as the positive control, to monitor PCR reactions.

Total RNA (500 ng) was reverse transcribed with random hexamer primers (GE Healthcare, Piscataway, NJ) to synthesize double-strand cDNA (SuperScript II; Invitrogen Corp.), according to the manufacturer’s instructions. A low-density array (Applied Biosystems, Inc. [ABI], Foster City, CA) containing a preset assay-on-demand mix of primers and FAM dye-labeled minor groove-binding probes (TaqMan; ABI) for target genes were used for real-time PCR. Low-density arrays are research tools for profiling gene expression using the comparative Ct method of relative quantification.32 33 18s rRNA were used as a calibrator in relative quantification calculations. Gene-specific PCR products were measured continuously by a sequence detection system (Prism 7900HT; ABI), according to the manufacturer’s protocols. The cycling parameters were incubation for 2 minutes at 50°C and 10 minutes at 94.5°C, 40 PCR cycles at 97°C for 15 seconds each, and annealing-extension at 59.7°C for 1 minute. Each sample was run in quadruplicate.

Statistical Analysis and Clustering Method
The microarray data were hierarchically clustered with average linkage clustering34 and visualized (DNASIS Stat software; Hitachi Software Engineering Co.). The Silhouette index and Dunn index in the clustering analysis were used to determine the number of clusters. Comparison of microarray analysis and quantitative RT-PCR experiments at respective time points was performed by t-test.35 P < 0.05 was considered statistically significant.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Reliability of the Microarray Technique
Figure 1A is a scatterplot of Cy3- versus Cy5-labeled intensity. Pair-wise comparisons of gene expression levels in three independent experiments displayed a correlation coefficient of R2= 0.995, indicating reliability. The reproducibility of the microarray technique was determined by comparing the cluster image of each analysis of independent amplification from the same RNA source. Figure 1B shows the cluster images from six independent experiments. In three independent experiments, total RNA derived from the eyes of the EAU and control mice was labeled with Cy3 and Cy5, respectively. In another three experiments, total RNA derived from the eyes of EAU and control mice was labeled with Cy5 and Cy3, respectively. Chip-to-chip normalization was performed by comparing the hybridization intensities of control probe sets spotted onto each microarray chip. Results of triplicate analysis of each sample were consistent.



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  		FIGURE 1. Reliability of the microarray technique. (A) Scatterplot of Cy3 versus Cy5. The Cy3 and Cy5 intensities were log2 transformed and are shown on the horizontal axis and vertical axis, respectively. The intensities of Cy3 and Cy5 displayed a strong linear relationship (R2 = 0.995). Red and blue lines: twofold and fourfold up- or downregulation, respectively. This result represents three independent experiments. (B) Comparison of cluster image of each experiment. Total RNA from EAU mice and normal mice was labeled with Cy3 and Cy5, respectively (lanes 1–3). In another experiment, total RNA from EAU mice and normal mice was labeled with Cy5 and Cy3, respectively (lanes 3–6). Cluster images of each experiment are simultaneously juxtaposed. The clustering pattern of the genes in each experiment shows the correlated pattern.

 
Microarray Data Results of Expression Changes in Developing EAU
Most analyzed genes were upregulated in EAU (Table 1) . Gene expression was considered consistently changed only if the average multiple of change between the control and experimental pools was higher than two in at least two hybridizations in three independent experiments. Under these conditions, of the 117 genes on the chips, 91 genes were significantly upregulated (more than twofold) during EAU; 17 genes had no significant changes. We could not evaluate nine genes because their expression was undetectable in the control animals. However, these nine genes were certainly upregulated during the development of EAU, although we could not calculate the extent to which this occurred.


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  		TABLE 1. Changes in Gene Transcription in EAU Mice

 
Most upregulated genes were Th1-type cytokines and chemokines and their receptors; however, some Th2-type genes were among those significantly upregulated. Notably, cytokine and chemokine receptors tended to be preferentially upregulated in developing EAU (8 of the top 10 genes).

Microarray Results of Expression Changes after Administration of PDS
Table 2 shows the functional categories and the changes (x-fold) in expression. The expression of 47 genes decreased significantly on day 1 after treatment, that of 10 genes on day 2, and that of 46 genes on day 3. Ten genes were upregulated on day 1, but none thereafter.


