September 2009
Volume 50, Issue 9
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Immunology and Microbiology  |   September 2009
Development of Experimental Autoimmune Uveitis: Efficient Recruitment of Monocytes Is Independent of CCR2
Author Affiliations
  • Athanasios Dagkalis
    From the Division of Applied Medicine, University of Aberdeen, Aberdeen, United Kingdom;
  • Carol Wallace
    From the Division of Applied Medicine, University of Aberdeen, Aberdeen, United Kingdom;
  • Heping Xu
    From the Division of Applied Medicine, University of Aberdeen, Aberdeen, United Kingdom;
  • Sebastian Liebau
    From the Division of Applied Medicine, University of Aberdeen, Aberdeen, United Kingdom;
  • Ayyakkannu Manivannan
    From the Division of Applied Medicine, University of Aberdeen, Aberdeen, United Kingdom;
  • Michael A. Stone
    Respiratory and Inflammation Research, AstraZeneca, Cheshire, United Kingdom; and the
  • Matthias Mack
    Department of Internal Medicine II, University of Regensburg, Regensburg, Germany.
  • Janet Liversidge
    From the Division of Applied Medicine, University of Aberdeen, Aberdeen, United Kingdom;
  • Isabel J. Crane
    From the Division of Applied Medicine, University of Aberdeen, Aberdeen, United Kingdom;
Investigative Ophthalmology & Visual Science September 2009, Vol.50, 4288-4294. doi:10.1167/iovs.09-3434
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      Athanasios Dagkalis, Carol Wallace, Heping Xu, Sebastian Liebau, Ayyakkannu Manivannan, Michael A. Stone, Matthias Mack, Janet Liversidge, Isabel J. Crane; Development of Experimental Autoimmune Uveitis: Efficient Recruitment of Monocytes Is Independent of CCR2. Invest. Ophthalmol. Vis. Sci. 2009;50(9):4288-4294. doi: 10.1167/iovs.09-3434.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Macrophages are major contributors to the damage occurring in the retina in experimental autoimmune uveitis (EAU). CCR2 may be needed for efficient recruitment of monocytes to an inflammatory site, and the aim of this study was to determine whether this was the case in EAU.

methods. EAU was induced and graded in C57BL/6J and CCR2−/− mice. Macrophage infiltration and CCR2 expression were assessed using immunohistochemistry. Retinas were examined for MCP-1 expression using RT-PCR. Rolling and infiltration of labeled bone marrow monocytes at the inflamed retinal vasculature were examined by scanning laser ophthalmoscopy and confocal microscopy, respectively. Effect of CCR2 deletion or blockade by antibody and antagonist was determined.

results. Expression of mRNA for MCP-1 increased as EAU developed and was localized to the retina. CCR2 was associated with infiltrating macrophages. However, EAU induced in CCR2−/− mice was not reduced in severity, and neither was the percentage of macrophages in the retina. CCR2−/− monocytes, 48 hours after adoptive transfer to mice with EAU, showed no significant difference in percentage rolling or infiltration into the retina compared to WT. CCR2-independent rolling of monocytes was confirmed by CCR2 neutralizing antibody and antagonist treatment.

conclusions. CCR2 does not have a primary role in the recruitment of monocytes to the inflammatory site across the blood–retina barrier in well-developed EAU. Therapeutics targeting CCR2 are unlikely to be of value in treating human posterior uveitis.

Monocyte trafficking to an inflammatory site is an important part of the immune response and essential to the resolution of bacterial infection. However, in some situations such as autoimmune disease, monocyte recruitment and differentiation into macrophages at the inflammatory site lead to pathogenic consequences. Endogenous posterior intraocular inflammation, or uveitis, is one such example. In experimental autoimmune uveitis (EAU), an animal model for the human condition which is induced by immunization with a uveitogenic peptide at a site distant to the eye, monocytes/macrophages are found to be the main effector cells, with neutrophils less common, and T cells, although important, acting more to initiate and maintain the response. 1 Macrophages cross the blood–retina barrier (BRB) and infiltrate the retina, where their release of mediators such as NO and TNFα can cause severe retinal damage and consequent loss of sight in humans. 2 Understanding the factors involved in regulation of monocyte recruitment to the inflamed retina is therefore key to identifying therapeutic targets. 
