Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis

Abstract

Photoreceptor apoptosis is a major cause of visual loss in retinal detachment (RD) and several other visual disorders, but the underlying mechanisms remain elusive. Recently, increased expression of monocyte chemoattractant protein 1 (MCP-1) was reported in vitreous humor samples of patients with RD and diabetic retinopathy as well as in the brain tissues of patients with neurodegenerative diseases, including Alzheimer's disease and multiple sclerosis. Here we report that MCP-1 plays a critical role in mediating photoreceptor apoptosis in an experimental model of RD. RD led to increased MCP-1 expression in the Müller glia and increased CD11b⁺ macrophage/microglia in the detached retina. An MCP-1 blocking antibody greatly reduced macrophage/microglia infiltration and RD-induced photoreceptor apoptosis. Confirming these results, MCP-1 gene-deficient mice showed significantly reduced macrophage/microglia infiltration after RD and very little photoreceptor apoptosis. In primary retinal mixed cultures, MCP-1 was cytotoxic for recoverin⁺ photoreceptors, and this toxicity was eliminated through immunodepleting macrophage/microglia from the culture. In vivo, deletion of the gene encoding CD11b/CD18 nearly eliminated macrophage/microglia infiltration to the retina after RD and the loss of photoreceptors. Thus, MCP-1 expression and subsequent macrophage/microglia infiltration and activation are critical for RD-induced photoreceptor apoptosis. This pathway may be an important therapeutic target for preventing photoreceptor apoptosis in RD and other CNS diseases that share a common etiology.

Photoreceptor apoptosis is the basis for permanent visual loss in a number of retinal disorders, including macular degeneration (1), retinal detachment (RD) (2, 3), diabetic retinopathy (4), retinopathy of prematurity (5), and retinitis pigmentosa (6). Physical separation of the photoreceptors from the underlying retinal pigment epithelium occurs in rhegmatogenous and in tractional and exudative RD, as well as in neovascular macular degeneration and central serous chorioretinopathy. In these conditions photoreceptors are highly vulnerable and undergo apoptosis (2, 3). In a rodent model of RD we have shown that electroretinograms sensitively reflect RD-induced functional changes and that these electroretinogram changes correlate highly with the amount of photoreceptor apoptosis (7). Although surgery is carried out for rhegmatogenous RD, visual acuity is not always restored because of photoreceptor apoptosis (2, 3). In the other conditions mentioned, serous RD may persist despite treatment, and vision loss progresses because of photoreceptor apoptosis. Therefore, new insights about the mechanisms that underlie photoreceptor apoptosis in RD would be of clinical interest and could lead to new treatments. Previously we demonstrated that caspase activation is associated with RD-induced photoreceptor apoptosis (8). However, suppression of caspases alone is not sufficient to prevent photoreceptor apoptosis (7), because caspase-independent pathways also appear to be involved, although the detailed mechanisms are currently unclear (9).

Monocyte chemoattractant protein 1 (MCP-1, CCL-2) contributes to the recruitment of leukocytes to sites of injury (10) in the pathogenesis of atherosclerosis (11), lung infection (12), angiogenesis (13, 14), and various CNS diseases (15–17). Vitreous samples from patients with RD contain significantly higher levels of MCP-1 than samples from patients with other retinal conditions, such as macular hole or idiopathic premacular fibrosis (18, 19). The MCP-1 receptor CCR2 is expressed on leukocytes, endothelium (20), glial cells, and neurons in the brain (21). Correspondingly, newly discovered functions of MCP-1 include neurodegeneration (22), neuroprotection (23), and increased vessel permeability (20). However, the mechanisms of MCP-1's effect on neurons, and the contribution of direct vs. indirect effects, for instance via macrophage/microglia recruitment, remain to be investigated.

