Accuri C6 Flow Cytometer System
Originally published online as doi:10.1189/jlb.0506342 on January 18, 2007

Published online before print January 18, 2007
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(Journal of Leukocyte Biology. 2007;81:1012-1021.)
© 2007 by Society for Leukocyte Biology

Interleukin-18 regulates pathological intraocular neovascularization

Hong Qiao*, Koh-Hei Sonoda*,1, Yasuhiro Ikeda*, Takeru Yoshimura*, Kuniaki Hijioka*, Young-Joon Jo*, Yukio Sassa*, Chikako Tsutsumi-Miyahara*, Yasuaki Hata*, Shizuo Akira{dagger} and Tatsuro Ishibashi*

* Department of Ophthalmology, Graduate School of Medicine, Kyushu University, Fukuoka, Japan; and
{dagger} Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

1 Correspondence: Kyushu University, Ophthalmology, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan. E-mail: sonodak{at}med.kyushu-u.ac.jp


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ABSTRACT
 
Recently, the proinflammatory cytokine IL-18 has been shown to have a role in angiogenesis. This study aimed to elucidate its role in abnormal neovascularization (NV) in an oxygen-induced retinopathy (OIR) mouse model of the retinopathy seen in human premature newborns. IL-18 was constitutively expressed in the retina in C57BL/6 mice, but expression transiently dropped on Day 17 after birth in mice exposed to 75% oxygen for 5 days between Days 7 and 12. Coincident with the IL-18 reduction in oxygen-treated mice, vascular endothelial growth factor was expressed in the retina, and OIR developed. By Day 24, NV in the retina had regressed to normal levels. By contrast, IL-18 knockout mice, exposed to elevated oxygen concentrations, developed more severe OIR on Day 17, and it is important that this persisted until Day 24. This suggested that IL-18 negatively regulated retinal NV. To investigate this further, we administrated recombinant IL-18 to C57BL/6 mice during the development of OIR but found no significant inhibition of retinopathy. However, when IL-18-binding protein was administered during the OIR recovery phase to neutralize endogenous IL-18, OIR was still apparent on Day 24. We therefore concluded that IL-18 regulates pathogenic retinal NV by promoting its regression rather than inhibiting its development. This suggests some useful, new approaches to treating retinopathy in humans.

Key Words: rodent • cytokine • inflammation


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INTRODUCTION
 
Angiogenesis is an important, normal process in many physiological situations, such as wound-healing, reproduction, and embryonic development. However, inappropriate angiogenesis—neovascularization (NV)—also occurs in many pathological situations. As new capillaries are fragile and prone to hemorrhage, NV, occurring in the eye, can trigger pathological conditions such as retinopathy of prematurity (ROP), diabetic retinopathy, retinal vein occlusion, and age-related macular degeneration. These changes can compromise transparency, an important characteristic of the eye, and can often lead to loss of sight. ROP is a disorder affecting premature infants, which can cause blindness [1 ]. To fight this serious condition, we sought to determine the mechanisms that trigger the development of intraocular NV in ROP using the oxygen-induced retinopathy (OIR) mouse model of angiogenesis, which closely resembles it [2 ].

Recently, IL-18, which was first described as an IFN-{gamma}-inducing factor (IGIF), has been recognized as a new proinflammatory cytokine [3 4 5 ]. It is a pleiotropic cytokine produced by activated monocytes, glial cells, and dendritic cells [6 ], and its immunoregulatory activities include inducing IFN-{gamma} production [7 ], enhancing NK cell activity, and promoting the proliferation of activated T cells [5 ]. The functions of IL-18 in vivo have been identified using knockout (KO) mice [8 ]. Although these mice develop normally and show no obvious signs of disease, they express lower levels of IFN-{gamma}, have impaired NK cell activity, and have defective Th1 responses [8 ].

