|Molecular Vision 2005;
Received 16 September 2004 | Accepted 27 October 2005 | Published 27 October 2005
Activation of microglia and chemokines in light-induced retinal degeneration
Cheng Zhang,1 Ji-kui
Shen,1 Tim T. Lam,2,3 Hui-yang Zeng,1,4 Samuel K.
Chiang,1 Fang Yang,1 Mark O. M.
1Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD; 2Department of Ophthalmology, Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA; 3Pharmacology, R&D, Bausch & Lomb, Rochester, NY; 4Beijing University Eye Center, Beijing, China
Correspondence to: Cheng Zhang, MD, Wilmer Eye Institute, Johns Hopkins Hospital, Woods Building, Room 457, 600 North Wolfe Street, Baltimore, MD, 21287-9238; Phone: (410) 614-0229; FAX: (410) 614-1114; email: firstname.lastname@example.org
Purpose: Microglial cells, which are activated and recruited by chemokines, have been shown to play crucial roles in neuronal degenerations of the central nervous system (CNS). This study investigated the activation and migration of retinal microglial cells and expression of chemokines in retinas in light-induced photoreceptor degeneration in mice.
Methods: Ninety-five Balb/cJ mice were kept in cyclic light for 1 week followed by dark adaptation for 48 h prior to light exposure of 3 h at 3.5 Klux. Animals were enthuanized at various times after light exposure. Terminal deoxynucleotidyl transferase-mediated dUTP nick end label (TUNEL) assay, rat-anti-mouse CD11b and 5D4 antibodies, isolectin-B4, and a chemokine-specific gene array were used to detect DNA fragmentation during retinal degeneration, to label retinal microglial cells, and to determine the expression of retinal chemokines and chemokine receptors, respectively. Reverse-transcriptase coupled polymerase chain reactions (RT-PCRs) were conducted on selected chemokine mRNAs to confirm the gene array findings.
Results: After intense light exposure, TUNEL-positive cells were noted in the outer nuclear layer (ONL) of the retina at 3 h, and their presence were noticeably increased at 1 day but declined at 3 days and 7 days after light exposure. In contrast, CD11b- or isolectin-B4-positive cells were seen in the ONL as early as 6 h and their presence increased significantly at 1 day and 3 days after light exposure. These cells displayed a round or ovoid morphology at 6 h and 1 day but assumed a more ameboid configuration at 3 days. By 7 day, the number of the microglial cells declined in the ONL and they became ramified, and were present mostly in the subretinal space. 5D4-positive cells with large cell bodies were only noted at 3 day and 7 day but not earlier. With chemokine-specific gene array analysis, we identified four chemokines and two chemokine receptors showing significant increases in their gene expressions. Among them, monocyte chemoattractant protein-3 (MCP-3), showed a remarkable 4.4 fold increase in its gene expression. RT-PCR confirmed a marked increase of MCP-3 expression in retinas at 3 h to 1 day, and a return to normal at 3 days following light injury.
Conclusions: Retinal chemokines such as MCP-3 and their receptors are involved in the activation and migration of retinal microglia in light-induced retinal degeneration, which in turn modulate the apoptotic loss of photoreceptor cells in the outer retina.
In photoreceptor degeneration, microglial cells have been shown to be activated and migrated from inner retinal layers to the outer nuclear layer (ONL) and the subretinal space of the retina. Examples of this microglial activation were reported in retinal degeneration of the retinal dystrophic Royal College of Surgeon (RCS) rats [1-3] and of the Type II photic injury to the retina , which is characterized by degeneration of only the photoreceptor, as described by Noell in 1980 . However, the roles and mechanisms of microglial activation and migration in retinal degeneration remain to be examined.