Previously, we and others demonstrated the presence of activated bone marrow-derived monocytes/macrophages and differentiated tissue macrophages, namely microglia, in the detached retina after RD (24, 25). Bone marrow-derived macrophages accumulated subretinally after RD and removed the debris of apoptotic photoreceptors (24). However, the chemoattractant that causes the accumulation of macrophage/microglia in the detached retina and its role in the neurodegeneration have remained unclear. Using an experimental model of RD, the current study demonstrates that MCP-1 plays a critical role in photoreceptor apoptosis by causing macrophage/microglia accumulation and generation of oxidative stress in the injured retina.

Results

MCP-1 Expression After RD.

To investigate the possible role of MCP-1 in RD we examined the expression of MCP-1 mRNA and protein in the retina 72 h after RD using quantitative PCR (TaqMan probe) and ELISAs, respectively. This time point was chosen because the number of TUNEL⁺ photoreceptors peaks by 72 h after RD (7, 26). Quantitative PCR revealed that MCP-1 mRNA levels increased 84-fold (Fig. 1A), whereas protein levels increased 10-fold over baseline (Fig. 1B). Whereas MCP-1 immunoreactivity was very weak in the normal controls (Fig. 1C), spindle-shaped cells, which morphologically resembled Müller glia, were strongly MCP-1-immunoreactive in the outer plexiform layer (OPL) after RD (Fig. 1 D and F). To verify the identity of the MCP-1⁺ cells, double immunostaining was performed by using antibodies against MCP-1 and glutamine synthetase, a Müller cell marker. The two signals colocalized in Müller cells in the OPL (Fig. 1H, arrowheads). These data demonstrate a dramatic increase of MCP-1 mRNA and protein in the mouse retina after RD, specifically in the Müller glial cells.

Fig. 1.

MCP-1 expression is up-regulated after retinal detachment (RD) in mice. (A–D) Up-regulation of MCP-1 after RD. (A) Quantitative real-time PCR data for MCP-1 mRNA 72 h after RD (n = 6). (B) ELISA to detect MCP-1 protein 72 h after RD (n = 6). ∗∗, P < 0.01. (C–E) Immunoreactivity of MCP-1 in control retina (C) or after RD in WT mice (D) or in MCP-1^−/− mice (E). (F–H) MCP-1 localization in Müller glia. Double IHC was carried out with antibodies against MCP-1 (F) and glutamine synthetase, a marker for Müller cells (G). Arrows indicate colocalization. (H) Overlay. (Scale bar: 50 μm.)

Acute Blockade or Genetic Deletion of MCP-1 Prevents RD-Induced Photoreceptor Loss.

To investigate whether MCP-1 is involved in RD-induced photoreceptor apoptosis, we injected a functionally blocking anti-MCP-1 F(ab′) fragment (0.1 μg/μl) subretinally at the time of RD induction. Photoreceptor apoptosis was quantified by TUNEL at 72 h (7, 26). MCP-1 blockade almost completely suppressed the appearance of TUNEL⁺ cells in the outer nuclear layer (ONL), whereas a control IgG1 F(ab′) fragment had no effect (Fig. 2). To understand the role of MCP-1 in this process better, we examined the consequences of RD in MCP-1-deficient (MCP-1^−/−) mice. In the absence of RD, the general appearance of the retina and the thickness of the ONL were similar in MCP-1^−/− and WT mice [supporting information (SI) Fig. 7F]. Seven days after inducing RD, the thickness of the ONL decreased significantly in WT animals (SI Fig. 7 B and F). In contrast, in MCP-1^−/− mice, the thickness of the ONL remained unchanged from baseline after RD (SI Fig. 7 D and F). As an additional parameter for quantifying the role of MCP-1 in RD-induced photoreceptor apoptosis, we performed TUNEL staining 72 h after inducing RD in WT and MCP-1^−/− mice. Under normal conditions, TUNEL⁺ cells are not detected in the ONL of WT or MCP-1^−/− mice (data not shown). After RD, TUNEL⁺ photoreceptors were detected in both groups, although the amount of cell death in MCP-1^−/− mice was ≈80% less than in WT mice (Fig. 3). Transmission EM (TEM) demonstrated cellular shrinkage, chromatin condensation, and apoptotic body formation, signs that were far more prevalent in the ONL of WT mice than in MCP-1^−/− mice (Fig. 3 C, arrows, and D). These data show that MCP-1 plays a critical role in RD-induced photoreceptor apoptosis.

