Constitutive Retinal CD200 Expression Regulates Resident Microglia and Activation State of Inflammatory Cells during Experimental Autoimmune Uveoretinitis
2002; Elsevier BV; Volume: 161; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)64444-6
ISSN1525-2191
AutoresCathryn Broderick, Robert M. Hoek, John V. Forrester, Janet Liversidge, Jonathon D. Sedgwick, Andrew D. Dick,
Tópico(s)Adenosine and Purinergic Signaling
ResumoRecent evidence supports the notion that tissue OX2 (CD200) constitutively provides down-regulatory signals to myeloid-lineage cells via CD200-receptor (CD200R). Thus, mice lacking CD200 (CD200−/−) show increased susceptibility to and accelerated onset of tissue-specific autoimmunity. In the retina there is extensive expression of CD200 on neurons and retinal vascular endothelium. We show here that retinal microglia in CD200−/− mice display normal morphology, but unlike microglia from wild-type CD200+/+ mice are present in increased numbers and most significantly, express inducible nitric oxide synthase (NOS2), a macrophage activation marker. Onset and severity of uveitogenic peptide (1-20) of interphotoreceptor retinoid-binding protein-induced experimental autoimmune uveoretinitis is accelerated in CD200−/− mice and although tissue destruction appears no greater than seen in CD200+/+ mice, there is continued increased ganglion and photoreceptor cell apoptosis. Myeloid cell infiltrate was increased in CD200−/− mice during experimental autoimmune uveoretinitis, although NOS2 expression was not heightened. The results indicate that the CD200:CD200R axis regulates retinal microglial activation. In CD200−/− mice the release of suppression of tonic macrophage activation, supported by increased NOS2 expression in the CD200−/− steady state accelerates disease onset but without any demonstration of increased target organ/tissue destruction. Recent evidence supports the notion that tissue OX2 (CD200) constitutively provides down-regulatory signals to myeloid-lineage cells via CD200-receptor (CD200R). Thus, mice lacking CD200 (CD200−/−) show increased susceptibility to and accelerated onset of tissue-specific autoimmunity. In the retina there is extensive expression of CD200 on neurons and retinal vascular endothelium. We show here that retinal microglia in CD200−/− mice display normal morphology, but unlike microglia from wild-type CD200+/+ mice are present in increased numbers and most significantly, express inducible nitric oxide synthase (NOS2), a macrophage activation marker. Onset and severity of uveitogenic peptide (1-20) of interphotoreceptor retinoid-binding protein-induced experimental autoimmune uveoretinitis is accelerated in CD200−/− mice and although tissue destruction appears no greater than seen in CD200+/+ mice, there is continued increased ganglion and photoreceptor cell apoptosis. Myeloid cell infiltrate was increased in CD200−/− mice during experimental autoimmune uveoretinitis, although NOS2 expression was not heightened. The results indicate that the CD200:CD200R axis regulates retinal microglial activation. In CD200−/− mice the release of suppression of tonic macrophage activation, supported by increased NOS2 expression in the CD200−/− steady state accelerates disease onset but without any demonstration of increased target organ/tissue destruction. It has long been recognized that resident macrophages isolated from different tissues and anatomical sites are heterogeneous and that resident macrophages are adapted to the local microenvironment.1Forrester JV McMenamin PG Immunopathogenic mechanisms in intraocular inflammation.Chem Immunol. 1999; 73: 159-185Crossref PubMed Scopus (50) Google Scholar, 2Laszlo DJ Henson PM Remigio LK Weinstein L Sable C Noble PW Riches DW Development of functional diversity in mouse macrophages. 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Leukocyte function, including macrophages, may be regulated by the integration of both activation and inhibitory signals received through cell surface receptors.9Lu Q Lemke G Homeostatic regulation of the immune system by receptor tyrosinekinases of the Tyro 3 family.Science. 