IL-18 has also been shown to have a role in angiogenesis [9 ]. Cao et al. [10 ] reported that it suppressed corneal NV induced by fibroblast growth factor and tumor angiogenesis and proposed that IL-18 acted as an angiogenesis and tumor suppressor. Kim et al. [11 ] reported that IL-18 acted as an important endogenous negative regulator of angiogenesis in herpetic stromal keratitis. In addition, IL-18 has been shown to inhibit angiogenesis by enhancing thrombospondin-1 (TSP-1) expression via the JNK pathway [12 ]. However, IL-18 has, in contrast, been shown to induce vascular endothelial growth factor (VEGF), promote endothelial cell migration, and increase vascular tube formation in vitro and in vivo [13 , 14 ]. The reason for the discrepancy in these results remains unclear, and the role of IL-18 in vascular development and whether it acts as an angiogenic or angiostatic factor warrant further investigation.

Although IL-18 gene expression has been detected in the normal iris and retina, the function of this protein in the eye remains unclear [15 ]. There have been no reports about the effects of IL-18 on intraocular NV; therefore, to try to understand its functions in vivo, we used a model of intraocular angiogenesis, OIR, induced in IL-18 KO mice or wild-type (WT) mice, treated with IL-18-binding protein (IL-18BP), a natural inhibitor of IL-18 activity [16 , 17 ]. In this report, we have demonstrated an important role in retinal NV for IL-18. However, it is interesting that it does not regulate the development of OIR but rather promotes the regression of abnormal vessels. The implications of these observations are discussed.


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MATERIALS AND METHODS
 
Animal model
C57BL/6 (B6) mice were obtained from SLC Japan (Shizuoka, Japan). IL-18 KO mice on a B6 background were generated at the Hyogo College of Medicine (Japan) [8 ]. All animals were treated humanely and were housed in specific pathogen-free conditions at Kyushu University (Japan). To induce retinal NV, litters at 7 days postpartum (P7) with nursing mothers were maintained in 75% ± 2% oxygen for 5 days and returned to normal air on P12 as described previously [1 , 18 ]. Mice of the same age kept in normal air were used as control animals. All experimental protocols using animals conformed to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by our institutional review board for animal experiments.

Conventional and real-time RT-PCR
The right eyes were removed from mice (n=5 for each time-point) under deep anesthesia, the corneas and lenses were removed, and the peripheral retinas were dissected. Total retinal mRNA was extracted using Trizol® (Life Technologies, Grand Island, NY, USA) and reverse-transcribed using the Gene Amp PCR System 9600 (Perkin-Elmer, Norwalk, CT, USA). First-strand cDNA was synthesized using avian myloblastosis virus RT (Boehringer Mannheim, Indianapolis, IN, USA), according to the manufacturer’s guidelines. Incubations were for 10 min at 25°C and 60 min at 42°C, and RT (Boehringer Mannheim) was denatured at 99°C for 5 min before PCR amplification. The primers used in the experiments were as follows: VEGF, sense 5'-GCG GGC TGC CTC GCA GT-3' and antisense 5'-TCA CCG CCT TGG CTT GTC A-3'; ß-actin, sense 5'-TCA GAA GGA CTC CTA TGT GG-3' and antisense 5'-TCT CTT TGA TGT CAC GCA CG-3'. PCR, using the cDNAs, was carried out in 20 µl vol containing 10 pmol of the primer pair and 2 µl Light Cycler (Roche, Indianapolis, IN, USA), followed by 32 amplification cycles. PCR products were separated on 2% agarose gels, and bands were visualized with 0.05% ethidium bromide and UV transillumination. Band intensities were measured with an image sensor (Densitograph, Atto, Tokyo, Japan) with a computer-controlled display.

