Arginase Activity Mediates Retinal Inflammation in Endotoxin-Induced Uveitis
2009; Elsevier BV; Volume: 175; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2009.081115
ISSN1525-2191
AutoresWenbo Zhang, Babak Baban, Modesto Rojas, Sohrab Tofigh, Suvika K. Virmani, Chintan Patel, M. Ali Behzadian, Maritza J. Romero, Robert W. Caldwell, Ruth B. Caldwell,
Tópico(s)Adenosine and Purinergic Signaling
ResumoArginase has been reported to reduce nitric oxide bioavailability in cardiovascular disease. However, its specific role in retinopathy has not been studied. In this study, we assessed the role of arginase in a mouse model of endotoxin-induced uveitis induced by lipopolysaccharide (LPS) treatment. Measurement of arginase expression and activity in the retina revealed a significant increase in arginase activity that was associated with increases in both mRNA and protein levels of arginase (Arg)1 but not Arg2. Immunofluorescence and flow cytometry confirmed this increase in Arg1, which was localized to glia and microglia. Arg1 expression and activity were also increased in cultured Muller cells and microglia treated with LPS. To test whether arginase has a role in the development of retinal inflammation, experiments were performed in mice deficient in one copy of the Arg1 gene and both copies of the Arg2 gene or in mice treated with a selective arginase inhibitor. These studies showed that LPS-induced increases in inflammatory protein production, leukostasis, retinal damage, signs of anterior uveitis, and uncoupling of nitric oxide synthase were blocked by either knockdown or inhibition of arginase. Furthermore, the LPS-induced increase in Arg1 expression was abrogated by blocking NADPH oxidase. In conclusion, these studies suggest that LPS-induced retinal inflammation in endotoxin-induced uveitis is mediated by NADPH oxidase-dependent increases in arginase activity. Arginase has been reported to reduce nitric oxide bioavailability in cardiovascular disease. However, its specific role in retinopathy has not been studied. In this study, we assessed the role of arginase in a mouse model of endotoxin-induced uveitis induced by lipopolysaccharide (LPS) treatment. Measurement of arginase expression and activity in the retina revealed a significant increase in arginase activity that was associated with increases in both mRNA and protein levels of arginase (Arg)1 but not Arg2. Immunofluorescence and flow cytometry confirmed this increase in Arg1, which was localized to glia and microglia. Arg1 expression and activity were also increased in cultured Muller cells and microglia treated with LPS. To test whether arginase has a role in the development of retinal inflammation, experiments were performed in mice deficient in one copy of the Arg1 gene and both copies of the Arg2 gene or in mice treated with a selective arginase inhibitor. These studies showed that LPS-induced increases in inflammatory protein production, leukostasis, retinal damage, signs of anterior uveitis, and uncoupling of nitric oxide synthase were blocked by either knockdown or inhibition of arginase. Furthermore, the LPS-induced increase in Arg1 expression was abrogated by blocking NADPH oxidase. In conclusion, these studies suggest that LPS-induced retinal inflammation in endotoxin-induced uveitis is mediated by NADPH oxidase-dependent increases in arginase activity. Uveitis is a damaging ocular condition that can lead to severe vision loss and blindness.1Read RW Uveitis: advances in understanding of pathogenesis and treatment.Curr Rheumatol Rep. 2006; 8: 260-266Crossref PubMed Scopus (52) Google Scholar Endotoxin-induced uveitis (EIU) is an experimental model that closely mimics human disease and is induced by administration of a single sublethal dose of lipopolysaccharide (LPS).2Kogiso M Tanouchi Y Mimura Y Nagasawa H Himeno K Endotoxin-induced uveitis in mice. 1. 