Fig. 2.

An MCP-1 blocking antibody prevents RD-induced photoreceptor loss. (A and B) TUNEL in retinal sections with subretinal injection of control antibody (A) or MCP-1 blocking antibody (B). (Scale bar: 100 μm.) (C) Quantification of TUNEL⁺ photoreceptors 72 h after RD (n = 8 each). ∗∗, P < 0.01.

Fig. 3.

Cytotoxic effect of MCP-1 on RD-induced photoreceptor apoptosis. (A and B) TUNEL 72 h after RD in WT mice (A) and MCP-1^−/− mice (B). (Scale bar: 50 μm.) (C and D) TEM photomicrographs through the ONL 72 h after RD in WT mice (C) and MCP-1^−/− mice (D). Note the increased presence of apoptotic photoreceptors in WT mice compared with MCP-1^−/− mice (arrows). (Scale bar: 10 μm.) (E) Quantification of TUNEL⁺ cells (n = 8 each). ∗, P < 0.05.

Role of CD11b⁺ Macrophage/Microglia in the Detached Retina.

Using an anti-CD11b antibody, immunohistochemistry (IHC) confirmed the previously reported accumulation of CD11b⁺ macrophage/microglia in the subretinal space and in the OPL 72 h after RD induction in WT (SI Fig. 8 C and F) (24, 25). Some CD11b⁺ macrophage/microglia were detected in the inner nuclear layer of normal mice even without RD, and the number of these cells did not changed after RD (data not shown). Double IHC revealed that MCP-1⁺ and most CD11b⁺ cells were in close apposition in the OPL (SI Fig. 8F). Confocal microscopy confirmed this colocalization and further revealed that macrophage/microglia processes were entwined around MCP-1⁺ Müller cells (SI Fig. 8G). To visualize the relationship between macrophage/microglia infiltration in the OPL and TUNEL⁺ photoreceptors in the ONL, we compared the number of CD11b⁺ macrophage/microglia and TUNEL⁺ cells at various time points after RD (SI Fig. 8I). These studies showed that the peak of macrophage/microglia infiltration coincided with the peak of TUNEL⁺ cells at 72 h. CD11b⁺ macrophage/microglia were seen to extend their processes into the ONL and to engulf TUNEL⁺ photoreceptors (SI Fig. 8H). These data suggest that the pathogenesis of RD involves MCP-1 expression, infiltration of CD11b⁺ macrophage/microglia, and RD-induced photoreceptor apoptosis.

MCP-1 Contributes to Macrophage/Microglia Infiltration and Disruption of the OPL in the Detached Retina.

To examine the role of MCP-1 in macrophage/microglia infiltration in more detail, we investigated whether deletion of the MCP-1 gene affects macrophage/microglia infiltration after RD. RD-induced infiltration of macrophage/microglia was strongly suppressed in MCP-1^−/− mice compared with WT mice (Fig. 4). Ultrastructural studies by TEM revealed that invading macrophage/microglia, which were much more prevalent in WT than in MCP-1^−/− mice, could be distinguished from photoreceptors by virtue of their larger somata and less electron-dense nuclei (Fig. 4E, white arrows). Synaptic structures, including cone pedicles and rod spherules between photoreceptors and bipolar cells, were more severely disrupted in the detached retinas of WT than MCP-1^−/− mice (Fig. 4 E and F, black arrows). These data suggest that MCP-1 is critical for the infiltration of macrophage/microglia to the subretinal space and the OPL after RD, and for the subsequent structural and functional disruption of these retinal layers.

Fig. 4.