2001; 293: 306-311Crossref PubMed Scopus (527) Google Scholar In common with most of the inhibitory receptors is the conservation of immunoreceptor tyrosine-based inhibitory motifs10Lanier LL Bakker ABH The ITAM bearing transmembrane adaptor DAP-12 in lymphoid and myeloid cell function.Immunol Today. 2000; 21: 611-614Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar in the cytoplasmic domains, although inhibitory molecules such as CTLA-4 lack this motif.11Ravetch JV Lanier LL Immune inhibitory receptors.Science. 2000; 290: 84-89Crossref PubMed Scopus (1046) Google Scholar Targeted disruption of such molecules leads to an increase in autoimmune disorders, which are frequently fatal.11Ravetch JV Lanier LL Immune inhibitory receptors.Science. 2000; 290: 84-89Crossref PubMed Scopus (1046) Google Scholar A paradigm has emerged in which the strength of the opposing activating and inhibitory signals determine the initiation, amplification, and termination of immune responses.12Lanier LL Face-off: the interplay between activating and inhibitory immune receptors.Curr Opin Immunol. 2001; 3: 326-331Crossref Scopus (126) Google Scholar More recently, investigations of CD200 (OX2), a 41- to 47-kd member of the immunoglobulin superfamily and its receptor (CD200R), have indicated that macrophages and granulocytes are restrained from tissue-damaging activation through CD200R signaling. In all species so far tested, CD200 has a wide distribution and expression on neurons, activated T cells, B cells, follicular dendritic cells, and endothelium.13Barclay N Ward H Purification and chemical characterisation of membrane glycoprotein from rat thymocytes and brain, recognised by monoclonal antibody MRC OX2.Eur J Biochem. 1982; 129: 447-452Crossref PubMed Scopus (64) Google Scholar, 14Wright G Puklavec M Willis AC Hoek RM Sedgwick JD Brown MH Barclay AN Lymphoid/neuronal cell surface OX2 glycoprotein recognises a novel receptor on macrophages implicated in the control of their function.Immunity. 2000; 13: 233-242Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 15Hoek R Ruuls SR Murphy CA Wright GJ Goddard R Zurawski SM Blom B Homola ME Streit WJ Brown MH Barclay AN Sedgwick JD Down regulation of the macrophage lineage through interaction with OX2 (CD200).Science. 2001; 290: 1768-1771Crossref Scopus (802) Google Scholar By contrast the CD200R is expressed predominantly by cells of the myeloid lineage14Wright G Puklavec M Willis AC Hoek RM Sedgwick JD Brown MH Barclay AN Lymphoid/neuronal cell surface OX2 glycoprotein recognises a novel receptor on macrophages implicated in the control of their function.Immunity. 2000; 13: 233-242Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 16Preston S Wright G Starr K Barclay N Brown M The leucocyte/neuron cell surface antigen OX2 binds to a ligand on macrophages.Eur J Immunol. 1997; 27: 1911-1918Crossref PubMed Scopus (72) Google Scholar including microglia (MG)14Wright G Puklavec M Willis AC Hoek RM Sedgwick JD Brown MH Barclay AN Lymphoid/neuronal cell surface OX2 glycoprotein recognises a novel receptor on macrophages implicated in the control of their function.Immunity. 2000; 13: 233-242Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar inferring a significant role for the CD200/CD200R axis within the central nervous system. Structurally, CD200R resembles CD200, with two IgSF domains, but with a larger cytoplasmic domain, which suggests CD200R engagement may lead to intracellular signals affecting macrophage function.16Preston S Wright G Starr K Barclay N Brown M The leucocyte/neuron cell surface antigen OX2 binds to a ligand on macrophages.Eur J Immunol. 1997; 27: 1911-1918Crossref PubMed Scopus (72) Google Scholar Regulation of macrophage function is supported by the following observations in CD200−/− mice: significant increase in numbers of myeloid-derived cells within lymphoid organs,15Hoek R Ruuls SR Murphy CA Wright GJ Goddard R Zurawski SM Blom B Homola ME Streit WJ Brown MH Barclay AN Sedgwick JD Down regulation of the macrophage lineage through interaction with OX2 (CD200).Science. 