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Arginase 1 (Arg1) is a cytosolic enzyme, while arginase 2 (Arg2) is mainly expressed in mitochondria. Arginase has a wide distribution in the body although Arg1 is strongly expressed in the liver and Arg2 is more evident in the kidney and prostate.20Jenkinson CP Grody WW Cederbaum SD Comparative properties of arginases.Comp Biochem Physiol B Biochem Mol Biol. 1996; 114: 107-132Crossref PubMed Scopus (498) Google Scholar Expression of arginase has been found in many cell types, including vascular endothelial cells, smooth muscle cells, and macrophages.21Erdely A Kepka-Lenhart D Clark M Zeidler-Erdely P Poljakovic M Calhoun WJ Morris Jr, SM Inhibition of phosphodiesterase 4 amplifies cytokine-dependent induction of arginase in macrophages.Am J Physiol Lung Cell Mol Physiol. 2006; 290: L534-L539Crossref PubMed Scopus (34) Google Scholar, 22Romero MJ Platt DH Tawfik HE Labazi M El-Remessy AB Bartoli M Caldwell RB Caldwell RW Diabetes-induced coronary vascular dysfunction involves increased arginase activity.Circ Res. 2008; 102: 95-102Crossref PubMed Scopus (300) Google Scholar, 23Wei LH Wu G Morris Jr, SM Ignarro LJ Elevated arginase I expression in rat aortic smooth muscle cells increases cell proliferation.Proc Natl Acad Sci USA. 2001; 98: 9260-9264Crossref PubMed Scopus (130) Google Scholar Given that l-arginine is also an indispensible substrate for nitric oxide synthase (NOS) in NO formation, arginase is recently recognized as a critical regulator for NO production by competing with NOS for l-arginine.19Durante W Johnson FK Johnson RA Arginase: a critical regulator of nitric oxide synthesis and vascular function.Clin Exp Pharmacol Physiol. 2007; 34: 906-911Crossref PubMed Scopus (404) Google Scholar Associated with this mechanism, increased arginase activity has been linked to several diseases, such as atherosclerosis, hypertension, asthma, and endothelial dysfunction in diabetes.22Romero MJ Platt DH Tawfik HE Labazi M El-Remessy AB Bartoli M Caldwell RB Caldwell RW Diabetes-induced coronary vascular dysfunction involves increased arginase activity.Circ Res. 2008; 102: 95-102Crossref PubMed Scopus (300) Google Scholar, 24Johnson FK Johnson RA Peyton KJ Durante W Arginase inhibition restores arteriolar endothelial function in Dahl rats with salt-induced hypertension.Am J Physiol Regul Integr Comp Physiol. 2005; 288: R1057-R1062Crossref PubMed Scopus (125) Google Scholar, 25Maarsingh H Zuidhof AB Bos IS van Duin M Boucher JL Zaagsma J Meurs H Arginase inhibition protects against allergic airway obstruction, hyperresponsiveness, and inflammation.Am J Respir Crit Care Med. 2008; 178: 565-573Crossref PubMed Scopus (90) Google Scholar, 26Ryoo S Gupta G Benjo A Lim HK Camara A Sikka G Lim HK Sohi J Santhanam L Soucy K Tuday E Baraban E Ilies M Gerstenblith G Nyhan D Shoukas A Christianson DW Alp NJ Champion HC Huso D Berkowitz DE Endothelial arginase II: a novel target for the treatment of atherosclerosis.Circ Res. 2008; 102: 923-932Crossref PubMed Scopus (195) Google Scholar Increased expression of arginase has been described in a rat model for EIU.27Koga T Koshiyama Y Gotoh T Yonemura N Hirata A Tanihara H Negi A Mori M Coinduction of nitric oxide synthase and arginine metabolic enzymes in endotoxin-induced uveitis rats.Exp Eye Res. 2002; 75: 659-667Crossref PubMed Scopus (35) Google Scholar However, the specific role of arginase in EIU and mechanisms underlying arginase expression in this disease are unknown. All procedures with animals were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the institutional animal care and use committee (Animal Welfare Assurance no. A3307-01). Experiments were performed with C57BL/6J wild-type mice, mice deficient in arginase 2 (Arg2−/−), mice deficient in both arginase 1 and 2 (Arg1+/−Arg2−/−), and mice deficient in NOX2 (NOX2−/−). The NOX2−/− mouse was produced by Dinauer and colleagues.28Pollock JD Williams DA Gifford MA Li LL Du X Fisherman J Orkin SH Doerschuk CM Dinauer MC Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production.Nat Genet. 1995; 9: 202-209Crossref PubMed Scopus (745) Google Scholar The C57BL/6J Arg1+/− and Arg2−/− mice developed by Cederbaum et al29Deignan JL Livesay JC Yoo PK Goodman SI O'Brien WE Iyer RK Cederbaum SD Grody WW Ornithine deficiency in the arginase double knockout mouse.Mol Genet Metab. 