Markedly reduced number of CD11b⁺ cells in MCP-1^−/− mice. (A and B) IHC with an antibody against CD11b 72 h after RD in WT mice (A) or MCP-1^−/− mice (B). Macrophage/microglia was recruited in the IPL (short arrows) and subretinal space (arrows). (Scale bar: 50 μm.) (C and D) Quantification of CD11b⁺ cells in the OPL (C) and in the subretinal space (D) (n = 8 each). ∗, P < 0.05. (E and F) TEM photomicrographs in the ONL 72 h after RD in WT mice (E) and MCP-1^−/− mice (F). Apoptotic photoreceptors (short white arrow) and invading cells (long white arrows) are more prevalent in WT mice than in MCP-1^−/− mice. The disruption of synaptic structures such as cone pedicles and rod spherules was more severe in WT mice than in MCP-1^−/− mice (black arrows). (Scale bar: 10 μm.)

Cytotoxic Effect of MCP-1 on Cultured Photoreceptors.

To investigate whether the increase in MCP-1 after RD contributes to photoreceptor apoptosis directly, we performed experiments using primary adult retinal cultures. Because these cultures contained a variety of retinal cells (60% photoreceptor), photoreceptors were identified by immunocytochemistry with an antibody against recoverin, a commonly used marker for photoreceptors in vitro (27). Primary cultures also contained CD11b⁺ macrophage/microglia (1%) (Fig. 5C, arrows). To remove the activated macrophage/microglia from these cultures, we carried out immunopanning in dishes precoated with rat anti-mouse CD11b antibody (Fig. 5B). After macrophage/microglia depletion from the cultures, MCP-1 had no effect on photoreceptor survival (Fig. 5 E, F, and I). In contrast, without depletion of CD11b⁺ cells form retinal cultures, the number of recoverin⁺ photoreceptors declined progressively with increasing MCP-1 concentration (Fig. 5 A, B, and I) and MCP-1 concentrations as low as 0.1 ng/ml had significant cytotoxic effects (Fig. 5I). These data suggest that MCP-1's cytotoxicity is mediated through resident macrophage/microglia but not through direct interaction with the cultured photoreceptors. Next, to investigate whether MCP-1's cytotoxicity for cultured photoreceptors was related to oxidative stress, a known cause of photoreceptor damage (28, 29), catalase was added to the culture medium. Catalase reduces oxidative stress through decomposition of hydrogen peroxide, a reactive oxygen species, into water and oxygen (28, 29). The cytotoxic effect of MCP-1 was significantly suppressed with the addition of catalase (Fig. 5I). To examine whether peripheral macrophages also have a cytotoxic effect for cultured photoreceptors, we added peripheral macrophages into retinal cultures after depletion of the resident macrophage/microglia with or without MCP-1. Addition of peripheral macrophages at a ratio of 1% of cultured cells restored MCP-1's cytotoxicity through oxidative stress (Fig. 5 F and G). Interestingly, peripheral macrophages derived from Mac-1 (CD11b/CD18) gene-deficient (Mac-1^−/−) mice did not have cytotoxic effects even after MCP-1 stimulation (Fig. 5J). These data suggest that the cytotoxic effect of MCP-1 on cultured photoreceptors is dose-dependent, is mediated through activated macrophage/microglia, and is likely due to oxidative stress.

Fig. 5.

CD11b⁺ cells mediate the cytotoxic effect of MCP-1 on cultured photoreceptors. (A and B) Recoverin⁺ photoreceptors in retinal primary culture with (B) or without (A) MCP-1. (C and D) CD11b⁺ cells before (C) or after (D) depletion by immunopanning. Arrows indicate CD11b⁺ cells. (E and F) Recoverin⁺ photoreceptors with (F) or without (E) MCP-1 (1 ng/ml) after depletion of CD11b⁺ cells. (G and H) Recoverin⁺ photoreceptors (red) and peripheral macrophage (green) in culture in the presence (H) or absence (G) of MCP-1 (1 ng/ml) after depletion of CD11b⁺ cells. (Scale bar: 100 μm.) (I) Dose–response curve of MCP-1 cytotoxicity on the cultured photoreceptors in the presence or absence of resident CD11b⁺ cells. ∗ (P < 0.05) and ∗∗ (P < 0.01) represent the significance when compared with controls without MCP-1. Catalase (2 μg/ml) suppresses MCP-1-induced photoreceptor loss. (J) Dose–response curve of MCP-1 cytotoxicity on cultured photoreceptors after added peripheral macrophage (PM).