IL-18 mRNA expression was quantified in real-time RT-PCR assays using 1 µl aliquots of cDNAs diluted 1:2 (40 µl final volume) and a Light Cycler (Roche). In brief, total RNA extracts from the retina were added to a master mixture. To detect the amount of IL-18 mRNA RT-PCR amplificon, target (IL-18) and control (ß-actin) hybridization probes were mixed with target or control PCR primers, respectively. These mixtures was transferred to thermal cycler tubes and transcribed at 42°C for 30 min, followed by 40 amplification cycles at 95°C for 15 s and 58°C for 10 s. The level of mRNA expression for IL-18 was estimated from the fluorescence intensity relative to ß-actin. The following PCR primers were used: IL-18 (sense) 5'-GAA GAA AAT GGA GAC CTG G-3' and IL-18 (antisense) 5'-TTC ACA GAG AGG GTC ACA-3'; ß-actin (sense) 5'-CCT GTA TGC CTC TGG TCG TA-3' and ß-actin (antisense) 5'-CCA TCT CCT GCT CGA AGT CT-3'. The probes used were as follows: IL-18 (3'-labeled with fluorescein) 5'-AAT ATC AGT CAT ATC CTC GAA CAC AGG C-3' and (5'-labeled with LCRed640, 3'-phosphorylated) 5'-GTC TTT TGT CAA CGA AGA GAA CTT GGT C-3'; ß-actin (3'-labeled with fluorescein) 5'-AGA TCC TGA CCG AGC GTG GCT ACA-3' and (5'-labeled with LCRed640, 3'-phosphorylated) 5'-AGA TCC TGA CCG AGC GTG GCT A-3'.

All IL-18 mRNA values were normalized to ß-actin mRNA levels (IL-18/ß-actin), and levels on different days were expressed relative to the value in untreated mice on P17 (relative index=IL-18 mRNA on the indicated day/IL-18 mRNA on P17 in untreated mice in the same experiment). Data represent the mean of three independent experiments.

VEGF mRNA was also quantified by real-time RT-PCR using SYBR Premix Ex Taq (RR041A, Takara Bio Inc., Japan) and a Light Cycler (Roche) without hybridization (see Fig. 1C ). We used the following primers: 5'-TTACTGCTGTACCTCCACC-3' and 5'-ACAGGACGGCTTGAAGATG-3' for VEGF and 5'-TGGAATCCTGTGGCATCCATGAAAC-3' and 5'-TAAAACGCAGCTCAGTAACAGTCCG-3' for ß-actin.


Figure 1
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  		Figure 1. Effect of oxygen treatment on IL-18 gene expression in the retina of B6 mice analyzed by real-time RT-PCR. In total, 15 newborn B6 mice were exposed to 75% oxygen from P7 to P12, and the retinas were removed from groups of five animals on P12, P17, and P24. Retinal RNA was isolated and analyzed by real-time PCR for IL-18 and VEGF gene expression. Equal numbers of control retinas were used from age-matched B6 mice with "no treatment" (naïve, without hypoxia). (A) Representative examples of RT-PCR amplification curves using mRNA from age-matched, naive mice and OIR (P17) retinas, and the final products are shown in the upper panel. (B) IL-18 gene expression in retinas from age-matched, untreated mice or mice exposed to 75% oxygen (OIR), measured on the indicated days. IL-18 mRNA levels were normalized to ß-actin levels in the same sample and expressed as a relative index compared with the level in P17-untreated samples (relative index=IL-18 value on the indicated day/IL-18 value on P17 in untreated mice) in the same experiment. Data are presented as the mean ± SD of three independent experiments. (C) VEGF gene expression in retinas from age-matched, untreated mice or mice exposed to 75% oxygen (OIR) measured on the indicated days. Total retinal RNA was extracted from pools of three or four eyes of B6 mice on the indicated days. VEGF mRNA levels were normalized to ß-actin levels in the same sample, and the data are presented as the mean ± SD (n=4). *, P < 0.05. n.s., Not significant. The experiment was repeated twice with similar results.

Fluorescein-dextran angiography
Angiogenesis was evaluated in flat-mounted retinas by angiography [1 , 18 ]. Mice were deeply anesthetized, and fluorescein-dextran (2x106 m.w.; 50 mg/ml solution; Sigma Chemical Co., St. Louis, MO, USA) was perfused through the left ventricle (0.03 ml/g body weight). Eyes were removed and fixed in 4% paraformaldehyde for at least 3 h. The cornea and lens were removed, and the peripheral retina was dissected and flat-mounted for examination by fluorescence microscopy. We quantified the extent of retinopathy using the characteristics shown in Table 1 , which were used additively to produce a retinal scoring system between 0 and 17 [18 ]. Each retinal flat-mount was scored independently by two ophthalmologists, and data shown are the mean values.