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  		TABLE 2. Gene Expression Changes after Prednisolone Treatment

 
Table 3 shows the 10 upregulated genes on day 1 and the top 10 downregulated genes at each time point. The top 10 downregulated genes differed greatly at each time point. The clinical score did not change significantly throughout the observation period (data not shown).


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  		TABLE 3. Top 10 Up- and Downregulated Genes

 
Pattern of Clustering of Gene Expression after Treatment with PDS
Figure 2B shows the cluster image of the different gene expression profiles. The number of clusters was determined by calculating the scores of the Silhouette index and Dunn index. The genes were clustered into four groups according to the highest score on each index (Silhouette index, –0.12; Dunn index, 0.51; Fig. 2A ).



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  		FIGURE 2. (A) The graphs of the Silhouette index and Dunn index display the valid clusters. The highest score for each index helps to determine the numbers of clusters. (Silhouette index, –0.12; Dunn index, 0.51). (B) By microarray analysis, cluster images show the different classes of gene expression profiles of 117 genes and the expression changes after PDS treatment. The subsets of genes are hierarchically clustered. The expression pattern of each gene in this set is displayed as a horizontal strip. For each gene, the ratio of the RNA levels in the eyes from the EAU mice at the indicated days after PDS treatment to the levels in the eyes of saline-treated mice is represented by a color, according to the scale at the bottom. The graphs show the representative expression profiles of the genes in the corresponding clusters 1 through 4.

 
Cluster 1 contained 28 genes with no change in expression (approximately onefold) on day 1 or 2, or both, after treatment. On day 3, some of them showed significantly decreased expression, but most of them did not. Therefore, the expression pattern of this cluster could be called flat (Fig. 2B) . A representative gene in cluster 1 was IL13ra2, the expression of which increased approximately 14-fold during EAU, but no significant change in expression was observed after administration of PDS.

Cluster 2 contained most of the examined genes (n = 67) with constantly decreased expression at all time points after treatment. Because the degree of downregulation was weak on day 2, the shape of this cluster resembled a mountain (Fig. 2B) . The representative genes in cluster 2 were CCR5; IL2ra; CCL11/eotaxin; and CCL5/regulated on activation, normally T-expressed, and presumably secreted (RANTES).

Cluster 3 contained five genes (IL7r, XCL1/lymphotactin, CXCR6/BONZO, CCR2, and lymphotoxin/TNFb) with significantly upregulated expression (>2.5-fold) on day 1 but dramatically decreased expression on days 2 and 3. This cluster had a steep downhill pattern.

Cluster 4 contained 17 genes, with slightly increased expression (twofold or less) on day 1 and gradually decreased expression on days 2 and 3 (less steep downhill pattern). Figure 3 shows the detailed information on each cluster.



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  		FIGURE 3. The expression patterns and detailed functional categories of inflammatory genes in each cluster. The subset of 117 genes encoding cytokines and chemokines and their receptors is clustered hierarchically into groups based on the similarity of the expression profiles. For each gene, the ratio of the RNA levels in the eyes of EAU mice at the indicated days after PDS treatment to its levels in the eyes of saline-treated mice is represented by a color, according to the scale at the bottom.

 
Quantitative RT-PCR Results
Because quantitative differences in analysis of relative mRNA levels are often observed when the Northern blot, array hybridization, and real-time PCR methods36 were used, we confirmed the exact quantitative changes during development of EAU and after treatment with PDS in selected genes by RT-PCR. Figure 4 shows the multiple changes in these genes in microarray analysis and quantitative RT-PCR experiments. The expression ratios of the genes correlated well between the microarray analysis and quantitative RT-PCR experiment, indicating experimental reliability.