Mac-1^−/− Mice Are Protected Against RD-Induced Photoreceptor Apoptosis.

To examine whether the cytotoxic effects of MCP-1 are mediated by macrophage/microglia in vivo, we induced RD in mice deficient for the Mac-1 integrin, a critical receptor for leukocyte recruitment and activation (30, 31). Without RD, the number and morphology of CD45⁺ cells in the OPL was similar in Mac-1^−/− and WT mice (SI Fig. 9 A, B, and E). However, 72 h after RD, significantly fewer CD45⁺ cells were found in Mac-1^−/− compared with WT mice (SI Fig. 9). Interestingly, the morphology of these macrophage/microglia after RD resembles that of resting cells. In contrast to WT mice, Mac-1^−/− mice did not show a decrease in ONL thickness (SI Fig. 10) or an increase in the number of TUNEL⁺ photoreceptors (Fig. 6), suggesting an important role for Mac-1-mediated infiltration and activation of macrophages/microglia during RD-induced injury.

Fig. 6.

Deletion of the Mac-1 gene prevents RD-induced photoreceptor apoptosis. (A and B) TUNEL in retinal sections of WT mice (A) or Mac-1^−/− mice (B). (Scale bar: 100 μm.) (C and D) TEM photomicrographs in the ONL 72 h after RD in WT mice (C) and Mac-1^−/− mice (D). Apoptotic photoreceptors (arrow) are more prevalent in WT mice than in Mac-1^−/− mice. (Scale bar: 10 μm.) (E) Quantification of TUNEL⁺ photoreceptors 72 h after RD (n = 8 each). (F) Quantification of TUNEL⁺ photoreceptors with or without PBN treatment. ∗∗, P< 0.01.

Role of Oxidative Stress in RD-Induced Injury in Vivo.

To examine the role of oxidative stress on RD-induced photoreceptor apoptosis, the antioxidant PBN (100 mg/kg per day) was administered for 3 days after RD. RD-induced MCP-1 expression was not changed by PBN treatment, nor was the number of recruited macrophage/microglia in the OPL (data not shown). i.p. administration of PBN significantly suppressed RD-induced photoreceptor degeneration (P = 0.007) (Fig. 6F), suggesting that the RD-induced photoreceptor apoptosis in vivo is likely due to oxidative stress.

Discussion

Using an experimentally induced model of RD in mice, we show that MCP-1 is a critical mediator of photoreceptor apoptosis, a major cause of visual loss in several retinal disorders. MCP-1 levels rapidly rise in Müller glial cells after RD and lead to an increased number of macrophage/microglia into the site of the injury. Acute blockade of MCP-1 with a functionally blocking antibody or deletion of its gene in mice almost completely eliminates RD-induced photoreceptor apoptosis. We further show that the cytotoxic effect of MCP-1 on cultured photoreceptors is not direct, but a consequence of oxidative stress produced by activated macrophage/microglia. Deletion of the gene for either MCP-1 or Mac-1 (CD11b/CD18) in mice almost completely eliminated infiltration of macrophage/microglia after RD and protected photoreceptors from RD-induced apoptosis.