Chemokines and their receptors are crucial to leukocyte trafficking, 3 and monocytes are reported to express various chemokine receptors, including CCR1, CCR2, CCR5, and CX3CR1. 4 CCL2 (MCP-1, JE in mice) and CCL5, ligands for CCR2 and CCR5 respectively, have been shown to trigger the arrest of monocytes under physiological conditions. 5 In inflammatory situations, it has been suggested that CCR2 in particular is important for the efficient recruitment of monocytes to an inflammatory site, 6 and this is supported by evidence from, for example, thioglycollate-induced peritonitis, 6 7 experimental autoimmune encephalomyelitis (EAE), 8 and atherosclerosis. 9  
However, although CCR2-positive monocytes may be recruited preferentially to an inflammatory site, CCR2-negative monocytes are also able to traffic to sites of inflammation, 10 11 12 13 and there are reports conflicting with those above that this is with equal efficiency. Thus, a confusing picture has emerged as to whether or not CCR2 is required for recruitment of monocytes to an inflammatory site, and this confusion is likely to result from differences in the stimulus, stage, and type of the inflammatory response and anatomic site. We have therefore investigated the requirement for CCR2 in the recruitment of monocytes to the inflamed retina in EAU. This is important to determine whether therapeutics targeting CCR2, which are presently in clinical trials, will be of value in the treatment of human endogenous posterior intraocular inflammation. We provide evidence that in this inflammatory situation at peak disease, monocyte recruitment is independent of CCR2 and that lack of CCR2 does not alter the severity of EAU. 
Materials and Methods
Animals and Retinal Inflammation Model
Eight- to sixteen-week-old C57BL/6J and CCR2−/− mice, generated as described elsewhere 7 and backcrossed onto a C57BL/6J background for 10 generations (Jackson Laboratory, Bar Harbor, Maine), were bred and maintained in the Medical Research Facility of the University of Aberdeen (Aberdeen, UK). All animals were housed under conditions as outlined in the Home Office Code of Practice. Procedures were approved according to the Home Office Regulations for Animal Experimentation (UK) and complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
To induce EAU, the animals were immunized with human IRBP peptide 1-20 (GPTHLFQPSLVLDMAKVLLD; 10 mg/mL; Sigma-Genosys, Cambridge, UK) emulsified 1:1 in complete Freund’s adjuvant (CFA; H37Ra [Difco Laboratories, Detroit, MI]) with additional 2.5 mg/mL Mycobacterium tuberculosis (H37Ra; Difco Laboratories). Each animal received a total of 500 μg peptide, given as 50 μL injected subcutaneously in each thigh. An additional 100 μL of Bordetella pertussis toxin (Health Protection Agency, Salisbury, UK) was administered IP at a concentration of 10 μg/mL. In this model, the first signs of retinal inflammation are observed at approximately day 15 postimmunization (pi) with peak disease between day 23 and day 28 pi. 14  
Histologic Evaluation of EAU
Eyes from animals were harvested on day 26 pi. Eyes were snap frozen in a water-soluble glycol and resin compound (OCT; Tissue Tek, Sakura, Zoeterwoude, The Netherlands) using isopentane and dry ice. Cryostat sections (8 μm thick) were prepared and air-dried for 48 hours. The dried sections were stained with hematoxylin and eosin. Histologic evaluation of EAU was done using our customized version of the grading system. 15 This method assigns a score of up to 5 both for cellular infiltration, by counting infiltrating cells and granulomas in different areas of the posterior chamber and assessing the degree of vasculitis, perivascular cuffing, and choroidal thickening; and for structural abnormalities, by assessing the degree of loss of rod outer segments and neuronal layers and the number of retinal folds and retinal detachment. Two independent examiners, masked as to treatment, determined the scores by observing the H&E-stained sections under ×20 objective lens. For each eye, six sections were graded. 
Analysis of MCP-1 mRNA Expression in the Retina
Retinas were dissected from the eyes of both naïve animals and those immunized with IRBP peptide after perfusion with 30 mL PBS containing 10 U/mL heparin under terminal anesthesia. Retinas from the same animal were pooled and RNA was isolated using a reagent (RNA-Bee; Biogenesis Ltd., Poole, Dorset, UK) according to the manufacturer’s protocol. 