2006; 89: 87-96Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar and O'Brien et al30Shi O Morris Jr, SM Zoghbi H Porter CW O'Brien WE Generation of a mouse model for arginase II deficiency by targeted disruption of the arginase II gene.Mol Cell Biol. 2001; 21: 811-813Crossref PubMed Scopus (117) Google Scholar were provided by Dr. Steven Cederbaum with the permission of Dr. O'Brien. The knockout mice have been backcrossed for at least 10 generations on C57BL/6 mice. EIU was induced by injection of lipopolysaccharide from Salmonella typhimurium (LPS, 4 mg/kg in PBS, i.p.; Sigma-Aldrich, St. Louis, MO). Control mice received vehicle alone. Another group of wild-type mice was treated with the arginase inhibitor [S]-[2-boronoethyl]-l-Cysteine-HCl (BEC, EMD Chemicals, Gibbstown, NJ). For this study mice were injected with BEC (20 mg/kg in 0.9% saline, i.v.) 1 hour before the injection of LPS. Rat Muller cells31Sarthy VP Brodjian SJ Dutt K Kennedy BN French RP Crabb JW Establishment and characterization of a retinal Muller cell line.Invest Ophthalmol Vis Sci. 1998; 39: 212-216PubMed Google Scholar were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. The cells were used from passages 2 to 6 and starved in serum-free medium overnight before treatment. Primary rat microglia were isolated and seeded at a density of 1 × 105 cells/well in 24-well plates in Dulbecco's modified Eagle's medium/F12 (Mediatech, Manassas, VA) that was supplied with 10% fetal bovine serum and 1% Penicillin-Streptomycin solution (Invitrogen) as described.7Liou GI Auchampach JA Hillard CJ Zhu G Yousufzai B Mian S Khan S Khalifa Y Mediation of cannabidiol anti-inflammation in the retina by equilibrative nucleoside transporter and A2A adenosine receptor.Invest Ophthalmol Vis Sci. 2008; 49: 5526-5531Crossref PubMed Scopus (98) Google Scholar One day after seeding, cells were washed with Cellgro Complete (Mediatech) and incubated in the Cellgro Complete with various treatments. Total RNA was isolated using an RNAqueous – 4PCR kit (Applied Biosystems, Austin, TX) according to the manufacture's suggestion (for retina tissue) or using TRIzol reagent (Invitrogen) (for cells). Total RNA was reverse transcribed with M-MLV reverse transcriptase (Invitrogen) to generate cDNA. Gene expression was determined by quantitative PCR with TaqMan Gene Expression Assays specific for 18S, Arg1, and Arg2 (Applied Biosystems), which was performed on a StepOne Plus thermocycler (Applied Biosystems). The cycle threshold, determined as the initial increase in fluorescence above background, was ascertained for each sample. 18S was used as internal control in the PCR reaction for normalization. Retinas were homogenized in a radioimmunoprecipitation assay lysis buffer (Millipore, Billerica, MA) supplemented with 10 mmol/L NaF, 10 mmol/L Na4P2O7, 1 mmol/L phenyl methyl sulfonyl fluoride and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Twenty-microgram protein samples were subjected to 10% SDS polyacrylamide gel electrophoresis. Proteins were transferred onto a nitrocellulose membrane and the membrane was incubated overnight at 4°C with primary antibodies against Arg 1 (1:1000, BD Biosciences, San Jose, CA), actin (1:3000), and tubulin (1:5000, Sigma-Aldrich), followed by horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were detected using the enhanced chemiluminescence system (GE Health care, Piscataway, NJ). Retinas or Muller cells were homogenized in ice-cold lysis buffer (50 mmol/L Tris-HCl, 0.1 mmol/L EDTA and EGTA, pH 7.5) containing protease inhibitors. The homogenate was centrifuged at 14,000 × g for 20 minutes and the supernatant was collected for the enzyme assay. Arginase activity was assayed as previously described.32Corraliza IM Campo ML Soler G Modolell M Determination of arginase activity in macrophages: a micromethod.J Immunol Methods. 