2001; 290: 1768-1771Crossref Scopus (802) Google Scholar accelerated onset of experimental autoimmune encephalomyelitis, and conversion of C57BL/6 mice from resistant to susceptible to collagen-induced arthritis.15Hoek R Ruuls SR Murphy CA Wright GJ Goddard R Zurawski SM Blom B Homola ME Streit WJ Brown MH Barclay AN Sedgwick JD Down regulation of the macrophage lineage through interaction with OX2 (CD200).Science. 2001; 290: 1768-1771Crossref Scopus (802) Google Scholar Increased central nervous system (CNS) macrophage/MG responses were also observed, including aggregate formation in the spinal cord, an effect normally associated with inflammation or neurodegeneration.15Hoek R Ruuls SR Murphy CA Wright GJ Goddard R Zurawski SM Blom B Homola ME Streit WJ Brown MH Barclay AN Sedgwick JD Down regulation of the macrophage lineage through interaction with OX2 (CD200).Science. 2001; 290: 1768-1771Crossref Scopus (802) Google Scholar Although it seems that CD200:CD200R engagement may modulate myeloid cell turnover as well as development of autoimmune disease, it is unclear from the reported experiments how this happens. In particular, it is unclear whether resident MG or perivascular macrophage activation in the CNS is the predominant event in predisposing CD200−/− mice to experimental autoimmune encephalomyelitis. We therefore wished to assess whether loss of CD200:CD200R engagement within the retina (a neural tissue containing comparatively few MG) also results in increased tissue destruction during experimental autoimmune uveoretinitis (EAU). As in the brain and spinal cord, CD200 is expressed on the axons of glial fibrillary acidic protein-negative neurons, and on choroidal and retinal vascular endothelium within human, rat, and mouse retina and we have described this previously.17Dick AD Broderick C Forrester JV Wright GJ Distribution of OX2 antigen and OX2 receptor within the retina.Invest Ophthalmol Vis Sci. 2001; 42: 170-176PubMed Google Scholar Murine EAU is a CD4+ T-cell-mediated destruction of the neuroretina and photoreceptors of the eye.18Caspi R Roberge FG Chan CC Wiggert B Chader GJ Rozenszajn LA Lando Z Nussenblatt RB A new model of autoimmune disease. Experimental autoimmune uveitis induced in mice with two different retinal antigens.J Immunol. 1988; 140: 1490-1495PubMed Google Scholar, 19Avichezer D Silver P Chan CC Wiggert B Caspi R Identification of a new epitope of human IRBP that induces autoimmune uveoretinitis in mice of the H-2b haplotype.Invest Ophthalmol Vis Sci. 2000; 41: 127-131PubMed Google Scholar EAU has the advantage of not only determining cellular infiltrate but the model also permits the semiquantitative documentation of the extent of tissue destruction.18Caspi R Roberge FG Chan CC Wiggert B Chader GJ Rozenszajn LA Lando Z Nussenblatt RB A new model of autoimmune disease. Experimental autoimmune uveitis induced in mice with two different retinal antigens.J Immunol. 1988; 140: 1490-1495PubMed Google Scholar, 20Dick AD McMenamin PG Korner H Scallon BJ Ghrayeb J Forrester JV Sedgwick JD Inhibition of TNF activity minimises target organ damage in experimental autoimmune uveoretinitis despite quantitatively normal activated T cell traffik to the retina.Eur J Immunol. 1996; 26: 1018-1025Crossref PubMed Google Scholar CD200-deficient mice (CD200−/−) of background strain C57BL/6 were generated by DNAX, Palo Alto, CA15Hoek R Ruuls SR Murphy CA Wright GJ Goddard R Zurawski SM Blom B Homola ME Streit WJ Brown MH Barclay AN Sedgwick JD Down regulation of the macrophage lineage through interaction with OX2 (CD200).Science. 