Poly A+ RNA from 5 μg total RNA was reverse transcribed with 200 U M-MLV reverse transcriptase (Promega UK, Southampton, UK). One microliter of this cDNA was used in the PCR. Each PCR was carried out in a total volume of 25 μL containing 12.5 μL master mix (Promega UK, Southampton, UK) and 2.5 μL primer (10 μM) mix. Mouse β-actin primers and MCP-1 primers 16 were obtained from ThermoFisher Scientific, Ulm, Germany (Table 1) . Primers were intron-spanned to allow discrimination of any genomic DNA. Thirty-three cycles of amplification were performed as described previously. 17 After amplification, samples were run on a 1.8% agarose gel (molecular biology grade, Promega) in TBE (0.045 M Tris-borate, 0.001 M EDTA) containing 0.4 μg/mL ethidium bromide. 
For quantitative real-time PCR, total retinal RNA was prepared from mice at different time points pi and reverse transcribed as above with 1 μL of the cDNA used in each reaction mix. The expression level of MCP-1 and GAPDH were determined using a probes master mix (Light Cycler 480 Probes Master Mix; Roche Diagnostics Ltd, East Sussex, UK). MCP-1 primers (Table 1)were designed using Roche universal probe library, and FAM-labeled UPL Probe 62 was used. Mouse GAPDH primers (Applied Biosystems, Foster City, CA) were VIC labeled. All results were normalized to the expression of GAPDH and expressed as the ratio of crossing point (Cp) for MCP-1 over Cp for GAPDH. Specificity of the PCR products was checked by determining the melting profile of each sample by heating from 60°C to 95°C at a linear rate of 0.10°C/s and measuring the fluorescence emitted to show that each pair of primers amplified a single product. Each run consisted of an initial denaturation time of 10 minutes at 95°C and 40 cycles at 95°C for 10 seconds and 60°C for 30 seconds. 
Immunohistochemistry
Eyes were frozen and 8 μm cryostat sections were cut, fixed, and stained as described previously. 18 Briefly, sections were incubated with primary antibody, either rat monoclonal antibody IgG-2b against murine CCR2 (MC21 19 ) or rat monoclonal antibody IgG2b to F4/80 (1:100; Serotec Ltd, Oxford, UK) for 1 hour at room temperature. Isotype control IgG2b antibody was from Serotec. Samples were then incubated for 1 hour, first with biotinylated secondary antibody and secondly with streptavidin, biotinylated alkaline phosphatase complex (both Dako Ltd, Cambridge, UK) before addition of fast red substrate solution. Six sections per eye and 3 to 4 fields of view per section (×20 objective BH-2 microscope; Olympus UK Ltd, Watford, UK) were examined and red and blue infiltrating cells counted. Infiltrating cells were determined by their position and morphology. 
Sections were prepared in the same way for confocal microscopy and stained dually for CCR2 and monocyte/macrophage marker (MOMA2), with antibody to MOMA2 (1:50, for 1 hour; Serotec), detected using FITC-conjugated antibody to rat Ig (1:100, for 30 minutes; Serotec), and biotinylated antibody to mouse CCR2 (MC21 as above), detected with streptavidin Texas red for 45 minutes (Amersham Biosciences UK, Bucks, UK). All incubations were at room temperature and were carried out in the dark from the addition of fluorochrome-conjugated antibody. Sections were mounted in mounting medium (Vectashield; Vector Laboratories, Peterborough, UK) and examined with a confocal laser scanning microscope (LSM 510; Carl Zeiss, Göttingen, Germany). 
Preparation of Bone Marrow Monocytes and Adoptive Transfer
Freshly isolated, lymphocyte-depleted bone marrow monocytes were collected from the femurs and tibiae of mice. The bone marrow was washed out of the bone with RPMI medium (obtained from PAA Laboratories Ltd, Somerset, UK), pushed through a 70 μm strainer (BD Biosciences) to form a single cell suspension, washed, and the red blood cells removed by ammonium chloride cell lysis. T and B cells were removed by magnetic depletion using CD4 and CD45R (B220) microbeads (Miltenyi Biotec, Surrey, UK) according to manufacturer’s instructions. For every 107 cells, 10 μL of each type of microbead were used. Bone marrow monocytes separated as above were labeled using a cell tracer kit (Vybrant CFDA-SE; Invitrogen, Paisley, UK) at the minimum working concentration of 0.5 μM, according to manufacturer’s instructions. Cells were transferred adoptively to recipient wild-type (WT) mice with EAU through the lateral veins of the tail, 107 cells in a volume of 150 μL per animal. 
