The studies were performed on 661W cells, an immortalized retinal cell line derived from mouse retinal tumors that were provided by Dr. Muayyad R. Al-Ubaidi (University of Houston, Houston, TX, USA). The 661W cells express SV40 T antigen, blue and green cone pigments, transducin, and cone arrestin, but not rod-specific antigens, such as opsin and arrestin or rod- and cone-specific proteins. The cells were incubated in Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque Inc, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Rockford, IL, USA), 100 U/mL penicillin (Meiji Seika Kaisha Ltd., Tokyo, Japan), and 100 μg/mL streptomycin (Meiji Seika) under a humidified atmosphere of 5% CO₂ at 37°C. The cells were released by trypsinization and passaged every 2 days.

For the western blot analysis, 661W cells were seeded at a density of 3 × 10⁴ cells/well in 12-well plates, and then incubated for 24 h under a humidified atmosphere of 5% CO₂ at 37°C. The medium was changed to DMEM with 1%FBS, and the cells were exposed to 2,500 lux of light. After 12 h, they were washed with PBS, lysed in RIPA buffer (Sigma–Aldrich, St. Louis, MO, USA) containing 1% protease inhibitor cocktail and 1% of the phosphatase inhibitor cocktails 2 and 3 (Sigma–Aldrich), and harvested. The lysates were centrifuged at 12,000 g for 15 min at 4°C. The protein concentration was measured with a BCA Protein Assay Kit (Thermo Fisher Scientific) with bovine serum albumin as a standard. An equal volume of protein sample and sample buffer was mixed, and the samples were boiled for 5 min at 100°C. The protein samples were separated by 5–20% SDS-PAGE gradient electrophoresis and then transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA, USA). The primary antibodies used for immunoblotting were rabbit anti-CCR3 monoclonal antibody (ab32512, Abcam, Cambridge, MA, USA), rabbit anti-phospho-nuclear factor-kappa B (NF-κB) (#3033, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-NF-κB (#8242, Cell Signaling Technology) and mouse anti-β-actin mouse monoclonal (A2228, Sigma–Aldrich) antibody. A horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (#32460, Thermo Fisher Scientific) and an HRP-conjugated goat anti mouse antibody (#32430, Thermo Fisher Scientific) were used as secondary antibodies. Immunoreactive bands were made visible by Immunostar-LD (Wako) and a LAS-4000 luminescent image analyzer (Fuji Film Co., Ltd., Tokyo, Japan). β-actin was used as the loading control.

661W cells were seeded at a density of 6 × 10⁴ cells/well in 6-well plates, and incubated for 24 h under a humidified atmosphere of 5% CO₂ at 37°C. The medium was changed to DMEM with 1% FBS. The cells were exposed to 2,500 lux of light for 6 h. The RNA was extracted with NucleoSpin^® RNA II [Takara Bio Inc. (Kusatsu, Japan)], and single-stranded cDNA was synthesized from the total RNA with the PrimeScript^TM RT reagent Kit. The results are expressed as values relative to the level of Gapdh which was used as the internal control. The primers used for amplification were Ccr3: 5′-TCA ACT TGG CAA TTT CTG ACC T-3′ and 5′-CAG CAT GGA CGA TAG CCA GG-3′; and Ccl11: 5′-CAG ATG CAC CCT GAA AGC CAT A-3′ and 5′-TGC TTT GTG GCA TCC TGG AC-3′; and Gapdh: 5′-TGT GTC CGT CGT GGA TCT GA-3′ and 5′-TTG CTG TTG AAG TCG CAG GAG-3′.

Polymerase chain reaction was performed using Blend Taq^® Plus with a thermal cycle program of 5 min at 94°C, and 3 PCR steps of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C, and finally 7 min at 72°C for a total of 45 cycles. The PCR product was transferred to 2% agarose gel for electrophoresis. The DNA bands were stained with ethidium bromide and quantified by a LAS-4000 luminescent image analyzer (Fuji Film Co., Ltd.).