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  		Table 1. Scoring System for OIR

Ocular fluid preparation and protein assay
Eyes were enucleated under deep anesthesia, the conjunctival tissue was removed, and the remaining eye tissues were homogenized using a Biomasher (Nippi Inc., Tokyo, Japan). After centrifugation at 12,000 g for 30 min, supernatants were collected, and the concentrations of IFN-{gamma}, IFN-{gamma}-inducible protein-10 (IP-10), and the monokine induced by interferon-{gamma} (MIG) were measured using a microbead-based ELISA system (Mouse Cytokine Twenty-plex antibody bead kit, Catalog #LMC0006, BioSource International, Camarillo, CA, USA), according to the manufacturer’s directions, using a Luminex Complete System 200 (Luminex, Austin, TX, USA). The concentration of IL-18 in ocular fluid and serum was measured by ELISA (mouse IL-18 ELISA kit, Code No. #7625, MBL, Nagoya, Japan), according to the manufacturer’s instruction.

IL-18BP and recombinant IL-18 (rIL-18) treatment
Oxygen-treated B6 mice (n=10) were given i.p. injections of IL-18BP [19 ] (0.5 µg/g body weight, R&D System, Minneapolis, MN, USA) or PBS in the control groups. Injections started on P17, when the animal’s own homeostatic processes have been shown to start to mediate a recovery in IL-18 levels in the OIR model and were repeated on P19, P21, and P23. Oxygen-treated B6 mice (n=10) were also inoculated i.p. with mouse rIL-18 (0.05 µg/g body weight, Medical and Biological Laboratories, Woburn, MA, USA) [10 ] or PBS as a control on P7, P12, P14, and P16 to compensate for falling IL-18 levels during this period.

Statistical analysis
Data were analyzed for significant differences between experimental groups by ANOVA and Scheffe’s test. A P value ≤0.05 was considered significant.


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RESULTS
 
IL-18 gene expression was down-regulated temporarily after exposure to a high oxygen concentration
In the OIR model, hyperoxic injury followed by retinal hypoxia produces retinal NV and retinopathy. After exposure to 75% oxygen for 5 days, between P7 and P12, C57BL/6 (B6) mice were returned to normal air to induce retinopathy, and IL-18 expression was measured using quantitative real-time RT-PCR (Fig. 1A ). We found that age-matched, untreated B6 mice showed a relatively constant level of IL-18 mRNA expression in the retina (P12, P17, and P24; Fig. 1B ). By contrast, the levels of IL-18 mRNA fell on P17 in oxygen-treated B6 mice (Fig. 1A and 1B) and then rose again to an equivalent value to that in untreated control mice by P24 (Fig. 1B) . We also measured the expression of VEGF in the retina by real-time PCR on P12, P17, and P24 in the OIR model. As reported previously [20 ], the level of retinal VEGF was raised significantly on P17 but fell to baseline levels by p24 (Fig. 1C) .

Significantly increased and prolonged NV between P17 and P24 in IL-18 KO mice
OIR was induced in newborn WT B6 and IL-18 KO mice, and retinal flat mounts were examined by fluorescein-dextran angiography on P12, P17, and P24 (Fig. 2A ). Changes in the total retinopathy scores over time are shown in Figure 2B . In WT mice with OIR, angiogenesis increased on P17 (after the development phase between P7 and P16) but returned to a similar level to that in untreated WT mice by P24 (after the recovery phase between P17 and P23), as described previously [1 ]. The result shown in Figure 1B and 1C , demonstrated that the OIR scores and VEGF expression were inversely related to the levels of IL-18 expression in the retina between P12 and P24 in WT mice.