2001; 290: 1768-1771Crossref Scopus (802) Google Scholar and breeding colonies established within the Biological Services Unit of Aberdeen University, and University of Bristol, UK, for further experimentation. All animals were specific pathogen-free, isolator-reared, and maintained in accordance to Home Office Regulations for Animal Experimentation, UK, and conformed to the Association for Research in Vision and Ophthalmology statement for the use of animals in Ophthalmic and Vision Research. C57BL/6 wild-type (CD200+/+) mice were purchased from Harlan Olac, UK. All animals were immunized between 6 to 8 weeks of age. For each experiment, groups of 24 CD200+/+ and 24 CD200−/− mice were immunized by a subcutaneous injection of 500 μg of peptide 1-20 interphotoreceptor retinoid-binding protein (IRBP) (Dr. Arthur Moir, University of Sheffield, Sheffield, UK) per mouse, emulsified in vol/vol complete Freund's adjuvant (2.5 mg/ml Mycobacterium tuberculosis). Mice were administered an additional intraperitoneal injection of 1.5 μg of Bordetella pertussis toxin. Mice were sacrificed by CO2 asphyxiation at days 0, 10, 16, and 21 or 23 after immunization. At the specified time points, eyes were enucleated for histological grading, immunohistochemistry, and flow cytometric analysis and lymphoid tissue dissected (spleen, inguinal and iliac lymph nodes) to determine proliferative and cytokine responses. Sex- and age-matched CD200−/− and CD200+/+ mice were sacrificed by CO2 asphyxiation. Eyes were dissected, and either snap-frozen in OCT and 6- to 8-μm cryostat sections prepared for immunocytochemistry (Leica, UK), or placed in neutral buffered formalin for paraffin embedding and hematoxylin and eosin staining for histological scoring. For grading, at least three sections from each eye were scored in a masked manner using a semiquantitative scoring system, which has been described18Caspi R Roberge FG Chan CC Wiggert B Chader GJ Rozenszajn LA Lando Z Nussenblatt RB A new model of autoimmune disease. Experimental autoimmune uveitis induced in mice with two different retinal antigens.J Immunol. 1988; 140: 1490-1495PubMed Google Scholar and combines the extent of the inflammatory infiltrate and tissue damage in the anterior and posterior chambers of the eye. All results are expressed as mean ± 1 SEM. Comparison of histological assessment of disease in CD200−/− and CD200+/+ were analyzed using the unpaired t-test (Graphpad Instat Software), and P values equal to or less than 0.05 were considered significant. For immunofluorescence, increased background staining because of endogenous avidin was prevented by preincubation with an avidin block (Vector Laboratories, Burlingame, CA) and 10% normal rabbit serum diluted in Tris-buffered saline. Primary monoclonal antibody (mAb) against F4/80, MOMA-1, MHC class II (all obtained from Serotec, UK), inducible nitric oxide synthase (NOS2) (clone 6, 1:100; Transduction Laboratories, UK), OX90 (anti-mouse CD200, 1:75, kindly supplied by G Wright, Oxford, UK) were used at previously determined optimized concentrations after dilution in biotin block (Vector kit). After a 1-hour incubation at room temperature slides were washed thoroughly in Tris-buffered saline. Primary antibodies were then detected with biotin-labeled rabbit anti-rat Ig (DAKO, Ely, UK), visualized using SA:ABC AP (DAKO) and fast red substrate, and lightly counterstained with hematoxylin. Apoptotic cells in the sections were detected using the diaminobenzidine- and fluorescein isothiocyanate (FITC)-labeled terminal dUTP nick-end labeling (TUNEL) method exactly according to the instructions of the kit manufacturer (Trevigen, AMS Biotechnological Ltd., UK, and Promega, Madison, WI). Retinal positive cells were counted in five fields in each of three sections per eye. Statistical significance was calculated using the unpaired t-test (Graphpad Instat Software). For whole-mount assessment, eyes were enucleated and fixed in 1% paraformaldehyde for 1 hour. Biomicroscopic dissection of whole retina was achieved after removal of the cornea and extirpation of the lens and vitreous body. The retina was washed three times for 5 minutes in 1% bovine serum albumin/phosphate-buffered saline (BSA/PBS) and