The following small interfering RNA (siRNA) sequences specific to murine Ccr3 were used:

Stealth RNAi Negative Control Medium GC Duplex #2 was used as a control. All of the siRNAs were synthesized by Thermo Fisher Scientific (Invitrogen^TM).

The 661W cells were transfected with 50 nM of the siRNA using Lipofectamine^TM RNAiMAX Reagent (Thermo Fisher Scientific) and Opti-MEM (Thermo Fisher Scientific) according to the manufacturer’s protocol. After 24 h of exposure, PCR was performed to determine the degree of transfection. The effect of the two siRNAs in protecting the 661W cells from light exposure was determined.

661W cells were seeded at a density of 3 × 10³ cells/well into 96-well plates, and incubated for 24 h under a humidified atmosphere of 5% CO₂ at 37°C. The medium was changed, and the cells were exposed to the two CCR3 antagonists, SB 328437 [N-(1-Naphthalenylcarbonyl)-4-nitro-L-phenylalanine methyl ester] (Abcam), and SB 297006 (N-Benzoyl-4-nitro-L-phenylalanine ethyl ester) (Abcam), N-acetylcystein (NAC) (Wako, Osaka, Japan), or to their vehicle separately. Then, the cells were exposed to 2500 lux of white fluorescent light that the wavelength peak is 403, 435, 546, and 577 nm (Nikon, Tokyo, Japan) for 24 h.

To transfect Ccr3 siRNA, 661W cells were seeded at a density of 1 × 10³ cells/well into 96-well plates without penicillin and streptomycin, and incubated for 24 h. The cells were then transfected with the negative control siRNA or Ccr3 siRNA. After 24 h, the cells were exposed to 2500 lux of visible light for 24 h.

After the light exposure, the cell death rate was calculated by double staining with two fluorescent dyes: Hoechst 33342 (Thermo Fisher Scientific) and propidium iodide (PI; Thermo Fisher Scientific). Hoechst 33342 stains the nuclei of all cells, whereas PI stains only dead cells. To determine the cell death rate, Hoechst 33342 and PI were added to the culture medium for 15 min at final concentrations of 8.1 and 1.5 μM, respectively, at the end of the culture period. Images were recorded with an Olympus IX70 inverted epifluorescence microscope (Olympus, Tokyo, Japan). The total number of cells was counted in a masked way (Y. K.) and the percentage of PI-positive cells was calculated.

For assay of apoptosis, TACS Annexin V FITC Apoptosis Detection kit (Trevigen Inc., Gaithersburg, MD, USA) are used according to manufacturer’s protocol. Briefly, Annexin V FITC (1:10 dilution), PI (1:1 dilution) and Hoechst 33342 (Invitrogen) (1:100 dilution) was diluted in 10x Binding Buffer. The buffer was added into the each wells and cells were incubated for 15 min. Images were recorded with KEYENCE Fluorescence Microscope BZ-X700 (KEYENCE, Osaka, Japan). The total number of cells was counted in a masked way (Y. K.) and the percentage of Annexin V-positive cells was calculated. We counted the nuclei of 300 cells (Hoechst-positive cells) at least for each sample in cell death analysis.

After 24 h of light exposure, the level of caspase-3/7 in the 661W cells was determined by the Caspase-Glo 3/7 Assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol. After the light exposure, caspase-Glo 3/7 reagent was added to the sample at 1:1 ratio, and the cells were incubated for 1 h at 37°C. The luminescence of each sample was measured with a microplate reader (Varioskan Flash 2.4; Thermo Fisher Scientific).

After light exposure, 10 μM of 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate and, acetyl ester (CM-H2DCFDA; Thermo Fisher Scientific), a free radical probe, was added to the cell cultures and incubated for 1 h at 37°C. The radical probe was converted to 2′, 7′-dichlorodihydrofluorescein (DCFH) by the intracellular esterase, and the intracellular DCFH (non-fluorescent) was oxidized to 2′, 7′-dichlorfluorescein (DCF, fluorescent) by intracellular ROS. The intensity of the fluorescence was measured by a Varioskan Flash 2.4 microplate reader (Thermo Fisher Scientific) at 485 nm (excitation) and 535 nm (emission).