Figure 2
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  		Figure 2. Assessment of retinopathy in the OIR mouse model. After exposure to 75% oxygen, retinas were isolated on the indicated days from WT B6 mice or IL-18 KO mice (n=5) and examined by fluorescein-dextran angiography to show areas of NV. (A) Representative examples of fluorescein-dextran angiography in WT and IL-18 KO mouse retinas. Areas of NV along the border between the perfused and the nonperfused regions of the retina are indicated by arrows, Original scale bar = 200 µm. (B) Development of OIR was quantified using retinopathy scores (see Table 1 ) in normoxic (no treatment) and hyperoxic (OIR) WT and IL-18 KO mice on P12, P17, and P24. Results are expressed as the mean ± SD. *, P < 0.05. The experiment was repeated three times with similar results.

As reported previously, retinopathy scores were high in untreated IL-18 KO mice compared with untreated WT mice, reflecting the abnormal retinal vascular development present [21 ]. It is more important that oxygen-treated IL-18 KO mice suffered a significantly more severe retinopathy on P17 than hyperoxic WT mice and untreated KO mice, which was accompanied by a neovascular response with an abundance of blood vessel tufts, a central loss of blood vessels or vasoconstriction, and blood vessel tortuosity (Fig. 2A and 2B) . The neovascular response apparently occurred at the junctions between the perfused and nonperfused regions of the retina (arrows, Fig. 2A ). Although the number of vascular tufts in the oxygen-treated WT mice was almost equal to the normal level in untreated WT mice by P24, the number of blood vessel tufts and the degree of blood vessel tortuosity clearly remained elevated in the oxygen-treated IL-18 KO mice. We concluded that OIR in IL-18 KO mice was significantly more severe on P17 and P24 than in age-matched OIR B6 mice and untreated IL-18 KO mice.

Expression of the angiogenesis-associated factor VEGF was up-regulated in IL-18 KO mouse retinas
Several lines of evidence indicate that VEGF is an important stimulator of the retinal NV, which occurs in ischemic retinopathies. It is up-regulated by hypoxia [22 ] and in the retina and vitreous of patients [23 ] or laboratory animals [20 ] with ischemic retinopathies. VEGF is necessary and sufficient to cause retinal NV, and although other stimulators might be involved [24 ], VEGF appears to play a central role.

After oxygen treatment, total retinal mRNA was extracted from WT B6 and IL-18 KO mice at P17 and P24, and we determined the levels of VEGF gene expression (Fig. 3 ). The levels of VEGF125 and VEGF165 mRNAs were up-regulated in the retinas of oxygen-treated WT mice compared with untreated mice, as described previously [20 ]. The retinal abnormalities induced by oxygen were correlated with the expression of VEGF. It is important that oxygen-exposed IL-18 KO mice expressed higher amounts of VEGF than WT mice, especially on P17. The local up-regulation of VEGF expression in IL-18 KO mice after ischemic injury has been reported previously [25 ]. Here, we demonstrated that VEGF gene expression was up-regulated in IL-18 KO mouse retinas following exposure to a high oxygen concentration.


Figure 3
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  		Figure 3. VEGF gene expression in hyperoxic retinas analyzed by RT-PCR. Newborn B6 and IL-18 KO mice (n=10) were exposed to 75% oxygen (P7–P12) and then returned to normal air. Total RNA was isolated from pools of five retinas on P17 and P24 and analyzed by RT-PCR for the expression of two VEGF isoforms. RNA levels were normalized to ß-actin levels. The experiment was repeated three times with similar results.

Restoring IL-18 in the development phase could not prevent OIR
Although the experiments in IL-18 KO mice (Figs. 2 and 3) suggested a regulatory role for IL-18 in retinal NV, one issue remained unclear. We have reported previously abnormal retinal vascular development in IL-18 KO mice and a role for IL-18 during the physiological development of the retinal vasculature [21 ]. Consistent with this, IL-18 KO mice, which had not been exposed to high oxygen concentrations, showed significant vessel abnormalities (Fig. 2B) . Although this effect was mild and transient [21 ], it could be argued that the deterioration of OIR in IL-18 KO mice could be a result of the augmentation of the developmental abnormality, rather than the active modification of an abnormal retinal NV process after oxygen exposure.