Chemokines are proteins of low molecular weight (8-10 kDa), and are major chemoattractants for microglial activation and migration in many diseases of the central nervous system (CNS). They are divided into two major subfamilies according to the arrangement of the first two N-terminal cysteines: the α-chemokines (CXC family), in which the two amino acids nearest the N-terminus of these proteins are separated by a single amino acid, and the β-chemokines (CC family) in which the two cysteines are adjacent. The α-chemokines primarily function as neutrophil chemoattractants, while the β-chemokines, such as monocyte chemoattractant proteins (MCP)s, macrophage inflammatory protein (MIP)-families, and RANTES (regulated upon activation, normal T cell expressed and secreted) function primarily as chemoattractants for monocyte and other leukocyte cell types. Profiles of chemokine expression in the CNS following autoimmune and post-traumatic inflammation have been shown to correlate well with the composition of leukocyte infiltration . Various chemokines have been shown recently to be involved in neuronal degeneration of the CNS and the retina [7-9]. For example, MCP-1, a member of the CC chemokine family, was first identified by its role in promoting the infiltration and accumulation of macrophages in tumors. In Alzheimer disease, MCP-1 was found in mature senile plaques and reactive microglia . After an ischemic insult, Wang et al.  observed in the ischemic cerebral cortex a significant increase in MCP-3 gene expression, which paralleled leukocytic infiltration and accumulation, suggesting a role for MCP-3 in recruiting these inflammatory cells into the ischemic tissue. It is believed that chemokine production may vary with different pathological paradigms. Even though many chemokines may elicit similar cellular infiltration profiles, only a few may be expressed in a particular setting .
This study was conducted to examine microglial responses using three microglial cell markers and to explore the possible mechanisms of microglial activation and migration in the Type I retinal photic injury, which shows damage to both photoreceptors and the retinal pigment epithelium, as defined by Noell's classic study  on photic injury to retinas.
Retinal photic injury model in mice
Photic injury to mouse retinas was carried out as described previously . Briefly, ninety-five albino Balb/cJ male mice (Jackson Laboratories, Bar Harbor, ME), aged 4-5 weeks, were housed under 12 h light (15 lux)/12 h dark cycles and were dark-adapted for 2 days before light exposure. The mice were exposed to green-filtered fluorescent light (Plexiglas number 2092 filter, Polycast Technology Corp., Stamford, CT) at an intensity of 3.5 Klux for 3 h. For immunohistochemical studies, the mice were euthanized at 0 h, 3 h, 6 h, 1 day, 3 days, or 7 days of dark recovery (three mice at each time). Three mice reared under cyclic light, followed by 48 h of dark adaptation were used as controls. All experimental procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care Committee at the Johns Hopkins University.
Animals were euthanized with an overdose of pentobarbital and their eyes were immediately enucleated and placed in freshly prepared 4% (w/v) paraformaldehyde fixative in PBS for 1 h. The anterior segments were removed and the posterior segments were further fixed in the same fixative for an additional period of 5 h. The tissue samples were transferred to 20% sucrose buffer overnight at 4 °C for cryoprotection and then embedded in OCT compound. Frozen sections were cut 8 μm thick with a cryostat and the sections were kept in a -80 °C freezer until use. For histology, the eyes were fixed in 2% paraformaldehyde and 1% glutaraldehyde fixative followed by 1% osmium tetroxide fixation and embedded in Araldite. Semi-thin sections were stained with toluidine blue for light microscopy.
Terminal dUTP transferase mediated nicked-end labeling (TUNEL) and immunohistochemistry
After rinsing in 0.1 M phosphate buffered saline (PBS) pH 7.0 and quenched with 0.3% H2O2 in methanol, retinal sections were incubated with terminal dUTP transferase and digoxigenin-dUTP in TdT buffer (TUNEL kit from Intergen Inc., Purchase, NY) for 60 min at 37 °C in a moist chamber. Following rinsing, the sections were reacted with anti-digoxigenin conjugate and developed in DAB.
For immunohistochemistry, the rat anti-mouse monoclonal antibody CD11b (1:50 dilution; Serotec, Raleigh, NC) and mouse monoclonal antibody 5D4 (1:800 dilution; Saikagaku, Tokyo, Japan) were used as primary antibodies. Immunoperoxidase staining was performed on frozen sections using ABC kits from Vector Labs (Burlingame, CA). For fluorescent immunostaining procedures, anti-mouse secondary antibodies conjugated with Cy-3 (1:100 dilution; Jackson ImmunoRes, West Grove, PA) were used. Briefly, the sections were incubated with a primary antibody overnight at 4 °C followed by a secondary antibody for 1 h at room temperature. Negative controls without the primary antibodies or with isotype controls using a corresponding IgG fraction were also performed. Biotinylated isolectin-B4 (Sigma, St. Louis, MO) was also used for labeling microglial cells. The slides were examined and photographed with a laser confocal microscope (Ultraview-2, Perkin Elmer, Wellesley, MA) or a conventional microscope with a digital camera (Axioskop2, Zeiss, Thornwood, NY).