incubated at room temperature for 15 minutes in hyaluronidase (10 μg/ml, Sigma, Dorset, UK) to digest any remaining vitreous on the surface of the retina. Tissue was then washed three times for 10 minutes in 1%BSA/PBS and 0.2% saponin and subsequently incubated for 1 hour at room temperature with unconjugated primary antibody-F4/80 (1:25, Serotec) diluted in 1%BSA/PBS and 0.2% saponin; each retina was individually placed in a 24-well culture plate. F4/80-positive cells were visualized by incubation with FITC-conjugated goat anti-rat IgG (1:100, Sigma, UK). Retina was finally mounted with great care to prevent folding using Vectashield (Vector Laboratories) and fluorescence was observed using appropriate filters using an Olympus BH2-RFC microscope. The phenotype of infiltrating and resident retinal leukocytes was investigated after isolation of cells at different phases of EAU. Retinas were dissected as above and single-cell suspensions prepared by passing through a 250-μm metal sieve. Cellular debris and nonviable cells were removed by a density gradient (Percoll; Amersham, UK) as previously described, which enriched for leukocytic populations.20Dick AD McMenamin PG Korner H Scallon BJ Ghrayeb J Forrester JV Sedgwick JD Inhibition of TNF activity minimises target organ damage in experimental autoimmune uveoretinitis despite quantitatively normal activated T cell traffik to the retina.Eur J Immunol. 1996; 26: 1018-1025Crossref PubMed Google Scholar Cell surface antigen expression was determined using mAb obtained from Pharmingen unless otherwise stated and included CD3, CD4, CD69, CD11b, CD45, MHC class II, CD86, CD40, F4/80 (Serotec, UK), MOMA-1 (Serotec, UK), and OX90 (G Wright, Oxford, UK) either unconjugated or conjugated to FITC, phycoerythrin, or biotin for subsequent two- and three-color analysis as previously described.20Dick AD McMenamin PG Korner H Scallon BJ Ghrayeb J Forrester JV Sedgwick JD Inhibition of TNF activity minimises target organ damage in experimental autoimmune uveoretinitis despite quantitatively normal activated T cell traffik to the retina.Eur J Immunol. 1996; 26: 1018-1025Crossref PubMed Google Scholar Intracellular staining for Ki67:FITC (Pharmingen, UK) required an additional permeabilizing step, protocol as suggested by the manufacturer. Biotin-labeled antibodies were detected by addition of SA:APC (1:400; Pharmingen, La Jolla, CA). OX90 (anti-mouse CD200) mAb was unconjugated and expression detected with goat anti-rat FITC (1:100; Serotec, UK). Staining was performed using fluorescence-activated cell sorting (FACS) buffer (PBS/BSA/10 mmol/L NaN3) for washes. All reagents, buffers, and incubations were performed and kept at 4°C. Negative isotype controls and single positive controls were performed to allow accurate breakthrough compensation and background limits. Cells were incubated sequentially with primary mAb, anti-rat IgG (whole molecule) FITC conjugate in the presence of 5% normal mouse serum (NMS) if unconjugated, blocked with 10% normal mouse serum/normal rat serum (NMS/NRS), biotinylated second antibody with phycoerythrin-conjugated third antibody, and finally SA-APC for three-color analysis. Acquisition was performed using a FACS Calibur (Becton Dickinson) and analysis using Cellquest software. A total of 10,000 events were collected and gates and instrument settings were set according to forward and side scatter characteristics. Fluorescence analysis was performed after further back gating to exclude dead cells and background staining. Splenocyte responses were obtained from single-cell suspensions after mechanical dissociation of the tissue through a 250-μm metal sieve. The cells were resuspended at a final concentration of 2 × 106 cells/ml. One hundred μl of cells were then added to a 96-well plate, containing 100 μl of either complete RPMI (cRPMI; RPMI plus 5% fetal calf serum, 1% l-glutamine, 1% sodium pyruvate, 1% non-essential amino acids (NEAA), 0.5% mercaptoethanol, 2% penicillin/streptomycin, unstimulated control population), cRPMI containing Con A (2.5 μg/ml, positive response control), 1-20 IRBP peptide (5 μg/ml), or cRPMI containing whole IRBP (10 μg/ml), which was prepared as previously described.21Adler AJ Evans CD Some functional characteristics of purified bovine IRBP.Invest Ophthalmol Vis Sci. 1985; 26: 273-282PubMed Google Scholar, 22Laliotou B Liversidge J Forrester JV Dick AD IRBP is a potent tolerogen in Lewis rat: suppression of EAU is retinal antigen specific.Br J Ophthalmol. 