To investigate whether IL-18 was actively involved in the pathological NV induced by oxygen in the developing retina, we supplemented endogenous IL-18 during the development of OIR in B6 mice (P7–P16; Fig. 4A ). As IL-18 was down-regulated during the development phase of OIR (Fig. 1) , we anticipated that OIR might be suppressed by this treatment. Oxygen-treated B6 mice (n=10) were injected i.p. with mouse rIL-18 at a sufficient dose to restore physiological concentrations [26 ]. However, no changes in the numbers of blood vessel tufts or the extent of blood vessel tortuosity were observed on P17 (Fig. 4B) , and no significant reduction in the OIR score was seen (Fig. 4C) . Contrary to our hypothesis, restoring IL-18 during the development phase did not affect OIR.


Figure 4
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  		Figure 4. rIL-18 did not suppress hyperoxia-induced, ocular NV. (A) Experimental scheme for the treatment of B6 mice (n=10) with OIR using rIL-18 (0.05 µg/g body weight) administered i.p. Retinas were evaluated by fluorescein-dextran angiography on P17. B6 mice inoculated with PBS on the same days were used as controls (n=10). (B) Representative examples of fluorescein-dextran angiography in retinas from untreated and rIL-18-treated mice. (C) OIR was quantified using a retinopathy score (mean±SD), and results are expressed as the mean ± SD. The experiment was repeated twice with similar results. (D) Confirmation of ocular delivery of systemically administered rIL-18. The concentration of IL-18 was measured by ELISA in ocular fluid collected 1 h after inoculation with rIL-18 on P17. Groups of 12 eyes (four eyes per tube) were collected from untreated, normal mice (P17 naïve), untreated, OIR mice (P17 OIR), and OIR, rIL-18-treated mice (P17 OIR rIL-18 i.p.).

In addition, we measured IL-18 concentrations in serum and ocular fluid of these mice by ELISA. The serum concentrations on P17 were 105.7 ± 3.3 pg/ml in untreated mice, 95.7 ± 3.3 pg/ml in OIR mice, and 15,394.1 ± 338.3 pg/ml in OIR mice inoculated with rIL-18. The ocular IL-18 concentrations for each group are shown in Figure 4D . As expected, the concentration of IL-18 in the eyes of untreated mice, under normal physiological conditions, was higher than in the serum (untreated P17, P<0.01). The ocular concentration of IL-18 on P17 in OIR mice was significantly lower than in untreated mice (Fig. 4D) , which was compatible with the reduction seen in IL-18 mRNA levels (Fig. 1B) . It is important that the ocular IL-18 concentration on P17 in OIR mice was increased significantly after administering rIL-18 systemically at a level sufficient to compensate for the reduction in endogenous IL-18 induced by hyperoxia (Fig. 4D) . Therefore the inability of exogenous IL-18 to prevent OIR during the development phase was not a result of the lack of local delivery to the eye.

Blocking endogenous IL-18 during the recovery phase prolonged OIR
Next, we examined the role of IL-18 in B6 mice during the recovery phase of OIR (P17–P23; Fig. 5A ). We anticipated that IL-18 might be able to reduce OIR and initially attempted to suppress retinopathy by restoring IL-18 during the recovery phase, as we had done in the induction phase. However, as endogenous IL-18 levels are rising anyway during this phase (Fig. 1) , we were afraid that the effects of additional, exogenous rIL-18 might be difficult to distinguish. We thus decided to look at the effects of neutralizing endogenous IL-18 instead.


Figure 5
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  		Figure 5. IL-18BP suppressed the antiangiogenic effect of IL-18 on OIR. (A) Experimental scheme for the treatment of B6 mice (n=10) with OIR with IL-18BP (0.5 µg/g body weight) administered i.p. during the development phase (P7, P12, P14, and P16) or recovery phase (P17, P19, P21, and P23). Retinas were evaluated by fluorescein-dextran angiography on P24. B6 mice inoculated with PBS during the recovery phase were used as controls (n=10). (B) Representative examples of fluorescein-dextran angiography in retinas from PBS-treated or mice treated during the recovery phase. (C) OIR was quantified using a retinopathy score (see Table 1 ), and the results are expressed as means ± SD. The experiment was repeated three times with similar results. (D) VEGF gene expression in retinas from OIR mice on P17 and P24, following PBS or IL-18BP treatment from P17. VEGF mRNA levels were normalized to ß-actin levels in the same sample, and the data are presented as the mean ± SD (n=4). *, P < 0.05. The experiment was repeated twice with similar results.