Increased MCP-1 expression has been reported in several other retinal disease models, including light damage (32), uveitis (33), diabetic retinopathy (34), retinitis pigmentosa (35), and ischemia–reperfusion (36). Thus, MCP-1 may be an important factor for macrophage/microglial responses during various acute and chronic retinal disorders. In the retinal ischemia–reperfusion model, MCP-1 up-regulation is detected only in the inner retina (36), corresponding to the area of retinal injury (37). In the current study MCP-1 protein expression was detected in Müller cells, especially in the OPL. The OPL has a rich vascular capillary bed, which would facilitate extravasation of leukocytes (38). The colocalization of MCP-1⁺ Müller cells and activated macrophage/microglia in the OPL, along with the decreased number of infiltrated macrophage/microglia in MCP-1^−/− mice after RD, indicates that increased MCP-1 in Müller cells attracts macrophage/microglia toward the outer retina. This finding is consistent with the fact that the outer retina is the main site of injury after RD. Shen et al. (39) have shown that Matrigel-induced angiogenesis and associated photoreceptor degeneration can be severe even in the absence of MCP-1, presumably because of the harmful effect of pathologic angiogenesis on photoreceptor viability. That study indicates an absence of macrophage/microglia recruitment in the absence of MCP-1, consistent with our findings.

Endothelin2 has previously been shown to play an important role in Müller cell activation in various types of retinal injury (40). We have confirmed that the expression of endothelin2 mRNA increased 4.5-fold 6 h after RD, although not at 3 h. Our recent data show that expression of MCP-1 mRNA was already elevated 1 h after RD and continued to be elevated over 3 days (41). These data suggest that the increased expression of MCP-1 occurs earlier than that of endothelin2, although it is possible that these two molecules act together to alter retinal responses after RD.

Photoreceptor apoptosis and degeneration of the ONL and OPL were nearly eliminated after RD when MCP-1 was blocked or its gene was deleted. These data indicate that suppression of MCP-1 has beneficial effects on RD-induced photoreceptor death and retinal degeneration in vivo. In line with our in vivo results, in primary retinal culture experiments we showed that MCP-1's cytotoxicity is mediated through macrophage/microglia activation and not through a direct effect on photoreceptors. MCP-1's cytotoxicity in this system was seen at a concentration as low as 0.1 ng/ml and peaked at 1 ng/ml (Fig. 5). A low concentration of MCP-1 similar to that which we find cytotoxic (i.e., 1 ng/ml) was also shown to be chemotactic for macrophage/microglia (42). In contrast, markedly higher concentrations of MCP-1 are required for other effects reported in vitro, including the modulation of Ca²⁺ dynamics in neurons (200 ng/ml) (43), degeneration or protection of neurons (10–100 ng/ml) (22, 23), endothelial cell migration (10–100 ng/ml), or a decrease in transendothelial cell electrical resistance (1 μg/ml) (20). These data suggest that macrophage/microglia are more sensitive to MCP-1 than other cells and that the cytotoxic effect of MCP-1 occurs primarily through activated macrophage/microglia both in vivo and in vitro.

The origin of the macrophage/microglia seen in the OPL after RD remains largely unknown. It is possible that resident macrophage/microglia translocate and proliferate in the OPL, based on previous reports showing the importance of resident monocytes in human, cat, rabbit, and ground squirrel RD (25). These cells may also represent newly recruited peripheral blood monocytes, consistent with our previous finding that bone marrow-derived macrophages are recruited to the subretinal space after RD (24). In normal mice we found that resident macrophage/microglia were detected mainly in the inner retina (GCL and IPL) but not in the OPL. Even after RD, the number of resident macrophage/microglia remained unchanged in the inner retina and very few macrophage/microglia, which had just migrated from the IPL to OPL through the inner nuclear layer, were detected in the inner nuclear layer (data not shown). Furthermore, after RD, some of the CD11b⁺ cells in the OPL showed immunoreactivity for CD68 and F4/80, markers for peripheral macrophages (data not shown). In vitro, both resident macrophage/microglia and isolated peripheral blood monocytes were cytotoxic to cultured photoreceptors after MCP-1 stimulation. Thus, although the exact origin of the CD11b⁺ macrophage/microglia in the OPL cannot be pinpointed, both resident and recruited macrophage/microglia may mediate MCP-1's cytotoxicity. However, our experiments show that CD11b⁺ macrophage/microglia are necessary for MCP-1's cytotoxic effect on cultured photoreceptors.