The data are presented as the means ± standard error of the means (SEM). Student’s t-test or one-way ANOVA followed by Bonferroni’s test, Dunnett’s test and Tukey’s test [STAT VIEW version 5.0 (SAS Institute, Cary, NC, USA)] were used to determine the significance of any differences. A P < 0.05 was considered to be statistically significant.

C-C chemokine receptor type 3 is expressed in normal murine retinas (Wang et al., 2016) and our western blotting results showed that CCR3 was expressed in 661W cells, a mouse photoreceptor-derived cell line. The expression of CCR3 protein was increased significantly at 12 h after light exposure (Figure 1A). Moreover, the level of the mRNA of Ccr3 was increased at 6 h after light exposure without the increase of Ccl11 which is the ligand of CCR3 (Figures 1B,C). These results suggest that CCR3 signaling may be activated by light exposure although the ligand was not changed.

We then examined whether CCR3 signaling was activated by light exposure and caused cell death. To do this, we investigated whether inhibition of CCR3 by Ccr3 knockdown or CCR3 antagonists will reduce the degree of cell death induced by light exposure. First, we tested the Ccr3 knockdown method on cells of the photoreceptor cell-line with Ccr3 siRNA #1 and #2. Exposure of 661W cells to Ccr3 siRNA #1 decreased the level of the mRNA of Ccr3 by 50% compared to the negative control siRNA. However, siRNA #2 did not alter the level of the mRNA of Ccr3 (Figures 2A,B).

Therefore, we investigated the effect of Ccr3 siRNA #1 on the light-induced death of cells of the photoreceptor cell line. This cell death assay was performed by double staining of the cells with Hoechst 33342 and PI (Nakanishi et al., 2013). The number of PI-positive cells (dead cells) was increased in the light-exposed group, and exposure to Ccr3 siRNA #1 significantly reduced the increase in the cell death compared to the negative control siRNA (Figures 2C,D). An antioxidant, N-acetylcystein (NAC) was used for positive control (Kuse et al., 2014).

Next, two types of CCR3 antagonists were used with the same cell death assay. SB 328437 and SB 297006 are selective antagonists of CCR3 (IC₅₀ for SB 328437 is 4 nM and for SB 297006 is 2.5 μM). SB 328437 is at least 2500 times more selective for CCR3 than for CXCR1, CXCR2, and CCR7, and SB 297006 is at least 250 times more selective for CCR3 than for CXCR1, CXCR2, CCR1, and CCR7. The two CCR3 antagonists reduced the rate of cell death induced by light exposure significantly (Figures 3A–C). Moreover, the rate of apoptosis was evaluated using Annexin V FITC Apoptosis Detection kit. The rate was increased in vehicle-treated group by light exposure. SB 297006 and NAC suppressed the apoptosis (Figures 3D,E). These results indicated that the inhibition of CCR3 signaling can reduce the rate of photoreceptor cell death induced by light exposure.

The activation of caspase-3/7 plays a role in the death of the cells of the photoreceptor cell line induced by light exposure (Wu J. et al., 2002). We evaluated the level of activation of caspase-3/7 with the Caspase-Glo 3/7 Assay. Light exposure activated caspase-3/7, and the CCR3 antagonist, SB 328437, suppressed the activation in a concentration-dependent manner (Figure 4). Oxidative stress caused by ROS is associated with photoreceptor cell death (Organisciak and Vaughan, 2010). We investigated whether the CCR3 antagonist will reduce the rate of 661W cell death through the inhibition of ROS production using a ROS detecting probe. Light exposure induced the production of ROS, and a CCR3 antagonist suppressed the production in a concentration-dependent manner (Figure 5A). Moreover, the decrease of ROS by NAC treatment suppressed the phosphorylation of nuclear factor-kappa B (NF-κB) induced by light exposure (Figure 5B). These results indicated that the inhibition of CCR3 reduced the rate of cell death induced by light exposure through the inhibition of ROS generation and activation of caspase-3/7.