1994; 174: 231-235Crossref PubMed Scopus (429) Google Scholar Briefly, the enzyme was activated by heating the lysate at 56°C in 25 mmol/L Tris buffer (pH 7.4) containing 5 mmol/L MnCl2. l-Arginine hydrolysis was then performed by incubating 50 μl of the activated lysate with 50 μl of 0.5 M/L l-arginine (pH 9.7) at 37°C for 60 minutes. The reaction was stopped in acid medium. The concentration of urea, which is the end product of l-arginine hydrolysis by arginase, was determined after adding 25 μl of 9% α-isonitrosopropiophenone. Protein concentration in the lysates was determined by bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL). Arginase activity was calculated as mM Urea/mg protein. Mice were sacrificed and eye balls were embedded in optimal cutting temperature (OCT) compounds after treatment with vehicle or LPS (4 mg/kg, i.p.) for 16 hours. The OCT-frozen sections (10 μm) were fixed using 4% paraformaldehyde solution in PBS for 5 minutes at room temperature. Sections were washed several times with PBS, treated with proteinase K for 3 minutes, then permeabilized with 0.5% Triton X-100 for 30 minutes and blocked with 20% donkey serum for 1 hour at room temperature. Then sections were reacted with mouse monoclonal anti-arginase 1 antibody (5 μg/ml, BD Biosciences) or mouse IgG1 isotype control antibody (5 μg/ml) overnight at 4°C, followed by Cy3.0-conjugated AffiniPure Donkey Anti-Mouse antibody (1:500, Jackson Immuno Research, West Grove, PA) at room temperature for 1 hour. Images were taken by fluorescence microscopy at × 200. Fluorescence was very low in sections stained with isotype IgG1 and exposure time was increased to show the retinal tissue layers. Retinas were dissected and pooled retina samples (3 to 4 retinas) were incubated in Dispase (Sigma Aldrich, 37°C, 10 minutes) and filtered through a 70 μm nylon mesh to obtain a single cell suspension. Cells were centrifuged (1500 rpm, 10 minutes) and washed twice with PBS. Then cells were fixed and permeabilized using Becton Dickinson CytofixCytoperm ready-to-use buffer (500 μl for 15 minutes on ice). Cells were then washed twice with PBS and blocked with 20% donkey serum before being double-stained for cell markers and arginase 1 or for cell markers and cytokines or for cell markers and isotype control IgGs. The antibodies used and corresponding isotype control are: mouse anti-arginase 1 and mouse IgG1 (2.5 μg/ml, BD Biosciences), rabbit anti-VEGF and normal rabbit IgG (2 μg/ml, EMD Chemicals), phycoerythrin (PE)-conjugated rat anti-TNF-α and PE Rat IgG1 isotype control (0.4 μg/ml, eBioscience, San Diego and, CA), rabbit anti-MCP-1 (1 μg/ml, PeproTech, Rocky Hill, NJ), and normal rabbit IgG (1 μg/ml, EMD Chemicals). To show arginase or cytokines in neuroglia, arginase 1 and TNF-α were double-stained with rabbit anti-glial fibrillary acidic protein (GFAP) (1:50, neuroglia marker, from Sigma Aldrich). VEGF and MCP-1 were double-stained with mouse anti-GFAP (ready-to-use, Abcam, Cambridge, MA). To show arginase or cytokines in macrophage/microglia, arginase 1 and cytokines were double-stained with biotinylated rat anti-F4/80 (1:800, marker for activated macrophage/microglia, a gift from Dr. Sally S. Atherton). Cells were incubated with primary antibodies for 1 hour at 4°C and washed. Then cells were incubated with second antibodies for 40 minutes at 4°C. The following second antibodies were used at 1:500 dilution: PE-donkey anti-mouse (for arginase 1), PE-donkey anti-rabbit (for MCP-1 and VEGF), fluorescein isothiocyanate (FITC)-donkey anti-rabbit (for GFAP), FITC-donkey anti-mouse (for GFAP), and FITC-streptavidin (for F4/80). After staining, samples were washed twice and analyzed using four-color flow cytometry (FACS Calibur, BD Biosciences) and CellQuestTM software. Samples double-stained with control IgG and cell marker were used to set up the compensation to subtract the spillover signal of FITC from PE detector (PE-%FITC). Proper compensation was set up to make sure the median PE fluorescence intensities of FITC negative cells and positive cells were identical and were both gated as PE-negative population. Due to the limited number of cells from mouse retinas, reciprocal compensation to subtract the spillover signal of PE from FITC detector (FITC-%PE) was not set up since this effect was minimal. Then the percentage of cells that express cytokines or arginase in each cell type was identified after gating to exclude dead cells and debris using