To further confirm the role of macrophage/microglia in RD-induced photoreceptor apoptosis, we studied mice with a deficit of macrophage/microglia recruitment. Mac-1 (CD11b/CD18) is a β2 integrin with an established role in monocyte recruitment to peripheral nonocular tissues (44). Mac-1^−/− mice show a decreased susceptibility to brain ischemia–reperfusion injury relative to WT mice (45). Activated macrophage/microglia express high amounts of Mac-1 in the retina (46). Here we show that Mac-1^−/− mice have significantly fewer macrophage/microglia and fewer signs of morphological activation both in the OPL and in the subretinal space than WT mice (SI Fig. 9). These data suggest that Mac-1 is an important adhesion molecule for infiltration and activation of macrophage/microglia after RD. Macrophage/microglia have recently been reported to promote apoptosis of developing Purkinje cells by engulfing and terminating these apoptotic cells producing superoxide ions (47, 48). Consistent with these findings, in the current study oxidative stress was found to mediate the cytotoxic effect of MCP-1 both in vitro (Fig. 5) and in vivo (Fig. 6F). Generally, oxidative stress is one of the major cytotoxic factors for photoreceptor death in various pathological conditions (28, 29). In contrast to the neurotoxic effects of oxidative free radicals, molecular oxygen (O₂) was found to be neuroprotective when given even 1 day after inducing RD in cats (49). Although the mechanism underlying the protective effect of molecular oxygen was not elucidated, it is possible that it prevented hypoxia-induced MCP-1 expression in retinal glia (50); this in turn would prevent the activation of macrophage/microglia and generation of oxidative free radicals, the final effectors of photoreceptor cell death. Thus, suppression of macrophage/microglia or antioxidant treatment may thus represent alternative strategies for neuroprotection against photoreceptor degeneration.

In conclusion, we demonstrate that MCP-1 up-regulation plays a critical role in inducing photoreceptor apoptosis after RD. The cytotoxic effect of MCP-1 on photoreceptors is mediated through its chemotactic properties and possibly macrophage/microglia-generated oxidative stress. Blockade of MCP-1 may open new therapeutic avenues to treat photoreceptor death in the setting of various retinal disorders as well as other CNS disorders that share a common etiology.

Materials and Methods

Animals.

All animal procedures were performed in accordance with the statement of the Association for Research in Vision and Ophthalmology and the protocol approved by the Animal Care Committee of the Massachusetts Eye and Ear Infirmary. Adult male MCP-1^−/− mice, Mac-1^−/− mice (C57BL/6 background, 20–25 g; The Jackson Laboratory, Bar Harbor, ME), and age- and sex-matched C57BL/6 mice were housed in covered cages. A total of 28 MCP-1^−/− mice, 28 Mac-1^−/− mice, and 128 WT mice were used for this study.

Surgical Induction of RD.

Induction of RD and subretinal injection of MCP-1 blocking antibody [11K2, mouse F(ab′) IgG1, 0.1 μg/μl; Biogen, Cambridge, MA] or isotype control (Southern Biotechnology Associates, Birmingham, AL) were performed as previously described (Fig. 4E) (7–9, 24, 41). The blocking effect for anti-MCP-1 F(ab′) antibody has been established in a mouse arteriosclerosis model (51). RD was created only in the right eye of each animal, with the left eye serving as a control.

RNA Extraction, RT-PCR, and Quantitative Real-Time PCR.

Total RNA extraction and reverse transcription were performed as previously reported (41, 52). PCR primers for MCP-1 used in this study are as follows: mMCP1 forward, 5′-ACTCACCTGCTGCTACTCATTCACC-3′; mMCP1 reverse, 5′-CTACAGCTTCTTTGGGACACCTGCT-3′; and mMCP1, VIC-ATC CCA ATG AGT AGG CTG GAG AGC TAC AAG AGG ATC-TAMRA. For relative comparison of each gene, we analyzed the Ct value of real-time PCR data with the ΔΔCt method normalizing by an endogenous control (18S ribosomal RNA) (41, 52, 53).