1997; 81: 61-67Crossref PubMed Scopus (15) Google Scholar Cells were incubated for 48 hours, at which time they were pulsed with 1 μl per well of 3H-thymidine (Amersham, UK) and further incubated for 18 hours. Cells were harvested onto glass filter paper and counts per minute recorded as mean of triplicate wells. Stimulation index (mean stimulation of triplicate wells of stimulant divided by mean stimulation of triplicate wells of controls) was used to present proliferation data because analysis was performed on different days. Statistical analysis was performed using the unpaired t-test and P < 0.05 was considered significant. At each time point, supernatants from splenocytes that were cultured (1 × 106/ml) for 48 hours, stimulated with either cRPMI or peptide (1-20 IRBP; 5 μg/ml) or IRBP (10 μg/ml) were removed and kept at −20°C until further analysis. Cellular interferon (IFN)-γ and interleukin (IL)-10 production was assessed using Quantikine ELISA (enzyme-linked immunosorbent assay) kits (R&D Systems, UK), following the manufacturer's instructions, with purified capture and biotinylated detector antibody pairs and standardized with recombinant cytokine to obtain a standard curve. The detection level of each kit was 2.0 pg/ml and 4.0 pg/ml, respectively. Results were presented as mean ± SD of triplicate wells of pooled samples of animals from each group and differences were considered significant if P < 0.05 using an unpaired t-test. In all cases, the nonstimulated cell supernatant cytokine levels were below detection. In concurrence with previous results we again show that CD200 is expressed widely on retinal vascular endothelium and glial fibrillary acidic protein-negative neurons and during EAU (Figure 1A).15Hoek R Ruuls SR Murphy CA Wright GJ Goddard R Zurawski SM Blom B Homola ME Streit WJ Brown MH Barclay AN Sedgwick JD Down regulation of the macrophage lineage through interaction with OX2 (CD200).Science. 2001; 290: 1768-1771Crossref Scopus (802) Google Scholar, 16Preston S Wright G Starr K Barclay N Brown M The leucocyte/neuron cell surface antigen OX2 binds to a ligand on macrophages.Eur J Immunol. 1997; 27: 1911-1918Crossref PubMed Scopus (72) Google Scholar, 17Dick AD Broderick C Forrester JV Wright GJ Distribution of OX2 antigen and OX2 receptor within the retina.Invest Ophthalmol Vis Sci. 2001; 42: 170-176PubMed Google Scholar CD200 is expressed throughout the neuronal layers of the retina (N, Figure 1A) and endothelium (E, Figure 1A) of inner retinal vasculature, which has been previously verified by two-color immunohistochemistry.17Dick AD Broderick C Forrester JV Wright GJ Distribution of OX2 antigen and OX2 receptor within the retina.Invest Ophthalmol Vis Sci. 2001; 42: 170-176PubMed Google Scholar As expected, concurrent immunodetection of CD200 on CD200−/− ocular tissue is negative (Figure 1B). The proportion of resident retinal CD45+CD11b+ cells (MG and perivascular macrophages) as determined by flow cytometric assessment of single cell suspension of retinal cell isolate, was increased significantly by 30% representing 1.7 ± 0.59% of retinal cells in CD200−/− compared to 1.3 ± 0.6% of retinal cells in CD200+/+mice (P = 0.04, Mann-Whitney test, n = 7; Figure 1C). Unlike CNS and rat retina,23Dick AD Ford AL Forrester JV Sedgwick JD Cytometric identification of a minority population of MHC class II positive cells in normal rat retina distinct from CD45low CD11b/c+ CD4low parenchymal microglia.Br J Ophthalmol. 