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From the Department of Ophthalmology,* University of Aberdeen, Aberdeen, United Kingdom; the Division of Ophthalmology, University of Bristol, Bristol, United Kingdom; the Department of Immunology, University Medical Centre, Utrecht, The Netherlands; and DNAX Research Incorporated, Palo Alto, California
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Abstract |
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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.13-15 By contrast the CD200R is expressed predominantly by cells of the myeloid lineage14,16 including microglia (MG)14 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.16 Regulation of macrophage function is supported by the following observations in CD200-/- mice: significant increase in numbers of myeloid-derived cells within lymphoid organs,15 accelerated onset of experimental autoimmune encephalomyelitis, and conversion of C57BL/6 mice from resistant to susceptible to collagen-induced arthritis.15 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.15 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.17 Murine EAU is a CD4+ T-cell-mediated destruction of the neuroretina and photoreceptors of the eye.18,19 EAU has the advantage of not only determining cellular infiltrate but the model also permits the semiquantitative documentation of the extent of tissue destruction.18,20
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Materials and Methods |
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CD200-deficient mice (CD200-/-) of background strain C57BL/6 were generated by DNAX, Palo Alto, CA15 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.
Immunohistochemistry and Retinal Whole-Mount Immunofluorescence
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 described18 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.
Flow Cytometric Analysis
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.20 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.20 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.
Proliferation Assay and Cytokine Analysis
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 x 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.21,22 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 x 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)-
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.
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Results |
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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)
.15-17
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.17
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,23
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)
.
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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).
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, particularly within resident cells of the photoreceptor layer and inner nuclear layers of the retina (Figure 5, B and C)
. This increase was highly significant and was maintained throughout the disease course (day 10, P < 0.0001; day 16, P < 0.0058; day 23, P < 0.001). Dual immunofluorescence confirmed that apoptosis occurred predominantly within non-T cell and macrophage populations (Figure 5, D and E)
. There was no increase in level of apoptosis in nonimmunized mice (data not shown).
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. NOS2 expression was significantly higher in CD200-/- mice both before peak disease (day 10, P < 0.004) and after peak disease (day 23, P < 0.035). However, despite the fact that retinal MG in CD200-/- mice constitutively express higher levels of NOS2 compared to CD200+/+ mice, they failed to achieve peak NOS2 expression levels during EAU at the time points tested (Figure 6)
.
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IRBP peptide 1-20 splenocyte proliferative responses were equivalent in both CD200+/+ and CD200-/- mice (Figure 7A)
. This was confirmed, as proliferation was also equivalent in both groups of mice, when T cells and APC were isolated (data not shown). In addition, IFN- and IL-10 production in splenocyte culture supernatants stimulated with IRBP peptide 1-20 was measured by ELISA (Figure 7B)
. IFN- was produced to equally high levels in both groups of animals at days 10 and 16 (548 (0.015), 554 (0.002) pg/ml at day 10 and 515 (0.011), 560 (0.06) pg/ml day 16, CD200+/+ and CD200-/-, respectively) but such a high level was maintained only in CD200-/- mice by day 23 after immunization (162 (0.035) pg/ml versus 474 (0.021) pg/ml; P < 0.0001, CD200+/+ and CD200-/-, respectively). IL-10 production was also higher in CD200-/- mice day 23 after immunization (2.9 (0.001) pg/ml versus 12.6 (0.008) pg/ml, P < 0.0001, CD200+/+ and CD200-/-, respectively).
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Discussion |
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This study confirms that depletion of CD200 also increases NOS2 expression in macrophages and MG in retina, a specialized neural tissue exhibiting lower numbers of MG than brain. In normal retina, MG are usually NOS2-negative (Figure 3)
and significant levels of expression of NOS2 are observed only during peak EAU subsiding again during resolution of inflammation.24
Correspondingly NOS2 inhibitors protect against photoreceptor destruction24
and suppress production of peroxynitrite and apoptosis of photoreceptors.29
MG are conditioned in situ and display characteristics of transforming growth factor-ß conditioned bone marrow-derived macrophages8
in that they neither spontaneously nor after stimulation with IFN-, produce NO but express ß-glucuronidase.30
It would seem therefore that in the absence of a functional CD200-CD200R axis, macrophages and retinal MG are activated. There was no specific anti-mouse CD200R mAb available, but within the confines of rat retina, where there is abundant expression of CD200, we have previously shown that CD200R was not detected.17
However, during EAU expression of CD200R is observed readily on both MG and infiltrating CD11b+ cells in the rat model.17
One explanation is that during CD200:CD200R tonic suppression of macrophage activation, MG down-regulate CD200R. It is not clear what the signals are that support up-regulation of CD200R expression in MG during inflammation. Certainly a Th1 response in EAU with pronounced IFN- and tumor necrosis factor-
activity31,32
within the retina, may generate signals, which override the CD200:CD200R axis and activate cells. Given that the data strongly supports that in CD200-/- mice MG are classically activated, we were surprised to observe that firstly; during peak disease, NOS2 expression was reduced compared to CD200+/+ mice and to that previously reported,24,33,34
and secondly there was no significant increase, in photoreceptor destruction observed histologically. One could assume, therefore, that as we have shown CD200-/- mice generate equivalent T cell responses and abundant IFN- production, deregulated activated MG and infiltrating macrophages are simply unable to respond further to a classical IFN--mediated increase in NOS2 expression.8
This may explain why tissue damage, despite our observed earlier disease onset and increased levels of macrophage infiltrate, was not enhanced in this model. Although indefinable in this model, the findings of sustained photoreceptor cell apoptosis, together with maintained higher NOS2 expression infer that chronic tissue loss and subsequent functional loss may result in the long term via an inability to recover homeostatic control. Despite increased levels of apoptosis in these current experiments we were unable to observe histologically differences in outer nuclear layer (ONL) cell loss, perhaps because the level of apoptosis precluded ability to observe differences in, for example, total number of cells remaining over rows of cells. We are presently attempting to establish electrophysiological assessment of retinal function to investigate whether our observed ONL apoptosis leads to retinal dysfunction. CD200 depletion alters local tissue control of both resident and myeloid cells, the persistence of cytokine production in CD200-/- (both IFN- and IL-10) observed during splenocyte responses also infers a peripheral activation of T cells (although not manifested as an increased proliferation) either directly or indirectly because of altered APC activation, both of which require further investigation.
Concluding Remarks
This data infers that CD200 acts primarily on effector responses that are mediated via interaction with CD200R-expressing macrophages. With respect to retina and CNS, CD200 is an important regulator of myeloid activation in situ during inflammation. Constitutive down-regulation of CNS and retinal macrophages have important implications on how potential deregulation of CD200:CD200R axis effects not only the course of autoimmune inflammation but also the generation of, and tissue response to, neurodegenerative conditions.
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Acknowledgements |
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Footnotes |
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Supported from grants from the Iris Fund for the Prevention of Blindness, the Royal College of Surgeons of Edinburgh, and Schering-Plough Corporation (to DNAX).
Accepted for publication July 22, 2002.
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