forward and side scatter plots. Adhesion of leukocytes to the wall of the retinal vessels was evaluated as described previously,17Al-Shabrawey M Rojas M Sanders T Behzadian A El-Remessy A Bartoli M Parpia AK Liou G Caldwell RB Role of NADPH oxidase in retinal vascular inflammation.Invest Ophthalmol Vis Sci. 2008; 49: 3239-3244Crossref PubMed Scopus (165) Google Scholar with some modification. After the induction of deep anesthesia, the chest cavity was carefully opened, and a perfusion cannula with 0.2 mm internal diameter was introduced into the aorta. Drainage was achieved by opening the right atrium. The animals were then perfused with 6 ml of PBS to wash out nonadherent blood cells. Next, the animals were perfused with 6 ml FITC-labeled concanavalin A lectin (40 μg/ml in PBS, Vector Laboratories, Burlingame, CA) to label the adherent leukocytes and vascular endothelial cells. Residual unbound concanavalin A was removed by perfusion with PBS. The eyeballs were removed and fixed with 4% paraformaldehyde. The retinas were then dissected, mounted flat on glass slides and observed by fluorescence microscopy at × 200 magnification. The total number of adherent leukocytes per retina was determined. Mice were sacrificed after treatment with vehicle or LPS (4 mg/kg, i.p, 16 hours) and eye balls were embedded in OCT compound. OCT-frozen sections (10 μm) were fixed and stained with H&E. Images were taken by using a Zeiss microscope at × 200. After the induction of deep anesthesia, aqueous humor was collected by anterior chamber puncture using a 30-gauge needle under a surgical microscope. The samples were used for either cell counting or protein concentration measurements. To quantify the number of infiltrated leukocytes, 1 μl of aqueous humor was mixed with 8 μl of 0.4% trypan blue stain solution and subjected to cell counting using a hemocytometer. Cells in five fields were counted, averaged and calculated as number of cells in 1 μl of humor. The protein concentration in the aqueous humor was determined by bicinchoninic acid assay and dilutions of bovine serum albumin as standards. Retinas were homogenized in PBS with a pestle. Then the lysates were centrifuged at 14000 rpm for 10 minutes at 4°C and supernatants were collected. The level of nitrite in the supernatants was analyzed using NO-specific chemiluminescence. In brief, samples containing nitrite were refluxed in glacial acetic acid containing sodium iodide. Nitrite is quantitatively reduced to NO under these conditions, which can be quantified by a chemiluminescence detector after reaction with ozone in a NO analyzer (Sievers). To measure the total level of nitrite plus nitrate, supernatants were incubated with PBS containing nitrate reductase (0.25 unit/ml), NADPH (13 μg/ml), and FAD-Na2 (4 μg/ml) at 30°C for 1 hour to reduce nitrate to nitrite. Then the level of nitrite was analyzed using NO-specific chemiluminescence. Protein concentration in the supernatant was determined by bicinchoninic acid assay. The level of nitrite or nitrite plus nitrate was normalized to the protein concentration in the supernatant and calculated as percentage of control. To evaluate production of superoxide in situ, the oxidative fluorescent dye dihydroethidium (DHE) was used. DHE is freely permeable to cells and in the presence of superoxide is oxidized to ethidium bromide, which binds to DNA and fluoresces red. Frozen sections were pre-incubated in NADPH (100 μmol/L) for 20 minutes followed by DHE (2 μmol/L, 20 minutes, 37°C). DHE images from serial sections were obtained using an Axiovision fluorescence microscope, DHE is excited at 488 nm with an emission spectrum of 610 nm. The images were analyzed for reaction intensity by using the Metamorph Image System (Molecular Devices). The results are expressed as mean ± SEM. Group differences were evaluated by using one way analysis of variance followed by posthoc Student's t-test. Results were considered significant at P < 0.05. To evaluate whether arginase plays a role in acute retinal inflammation, the expression and activity of arginase were evaluated in a mouse model for EIU. Quantitative PCR analysis of