Gene array and real-time PCR
Chemokine-specific membranes (NonRad GEArray Q series; SuperArray, Bethesda, MD), which contained 96 chemokines and their receptor genes, were used for gene array analysis with retinal samples obtained at 0 h and 3 h after retinal photic injury. Total RNA was isolated from mouse retinas using RNeasy Mini kits (Qiagen, Valencia, CA). Total RNA (3 to 5 μg) was isolated from four pooled retinas. Analysis was performed by following the manufacturer's protocol. Briefly, 1 μg total RNA was reverse transcribed to cDNA probe. Hybridization was performed in a custom-made hybridization cylinder and the signal was detected using a chemiluminescent method. The exposed X-ray film was scanned and analyzed by the GEArrayAnalyzer software from SuperArray. The study was performed using pooled retinal samples and the reported values are the averages of two trials. Detailed gene array technical information was listed at SuperArray (mouse chemokine gene array membrane, Catalog number MM-005N).
We performed a reverse-transcriptase coupled polymerase chain reaction (RT-PCR) assay for MCP-3 to confirm the results of the chemokine-specific gene array study after retinal photic injury. Briefly, 0.5 μg total RNA was used for reverse transcription using the kits from Invitrogen (SuperScript II First-Strand Synthesis System for RT-PCR) following the manufacturer's instructions. PCR amplification was performed with 30 cycles of 30 s denaturation (94 °C), 30 s annealing (for individual temperatures see Table 1) and 40 s elongation (72 °C) using Taq DNA polymerase (Invitrogen). PCRs were performed in duplicate samples with a Genius thermocycler (Techne Ltd., Duxford, Cambridge, UK). PCR products were separated by electrophoresis in a 1% agarose gel containing ethidium bromide (500 ng/ml) and photographed.
Quantitative real-time PCR after reverse transcription was performed to verify the increase of MCP-3 gene expression identified in the preceding chemokine gene array study and RT-PCR. RNAs isolated from normal mice and animals at 24 h after light damage were used for reverse transcription. GAPDH was used as the reference standard (normalizer). Real-time PCR was performed using a LightCycler rapid thermal cycler system (Roche Applied Bioscience, Indianapolis, IN) according to the manufacturer's instructions. Reactions were performed in 20 μl with 0.5 μM primers and 2 mM MgCl2. Nucleotides, Taq DNA polymerase, and buffer were included in the LightCycler-DNA Master SYBR Green I mix (Roche Diagnostics Ltd.). Murine MCP-3 and GAPDH standards were made by PCR amplification using Pfu Taq enzyme (Stratagene, La Jolla, CA), and the PCR products were purified by a QIAquick Gel Extraction Kit (Qiagen). The absolute mRNA copy numbers were calculated according to Roche's procedures in their absolute quantification technique manual. Standard curves for each gene were plotted with quantified cDNA templates during each real-time PCR. MCP-3 gene mRNA copy numbers were then normalized to 106 copies of the control GAPDH gene. PCRs were run in duplicate and were repeated once.
Photoreceptor degeneration in retinal photic injury model
At 24 h after 3 h of intense light exposure, the RPE cells were swollen, the inner and outer segments were disorganized and the outer nuclear layer (ONL) showed abundant pyknotic nuclei (Figure 1A). Considerable photoreceptor cell loss was noticeable at 3 days following light exposure (Figure 1). Quantitation of the progressive loss 7 days after photic injury was previously described . TUNEL-positive cells were seen in the ONL as early as 3 h of dark recovery (not shown), and their numbers reached a peak at 1 day of dark recovery. The number of these cells gradually decreased at 3 days and 7 days of dark recovery, exhibiting a thinning of the ONL (Figure 2).
Microglial activation and migration in retinal photic injury
Three cell markers, CD11b and 5D4 antibodies and isolectin-B4, were used to identify microglia. In normal retinas, a few ramified CD11b-positive microglial cells with thin, long cellular processes were scattered in the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), but no CD11b-positive cells were seen in the ONL or subretinal space (Figure 3). Six hours after photic injury there was an early appearance of CD11b-positive cells in the ONL, followed by a sharp increase in numbers at 1 day and 3 days after photic injury with a few cells in the subretinal space. These cells appeared round or oval at 6 h and 1 day but became amoeboid at 3 days. By 7 days, a decrease of CD11b-positive cells was noted in the ONL with some remaining ramified microglial cells in the subretinal space.