1995; 79: 834-840Crossref PubMed Scopus (103) Google Scholar it is not possible in mouse retina to distinguish between MG and perivascular macrophages flow cytometrically because both populations are CD45high. Morphologically MG in CD200−/− mice were identical, displaying normal ramified appearance with no loss of processes (Figure 2A). Flow cytometric analysis also demonstrated that these cells expressed equivalent low amounts of CD86 and CD40 and MHC class II as seen in CD200+/+ mice (Figure 2B). However, despite these normal morphological features, MG in CD200−/− mice expressed NOS2, normally low or absent in mouse retina (Figure 3).Figure 2CD200−/− MG have normal morphology and express low amounts of MHC class II and co-accessory molecules. A: Retinal whole-mount immunofluorescence demonstrating F4/80 expression shows that the morphology of CD200−/− MG do not conform to an activated phenotype and resemble CD200+/+ retinal MG. Both characteristically display ramified forms with no loss of processes. B: Three-color flow cytometric assessment of CD45+CD11b+ MG show that in both animals there is an equivalent low level of MHC class II, CD40, and CD86 expression (mean fluorescent intensity). The low levels depicted are known to be distinct from background because experiments showed consistent, repeated mean fluorescent intensity levels greater than the limits defined during set up using isotype-positive and -negative controls (mean geometric fluorescent intensity, 2.3 ± 0.9). Original magnifications: ×750 (Ai); ×700 (Aii).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3CD200−/− MG display increased constitutive NOS2 expression. Immunohistochemical sections demonstrating NOS2 expression detected with SA:ABC-AP. NOS2-positive MG cells were detected within normal CD200−/− retina (A), and primarily absent in CD200+/+ retina (B). C shows that within the CD200+/+ eye NOS2 expression was confined primarily to nonretinal sites such as ciliary body (CB) (arrowhead; CE, ciliary body epithelium that is pigmented). Counting confirmed that within the retina there is a significant increase in CD200−/− MG NOS2 expression. For quantification, mean NOS2-positive retinal cells were calculated from a minimum of three sections per eye at each time point (n = 9; groups of three, three sections per eye). Original magnifications: ×600 (A); ×650 (B); ×300 (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT) After immunization with IRBP peptide 1-20, both CD200−/− and CD200+/+ animals developed EAU with an incidence of 100%. Greatest disease activity was observed day 16 after immunization in CD200−/− mice compared to day 23 in CD200+/+ mice (Figure 4A). During peak disease, histology of both groups of animals showed features typical of EAU, including vasculitis, leukocytic infiltrate of the vitreous and retina, formation of retinal folds, and ultimately photoreceptor cell and ganglion cell death (Figure 4, B and C). The accelerated and increased leukocytic infiltration observed in CD200−/− mice was confirmed by both flow cytometry and immunohistochemistry (Figure 4; D to J). Moreover, in later experiments, there was no significant difference in disease scores histologically by day 35 after immunization (data not shown), and no evidence of relapsing disease. Flow cytometric analysis of whole retina showed that CD200−/− retina contained significantly higher numbers of CD11b+ cells compared to CD200+/+ mice at days 10 (P < 0.04) and 23 (P < 0.03) (Figure 4D). There was no evidence on morphology or scatter profile during flow cytometry to infer a significant CD11b+ granulocyte infiltration, or any significant difference in numbers or distribution of T cell infiltrate in both groups throughout the course of EAU. No proliferation, as determined by detection of Ki67, a nuclear-associated antigen present at all stages of active cell cycle, was detected within retinal cells by flow cytometry (data not shown). Given the prominence of macrophage infiltration in CD
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