Oxygen-treated B6 mice were inoculated i.p. with PBS or IL-18BP, which is a natural inhibitor of IL-18, between P17 and P23 (Fig. 5A) . As a control, we also inoculated mice with IL-18BP during the development phase, between P7 and P16 (Fig. 5A) . The number of vascular tufts on P24 was low in the PBS-treated, control mice, similar to the levels in the normal retina, but some blood vessel tufts and blood vessel tortuosity were still apparent in the IL-18BP-treated mice during the recovery phase (Fig. 5B) . The total retinopathy scores in the IL-18BP-treated mice at this stage were significantly higher than in the control mice (Fig. 5C) . In contrast, IL-18BP treatment during the development phase had no effect on the OIR scores at P24. We also confirmed that treatment during the prerecovery phase did not affect the OIR scores on P17, compared with control mice (data not shown). IL-18BP treatment during the development phase did not reduce retinal VEGF expression until P24 (Fig. 5D) .

Expression of IFN-{gamma}-related angiostatic cytokines in the OIR model
To investigate the mechanisms by which IL-18 mediated the regression of abnormal vessels, we considered its ability to induce IFN-{gamma} and subsequently, the IFN-inducible CXC chemokines, which lack the Glu-Leu-Arg (ELR) motif [27 ]. IFN-{gamma} is known to be angiostatic [28 , 29 ], not only as it induces the expression of the non-ELR CXC chemokines, IP-10 and MIG, but also as it suppresses the expression of angiogenic CXC chemokines [29 ]. We therefore measured the protein concentration of the angiostatic cytokines IFN-{gamma}, IP-10, and MIG in the intraocular fluid, using the multiplex Luminex assay in B6 OIR mice on P12, P17, and P24. This assay allows us to measure a series of cytokines in the same sample at the same time. We confirmed that VEGF protein increased on P16 and then regressed by P24, in agreement with the PCR results in Figure 1B (lower panel). However, IFN-{gamma} was undetectable in ocular fluid at all time points (<5 pg/ml), and IP-10 and MIG levels were not elevated on P24 during the vascular regression phase (Fig. 6 ).


Figure 6
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  		Figure 6. Angiostatic cytokine productions in the oxygen-exposed eye. Newborn B6 mice (n=12) were exposed to 75% oxygen (P7–P12) and then returned to normal air. Ocular fluid was collected from pools of four eyes on the days indicated, and the concentrations of the angiostatic cytokines (A) VEGF, (B) IP-10, and (C) MIG were measured using the Luminex protein assay. The cytokine concentrations were compared with those in age-matched, normoxic B6 mice (control, n=18). *, P < 0.05. The experiment was repeated twice with similar results.


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DISCUSSION
 
In summary, the pathology seen in OIR was most severe in WT mice on P17 and had normalized by P24. It is notable that the levels of endogenous IL-18 varied inversely with the severity of OIR, reaching a minimum on P17 and returning to normal by P24. IL-18 KO mice exposed to oxygen developed pronounced retinal NV compared with WT mice, which did not regress by P24. We also demonstrated that although restoring IL-18 did not affect the development of retinal NV, neutralizing IL-18 in vivo during the recovery phase was inhibitory in WT mice. Taken together, these findings suggest a role for IL-18 in regulating retinal NV by promoting the regression of abnormal vessels, rather than by inhibiting the development of OIR. To our knowledge, this is the first report to demonstrate that IL-18 is an important molecule in the regulation of pathogenic retinal NV. In contrast to our data about intraocular events, a recent study reported that IL-18 had a proangiogenic effect in a model of rheumatoid arthritis, inducing endothelial cell migration [30 ]. The reason for this difference remains unclear, but there might be the differences between the eye and a joint and therefore the tissues in which NV is occurring or between the type of inflammatory processes in the different pathologies.