In normal retinas, few 5D4-positive cells, a subset of ramified microglial cells in CNS and retina, were seen in the inner retina and only occasionally observed in the outer retina. In contrast to the early appearance of CD11b-positive cells in the outer retina, no 5D4-positive cells were observed in the outer retinas of light-exposed animals (Figure 4B) after 1 day of dark recovery. Plump 5D4-positive microglial cells with distinct cellular processes were noted in the subretinal space at 3 days of dark recovery (Figure 4C) and persisted at 7 days (Figure 4D).
No isolectin-B4-positive cells (Figure 5) were seen in the ONL in normal retinas, except the labeling of some small retinal vessels. At 1 day of dark recovery, round isolectin-B4-positive cells were seen in the ONL in clusters. At 3 days of dark recovery, more positive cells were seen in the ONL. The number of isolectin-B4-positive cells began to decrease at 7 days of dark recovery as the photoreceptor degeneration subsided.
Chemokine gene expression
Using a chemokine-specific gene array, we identified markedly increased gene expressions (greater than or equal to 2 fold) of five chemokines: monocyte chemoattractant protein-3 (MCP-3, Scya7), small inducible cytokine A21a (leucine, Scya21a), small inducible cytokine A6 (C10, Scya6), small inducible cytokine subfamily B member 15 (Scyb15), and erythropoietin (Epo), and two chemokine receptors: Burkitt lymphoma receptor (Blr1, CXCR5), and chemokine (CC) receptor 7 (Cmkbr7, CCR-7) at 3 h after retinal photic injury (Table 2). Compared to their respective normal values, Scya21a, CXCR5, and MCP-3 showed the biggest changes (>4.2 fold) at 3 h after light exposure.
MCP-3 of the β-chemokine (CC chemokine) family was selected for RT-PCR and real-time PCR to confirm the gene array study. With RT-PCR (Figure 6A), MCP-3 gene expression was undetectable in the normal retinas (Lane N) but significantly increased in retinas at 3 h and 24 h of recovery after photic injury, and sharply decreased at 72 h. With real-time PCR, the ratio of MCP-3 transcripts per million GAPDH transcripts was 714.5±185.6 (mean±SD, n=6) in normal retinas, and was significantly increased to 2065.9±657.2 (n=4) in retinas after 24 h of recovery from photic injury (p=0.011, compared to normal; Figure 6B).
Our experiments demonstrated that retinal microglial cells were activated and migrated to the outer retina in the light-induced Type I photoreceptor degeneration. In addition, we noted that the appearance of photoreceptor degeneration as indicated by TUNEL paralleled the increased level of the chemokine MCP-3 and preceded microglial infiltration. We hypothesize that damaged photoreceptor cells produce chemokines that attract and activate retinal microglial cells, which may modulate retinal degeneration through many bioactive molecules.
In Noell's classic study  on photic injury to the retina, he described two types of injuries: Type I was induced by exposure to high intensity light for 24-48 h resulting in extensive degeneration of both photoreceptors and retinal pigmented epithelium (RPE); and Type II was induced by long-term exposure to moderate light intensity resulting in exclusive degeneration of photoreceptors. Similar to Ng and Streilien's study  on the Type II photic injury, we also noted an invasion of microglial and macrophagic cells into the ONL and the subretinal space during the retinal degenerative process. Similarly, an earlier study on the retinal dystrophic retinas of the RCS rat also showed the presence of these cells in the outer retina and the subretinal space as photoreceptors degenerated [1-3]. A recent study in our laboratory showed a neuroprotective effect of minocyline on photic injury with a concomitant suppression of microglial activation and invasion in the outer retina . These observations suggest that microglia may play crucial roles in photoreceptor degenerations. Earlier studies showed that the activation and invasion of microglia in Type I photic injury and the retinal degeneration of RCS rats [15-17], but the roles of microglia in apoptotic degeneration of photoreceptors need to be clarified. In many neuronal degenerative diseases such as multiple sclerosis, Parkinson, and Alzheimer diseases, the microglial cells were activated by the injured neurons [18-20]. It is believed that the activated microglial cells phagocytosed the diseased neurons in the CNS and the retina as well as secreted many pro-inflammatory cytokines, which might be cytotoxic to neurons. Whether these cells in the retina have similar functions and the relationship between microglial activation and apoptotic loss of photoreceptors remain to be examined.