mRNA revealed that both Arg1 and Arg2 were expressed in the retina. The mRNA level of Arg1 was significantly increased following LPS injection. It reached a peak at 12 hours (2.21 ± 0.14-fold of control) and was slightly decreased at 24 hours (1.94 ± 0.06-fold of control) (Figure 1A). The mRNA level of Arg2 was not changed (or even slightly decreased) (Figure 1B). Consistent with the increase in Arg1 mRNA, Arg1 protein was also increased 16 hours after the LPS injection as revealed by immunoblotting with a specific antibody for Arg1 (Figure 1C). The level of arginase activity in the retina was determined by measuring the amount of urea produced from l-arginine hydrolysis. LPS treatment significantly increased arginase activity by 1.9 ± 0.3-fold (Figure 1D). To assess the generality of this effect, studies were also performed in rats treated with LPS (1 mg/kg, i.p.). These experiments showed that arginase activity was increased by ∼1.8-fold within 12 or 24 hours following the LPS treatment (data not shown). Prompted by the observation that Arg1 expression was up-regulated in EIU, we next determined the localization of Arg1 in retina sections. Immunolocalization analysis showed that immunoreactivity for Arg1 was localized to the nerve fiber layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, and outer limiting membrane in a pattern that corresponded to the processes of Muller cells, astrocytes, and microglia cells (Figure 2A). Immunofluorescence with an isotype control IgG was very weak and did not show any specific structure (Figure 2A), indicating specificity of the arginase antibody. To further investigate the expression of Arg1 in specific cell types, flow cytometry analysis was performed after retinal cells were separated into a single cell suspension by Dispase digestion and costained with both Arg1 antibody and cell type markers. GFAP was used as neuroglial marker. Retinal astrocytes express GFAP under both resting and activated conditions and Muller cells express GFAP after treatment with LPS. F4/80 was used as a marker for activated macrophage/microglia. As shown in Figure 2B, in the retina from vehicle-treated mice, a small fraction of GFAP-positive cells were located in quadrants 2 and 4 and a small fraction of Arg1-positive cells were located in quadrants 1 and 2. In the GFAP-positive population (quadrants 2 and 4), only 5% of cells were as also positive for Arg1 (quadrant 2). Parallel experiments using a Muller cell-specific antibody, CRALBP, showed that Muller cells are also largely negative for Arg 1 (data not shown). In contrast with the vehicle-treated controls, LPS treatment resulted in a prominent increase in the GFAP-positive cell population, which is consistent with previous findings that LPS treatment increases GFAP expression in both astrocytes and Muller cells.33Brahmachari S Fung YK Pahan K Induction of glial fibrillary acidic protein expression in astrocytes by nitric oxide.J Neurosci. 2006; 26: 4930-4939Crossref PubMed Scopus (251) Google Scholar, 34Jang S Lee JH Choi KR Kim D Yoo HS Oh S Cytochemical alterations in the rat retina by LPS administration.Neurochem Res. 2007; 32: 1-10Crossref PubMed Scopus (17) Google Scholar Interestingly, the percentage of Arg1-positive cells in the GFAP-positive population (neuroglia) also was robustly increased to 45% after LPS treatment, suggesting an increase of Arg1 expression in astrocytes and Muller cells during EIU. Similarly, analysis of Arg1 expression in the F4/80-positive population (activated microglia and macrophages) also revealed that Arg1-positive cells were increased from 5% to 48% following LPS administration (Figure 2B). Analysis of Arg1 expression in combination with CD11b, which is a marker for the entire macrophage/microglia population, also revealed that Arg1 positive cells were increased from 3% to 40% following LPS administration (data not shown). This observation is consistent with the report that there is an 80% overlap between CD11b-positive cells and F4/80-positive cells in the retina.35Gregerson DS Yang J CD45-positive cells of the retina and the
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