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  		FIGURE 4. Comparison of expression changes of microarray analysis ({cjs2100}) and quantitative RT-PCR ({square}) experiments. The same RNA sources were used for both experiments. (A) IL21r, TNFa, and CXCL1 are the representative genes with upregulated patterns in the eyes of EAU mice. For microarray analysis, samples were analyzed in triplicate. In the RT-PCR experiments, x-fold change was determined by averaging the changes obtained for the four replicates. Comparisons were performed by t-test. The change was considered significant at P < 0.05 (*P < 0.01) (B) A representative gene in each cluster was selected and the sequential expression changes analyzed after PDS treatment: CXCL4 (cluster 1), CXCL5 (cluster 2), CXCR6 (cluster 3), and IL16 (cluster 4). Comparisons between quantitative RT-PCR data and microarray data were performed by t-test. Change was considered significant at P < 0.05 (*P < 0.01 by t-test).

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
To the best of our knowledge, this article is the first to report a comprehensive expression analysis of inflammatory genes after corticosteroid administration in an EAU murine model. The results demonstrate that administration of a corticosteroid suppressed gene expression of many cytokines and chemokines and their receptors, the expression of which was upregulated at the inflammatory peak in the EAU model. This study also showed that gene expression profiles after corticosteroid treatment differed depending on the observation time. One day after administration of PDS, 47 genes were significantly downregulated; however, only 10 genes were downregulated on day 2, and then 46 genes on day 3. In addition, the top-10 downregulated genes differed remarkably at each time point. Therefore, to clarify the gene regulation pattern after treatment with PDS, we estimated these results in a hierarchical clustering format, which is believed to be a powerful method to analyze the multiple and sequential changes in microarray data. We also found that the genes were clustered into four groups by pattern: flat, mountain, steep downhill, and less steep downhill patterns.

In cluster 1, which contained 28 genes, the gene expression change remained almost constant (flat pattern), and the degree of change was unremarkable (twofold or less). GAPDH and ß-actin were in this cluster, suggesting that these genes may not be the main target of PDS. Of interest, IL2 was also in this cluster. The mRNA expression of IL2 reaches a peak during the active phase of EAU and declines parallel to disease resolution.20 We also observed a significant increase in gene expression of this cytokine at the inflammatory peak; however, the expression pattern did not change dramatically after treatment with PDS. Several studies also showed that corticosteroids failed to inhibit IL-2 production significantly.37 38 39 However, cyclosporine, a representative T-cell immunosuppressant, downregulates IL-2 gene expression.40 41 42 Recent investigations have shown the effectiveness of cyclosporine in treating uveitis refractory to corticosteroid therapy.43 44 45 The difference between PDS and cyclosporine in regulating IL-2 may explain those recent clinical experiences.

Among 117 cytokines and chemokines and their receptors, more than half (n = 67) were in cluster 2, which had a mountain pattern. The number of significantly downregulated genes was fewer only on day 2. Considering all evidence, cluster 2 could represent the main pattern of gene expression changes after PDS treatment. This cluster contained IFNg, IL-6, TGFb, IL2r, CXCL9, and RANTES/CCL5, all well-known participants in ocular inflammation.18 19 20 21 46 47 48 49 Kodama et al.14 recently showed that the level of intraocular IFN-{gamma} in EAU rats decreased after dexamethasone was injected into the anterior chamber, which agrees with our data. The reason downregulation of gene expression by PDS occurred on day 2 to a lesser extent than at the other time points remains unclear. One possibility is that there is a temporary physiologic compensatory mechanism during PDS treatment.

A noteworthy finding was the existence of cluster 3. Five genes, IL7r, XCL1/lymphotactin, CXCR6/BONZO, CCR2, and lymphotoxin A/TNFb, were significantly upregulated by PDS on day 1 but sequentially downregulated on days 2 and 3 (steep downhill pattern). IL-7 plays a role in stimulating the proliferation of murine pre-B cells.50 Investigators have shown that B cells develop in the bone marrow from progenitor cells.51 Differentiation of progenitor cells requires signaling through IL-7r, mediated by the growth-signaling factor, consisting of IL-7 and 30-kDa protein cofactor.52 The gene of CXCR6/BONZO, the receptor for CXCL16, is important in the trafficking of effector T-cells mediating type-1 inflammation.53

XCL1/lymphotactin is chemotactic for CD4+ and CD8+ T-cells but not for monocytes and induces a rise in intracellular calcium in peripheral blood lymphocytes.54 55 These genes are not associated with EAU development and/or the effect of corticosteroid treatment on ocular inflammation.