In our study, TUNEL-positive cells were seen at 3 h and CD11b- and isolectin-B4-positive cells were seen at 6 h following light exposure. CD11b antibody and isolectin-B4 are the most common markers for resting ramified and activated ameboid microglia in the CNS . 5D4 antibody (an epitope of keratan sulfate proteoglycan, KSPG) is a recently identified marker for a subpopulation of ramified microglial cells in the brain [22,23]. The 5D4-positive cells are present throughout the CNS parenchyma, but their number is fewer than that observed with isolectin-B4 or with CD11b. In the normal retina, 5D4-positive cells were rarely seen in the subretinal space, which may be due to the lower light intensity of cyclic light condition in our experimental setting compared to the study by Ng and Streilein . However, many 5D4-positive cells were seen in the subretinal space at a late phase of photoreceptor degeneration, while CD11b- and isolectin-B4-positive cells appeared at an early stage following retinal photic injury. These findings seem to suggest that in the retina there are heterogenous populations of microglial cells that have different roles during photoreceptor degeneration.
The origin of retinal microglia in photoreceptor degeneration is in dispute. Several studies have demonstrated that activated retinal microglial cells migrated into the ONL and subretinal space in the photoreceptor degeneration in RCS rats [1-3,24]. Using retrolabeling technique, Moore and Thanos  showed these microglial cells come from the inner retina. Using the phosphotyrosine and ED2 immunolabeling, Roque et al.  showed the microglia in the outer retinas in RCS rats are resident microglial cells and blood-born macrophages were not involved in the retinal degenerative process. In contrast, in another study of eyes with the breakdown of blood retinal barrier induced by needle puncture of the globe, many large round macrophages were present in the retina with intense ED2-positivity. However, Akaishi et al.  showed that some MHC-II positive cells also express ED1 but not OX42 in the outer retinas of RCS rats, suggesting that blood-born macrophage might be involved in the retinal degeneration. Ng's recent study  suggested that in photic injury microglia migrate from inner to the outer retinal layers. In our study, we did not plan to demonstrate the origin of the activated retinal microglia, but the infiltrated microglia first appeared along the inner part of ONL suggesting that they might have migrated from the inner retinal layers.
Chemokines in microglial activation and migration
Among the MCP-family members, which are considered to be the principal chemokines in the recruitment of monocytes and macrophages, MCP-1 is the most extensively studied. There are at least five members of the MCP family, namely MCP-1 through MCP-5. MCP-2 and MCP-3 are closely related to MCP-1 with 70% homology amino acid sequence. Both MCP-2 and MCP-3 show activities similar to MCP-1 with the ability of attracting microglia and monocytes, with the exception that MCP-3 has also been implicated in the chemotaxis of dendritic cells and eosinophils [26,27]. Recent in vivo studies demonstrated MCP-1 is involved in the neuronal degenerative, ischemic and inflammatory diseases in the brain [28-31]. However, in our chemokine gene array study, MCP-1 gene expression was not detected. Other β-chemokines, which may activate microglia and monocytes, such as MIP-1α and RANTES, were also undetected in our study. Of the 4 chemokines we identified, MCP-3 was the only one noted in the retina after photic injury and has been demonstrated to be one of the most pluripotent chemokines that activate most types of leukocytes. In spite of its broad range of chemoattractant activities for monocytes, T lymphocytes, eosinophils, basophils, dendritic cells, and natural killer cells, the most potent effect of MCP-3 is to activate monocytes and dendritic cells [12,32]. We hypothesize that MCP-3 may have a crucial role in the activation and migration of retinal microglia after light injury.
It is important to note that the elevated level of chemokine MCP-3 gene expression preceded microglial infiltration into the outer retinas. The peak of TUNEL-positive cells (1 day) paralleled to that of MCP-3, and both were ahead of the peak of microglial infiltration (3 days). MCP-3 may therefore play an important role in the activation and migration of microglial cells in light-induced photoreceptor degeneration. Taking these observations together, we hypothesize that, after intense light exposure, chemokines are initially produced by damaged photoreceptors, possibly RPE or Müller cells, and attract microglia/macrophage to the outer retina. These microglia secrete additional chemokines and amplify the post-injury inflammatory response. However, it is possible that degenerated photoreceptor cells, activated microglia, and RPE cells were all involved in the marked increase of MCP-3 gene expression. Further immunohistochemical study of MCP-3 protein expression will be needed to identify the types of cells that produce MCP-3. The significance of the upregulation of these chemokines and chemokine receptors as shown here remain to be further examined.
Supported by Michael Panitch Research Fund, RPB Foundation, and the Oliver Birckhead Research Fund.
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