ELISA.

The posterior lens capsule, vitreous and neural retina combined, was collected 72 h after RD. Protein extraction and ELISA were performed as previously described (41). One hundred micrograms of total protein was used for ELISA of MCP-1 (BioSource, Camarillo, CA).

IHC.

IHC was performed as previously reported (41, 54). Rabbit anti-MCP-1 (1:100; PeproTech, Rocky Hill, NJ), rat anti-mouse CD11b (1:50; Serotec), rat anti-mouse CD45 (1:50; Pharmingen), or mouse anti-glutamine synthetase (1:100; Chemicon, San Diego, CA) were used as primary antibodies.

TUNEL.

TUNEL and quantification of TUNEL⁺ cells were performed as previously described (41) by using the ApopTag Fluorescein In Situ Apoptosis Detection Kit (S7110; Chemicon International, Temecula, CA). The center of the detached retina was photographed, and the number of TUNEL⁺ cells in the ONL was counted in a masked fashion. The area of the ONL was measured with OpenLab software.

TEM.

TEM was performed as previously described (7, 24). Eyes were fixed in 1% glutaraldehyde/1% paraformaldehyde in PBS and postfixed in veronal acetate buffered osmium tetroxide (2%), dehydrated in ethanol and water, and embedded in Epon. Ultrathin sections were cut from blocks and mounted on copper grids. The specimens were examined by using a Philips CM10 electron microscope.

Adult Mouse Retinal Primary Cultures.

Adult primary retinal cultures were prepared as previously described (54) with minor modifications. Neural retinas were incubated at 37°C for 20 min in a CO₂ incubator in digestion solution containing papain (10 units/ml; Worthington) and l-cysteine (0.3 mg/ml; Sigma, St. Louis, MO) in Hanks' buffered saline solution (HBSS). Cell density was adjusted to 3.5 × 10⁴ cells per well of an eight-well chamber (Nunc) with Neurobasal A medium (Invitrogen) containing B27 supplement without antioxidants (NBA/B27AO−; Invitrogen) and 1 μg/ml insulin, 2 mM l-glutamate, and 12 μg/ml gentamicin. One hour later, MCP-1 at specified concentrations was added to culture medium, and incubation was continued for 24 h. To assess the viability of photoreceptors, we performed immunocytochemistry with rabbit anti-recoverin antibody (1:500 dilution, AB5585; Chemicon). The number of recoverin⁺ photoreceptors was counted at 10 random fields per well in a blind fashion by using ImageJ software. Values are given as the mean ± SEM of four replicate wells. An immunopanning dish was prepared by incubating 10-cm culture dishes (Falcon) with 50 μg/ml rat anti-CD11b antibody (Serotec) in 4 ml of HBSS overnight. The panning dish was blocked with 4 ml of HBSS/0.1% BSA for 1 h, and dissociated cells were incubated in the dish for 30 min, with slow agitation of the dish every 10 min. Peripheral CD11b⁺ macrophage was collected as previously described (55) and added to retinal primary culture after depletion of resident macrophage/microglia.

Statistical Analysis.

The statistical significance of RT-PCR and ELISA results was determined by using unpaired t tests. The data from the TUNEL and in vitro survival assays were analyzed with the Scheffé post hoc test by using StatView 4.11J software for Macintosh (Abacus Concepts, Berkeley, CA). The significance level was set at P < 0.05 (* in figures) and P < 0.01 (** in figures). The data represent mean ± SD except for primary culture results.

Abbreviations

RD

retinal detachment

MCP-1

monocyte chemoattractant protein 1

OPL

outer plexiform layer

IHC

immunohistochemistry

ONL

outer nuclear layer

TEM

transmission EM.