Other representative genes in cluster 3 were CCR2 and lymphotoxin A/TNFb. CCR2, a major receptor for CCL2/monocyte chemotactic protein (MCP)-1, was present at high levels in infiltrating lymphocytes in an experimental autoimmune encephalomyelitis (EAE)/EAU rat model.56 57 58 An earlier study has shown that CCL2/MCP-1 enhances the Th1 immune response in an EAE model.58 Keino et al.21 also reported that upregulated CCR2 is involved in recruiting inflammatory cells, such as Th1-type T-cells, into the eyes of EAU mice.21 Lymphotoxin A, a member of the TNF family, plays important roles in lymphocyte activation, immune regulation, and lymphoid tissue development.59 60 61 It is secreted by lymphocytes and implicated in inflammatory diseases such as Behçet’s disease.62 The level of lymphotoxin A in patients with Behçet’s disease is higher than that in healthy control subjects.62 Considering those studies, cluster 3 contains genes that play important roles in lymphocyte behavior. Further investigation of this cluster may lead to a new therapeutic approach to the control of ocular inflammation.

The expression pattern of cluster 4 was similar to that of cluster 3, but the extent of up- and downregulation was less than in cluster 3 (less steep downhill). The five genes in this cluster were significantly upregulated on day 1, but the upregulation was <2.5-fold. The representative genes in this cluster were CCR8 and CXCL11/interferon-inducible T-cell {alpha}-chemoattractant (ITAC). CCR8, expressed in natural killer cells, T lymphocytes, and monocytes, increases strongly in the presence of inflammation.63 64 Previous results have indicated that CCR8 and CXCL11/I-TAC are potent regulators of endothelial cell function and act as angiogenic molecules such as fibroblast growth factor 2 and vascular endothelial growth factor.65 The formation of focal subretinal neovascularization is an ocular manifestation of EAU66 and it is well known that corticosteroids suppress angiogenesis.67 68 Therefore, we can speculate that corticosteroids control the angiogenesis through CXCL11 and CCR8.

Several cytokines and chemokines have been identified as upregulated genes by quantitative PCR in development of EAU.17 Whereas the pathogenesis of EAU has been related to an elevation in the amount of Th1-type cytokines during the acute phase,18 Th2-type cytokines also were reported to increase simultaneously in EAU.17 Our comprehensive gene expression analysis in EAU development confirmed these previous studies. Our results also revealed that most genes of cytokines/chemokines and their receptors (91/117 genes) were significantly upregulated at the peak of inflammation. In the top 10 upregulated genes especially, we found eight cytokine/chemokine receptors, suggesting that cytokine/chemokine receptors may play a pivotal role in the pathogenicity of EAU.

Studies have indicated that corticosteroids decrease accumulation of activated T-cells, neutrophils, and macrophages in inflamed tissue.9 Investigators have also demonstrated that the chemokines and cytokines produced by both infiltrated cells and ocular resident cells play a role in development of EAU.17 However, we could not show whether the expression changes after treatment with PDS represent changes in infiltration of lymphocytes, resident inflamed ocular cells, or both, and thus further studies are needed. In addition, as previously reported, the clinical score did not change significantly throughout the observation period.27 Therefore, we could not correlate the gene expression changes observed in this study with the improvements in the clinical scores in EAU mice.

In summary, we performed hierarchical cluster analysis based on comprehensive gene expression profiles of the cytokines and chemokines and receptors after corticosteroid treatment in a murine EAU model. The genes clustered into four patterns. Although the implications of each cluster remain unclear, these findings may open a new avenue to identify new therapeutic targets in ocular inflammation.


   Footnotes
 
Supported by Grant in Aid for Scientific Research C-15591852 from the Japanese Ministry of Education, Culture, Sports, Science, and Technology.

Submitted for publication March 18, 2005; revised June 15, 2005; accepted September 13, 2005.

Disclosure: N. Hashida, None; N. Ohguro, None; K. Nakai, None; M. Kobashi-Hashida, None; S. Hashimoto, None; K. Matsushima, None; Y. Tano, None

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

Corresponding author: Nobuyuki Ohguro, Department of Ophthalmology, Osaka University Medical School, E7, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan; nohguro{at}ophthal.med.osaka-u.ac.jp.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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