Several lines of evidence indicate that VEGF is an important stimulator of the retinal NV associated with ischemic retinopathies. It is up-regulated by hypoxia [31 , 32 ], and its levels are increased in the retina and vitreous of patients [33 ] and laboratory animals [34 ] with ischemic retinopathies. VEGF is necessary and sufficient to cause retinal NV, and although other stimulators may be involved [35 ], VEGF appears to play a central role. The local up-regulation of VEGF expression in IL-18 KO mice after ischemic injury has already been demonstrated [25 ]. In this study, we also confirmed that VEGF gene expression was up-regulated in the retinas of IL-18 KO mice following high oxygen treatment (Fig. 3) , and this increase could stimulate retinal NV. We found higher concentrations of IL-18 in ocular fluid than in serum in untreated B6 mice on P16, that is, under normal physiological conditions (Fig. 4D) . The murine retinal vascular system develops after birth, as the capillary vessels reach the peripheral retina on P10 and then terminate their further expansion automatically. Consistent with this, the level of retinal VEGF reached a maximum on P3 and decreased until P15 under normal physiological conditions, preventing the synthesis of further vessels [36 ]. As IL-18 did not inhibit development but promoted regression of new vessels, we speculated that ocular IL-18 might also play a role in the self-limiting nature of the normal development of the retinal vasculature.

Not only the physiological development of retinal vasculature but also, pathologic OIR is self-limiting in B6 mice (Fig. 2B) . It is because a number of factors have been implicated in pathological changes in vessel formation and regression in the OIR [37 ]. The relative expression of angiostatic factors (including TSP-1 and endotsatin) became dominant in the recovery phase rather than the developing phase [38 , 39 ], and they might play an important role in the self-limiting process of pathogenic retinal NV. It is interesting that IL-18 is known to enhance TSP-1 production [40 ]. We thus currently postulate that endogenous IL-18 can selectively enhance the effects of these late-phase, dominant, angiostatic factors but not directly regulate development-phase, dominant, angiogenic factors. This might be the reason supplementation of rIL-18 in the development phase did not inhibit OIR.

In addition, several other possible mechanisms by which IL-18 regulates ocular angiogenesis could be considered. IL-18 was first described as IGIF [3 4 5 ], and IFN-{gamma} and its related cytokines are known to regulate angiogenesis [11 , 41 ]. However, we have shown that IFN-{gamma} and the IFN-{gamma}-related angiostatic cytokines IP-10 and MIG did not increase during the OIR recovery phase (Fig. 6) . Thus, these three cytokines may not mediate the effect of IL-18 on vessel regression. Another possible mechanism is that IL-18 can enhance Fas/Fas ligand (FasL) pathways and so induce apoptosis [42 43 44 ] and inhibit endothelial cell proliferation and migration [10 ]. Activated Fas/FasL has been reported to control retinal angiogenesis [45 , 46 ] and induce endothelial cell apoptosis [47 ], thus suggesting a potential mechanism by which IL-18 could regulate intraocular angiogenesis. It is interesting that the antiangiogenic activity of TSP-1 and pigment epithelium-derived factor was dependent on the induction of Fas/FasL and the resulting apoptosis [47 ].

An understanding of how the cytokine network regulates the delicate balance of angiostatic and angiogenic chemokines must be an important approach to improving the treatment of chronic inflammatory diseases and neoplasias. Our results suggest new approaches to therapies for maladies, such as ROP, diabetic retinopathy, retinal vein occlusion, and age-related macular degeneration, which involve retinal NV.


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ACKNOWLEDGEMENTS
 
This work was supported, in part, by grants to K-H. S. from the Ministry of Education, Science, Sports and Culture, Japan (B2 No. 14770962), and the Japan National Society for the Prevention of Blindness. We especially thank Ms. Michiyo Takahara for her excellent technical support with all these experiments.

Received May 22, 2006; revised November 28, 2006; accepted December 4, 2006.


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