Scanning Laser Ophthalmoscopy (SLO)
To examine the role of CCR2 in monocyte trafficking at the blood–retina barrier in real time, we used SLO, which can determine the percentage of rolling cells in retinal venules and their velocity. For SLO analysis, EAU was induced in WT recipient animals and monocytes adoptively transferred at day 25 pi. We have previously shown that adoptively transferred monocytes only traffic efficiently into the inflamed retina 24 to 48 hours after transfer 20 so monocyte trafficking in the retina was studied using the SLO technique (available online at http://bjo.bmijournals.com/misc/eyemov.shtml) 21 22 48 hours after their transfer. The animals were anesthetized with an anesthetic cocktail of 130 μg/kg fentanyl (Janssen-Cilag Ltd., Bucks, UK), 10.25 mg/kg midazolam (Roche), and 3.8 mg/kg acepromazine (ACP; Novartis Animal Health Ltd., Essex, UK), injected intraperitoneally, producing deep anesthesia for 45 minutes. SLO images were recorded simultaneously on DVD-R and captured digitally at 150 frames per second. For each eye, a region of interest containing two to four veins/venules was recorded. Rolling leukocytes and those not interacting with the endothelium were counted in each venule. Rolling cells were defined as those cells with a velocity below the critical velocity. 23 24 25 The rolling efficiency was calculated over 15 minutes as the percentage of labeled rolling cells among the total number of labeled cells that entered a venule. Rolling velocities and the length of time a cell rolled along the endothelium were calculated from digital images for randomly chosen, rolling, labeled cells in retinal venules. 22  
The effect of CCR2 blockade on monocyte trafficking was studied using an antibody and an antagonist against CCR2. The inhibitors were injected through the tail vein on separate occasions, 15 minutes after the start of SLO recording. For each animal, a further recording of 15 minutes was done. Antibody to CCR2, MC21 19 was at a concentration of 15 μg/mL blood (30 to 37.5 μg in 150 μL PBS). MC-21 shows excellent binding to CCR2 and does not cross-react with murine CCR5 or human CCR2 and CCR5. 19 MC-21 binds CCR2 with a neutralizing effect and has been shown to prevent inflammatory influx of monocytes. 19 26 Control antibodies were 30 μg anti-mouse CD62L, rat IgG MEL-14 27 and isotype control rat IgG (BD Biosciences). The antagonist used was JE (9-76) (kind gift of AstraZeneca, UK) an N-terminal truncated form of JE (mouse MCP-1). JE (9-76) is a potent inhibitor of the murine CCR2 receptor with a binding Ki of 6.4 nM (data not shown). To inhibit JE-induced chemotaxis of murine WEHI 274.1 cells, a concentration of 20 nM was needed (data not shown). At 1 mg/kg IV it exhibits a Cmax of 250 nM at 0.5 hour post-dose. The antagonist was therefore administered at 1 μg per 1 g of mouse body-weight (20–25 μg in 150 μL PBS). 
After SLO, mice were injected IV with 100 μL Evans blue 2% (w/v) in PBS (Sigma Aldrich, Cambridge, UK) which binds albumin. Mice were humanely killed 10 minutes later by CO2 inhalation and eyes immersed in 2% (w/v) paraformaldehyde for 1.5 hour. Retinal wholemounts were prepared by removing the retinas as described, 28 washing them twice in PBS for 15 minutes, spreading on clean glass slides and mounting vitreous side up. Fluorescent cells in the whole retina which had exited vessels were counted using a confocal laser scanning microscope (LSM 510; Carl Zeiss) for detection. As there can be a range of disease in animals at each time point, recipient mice were only included in the study if EAU was of comparable severity (50% of veins/venules showing more than one point of leakage of Evan’s blue). 
Data Analysis
Probability values were calculated using an unpaired two-tailed Student’s t-test. Probability values of <0.05 were considered significant. 
Results
MCP-1 Expression in Retina During EAU
The expression of mRNA for the CCR2 ligand MCP-1 (CCL2) in perfused retina was determined by routine RT-PCR. In normal retina from nonimmunized animals, only very low levels of MCP-1 mRNA could be detected, whereas in animals with EAU MCP-1 was strongly expressed (Fig. 1A) . Q-PCR was used to examine this finding quantitatively over specific time points pi. The results were normalized using the expression of GAPDH and expressed as the ratio of crossing point (Cp) for MCP-1 over Cp for GAPDH. The ratio decreased with days pi and was significantly lower at day 21 pi than for the non-immunized mice, indicating a significant increase in MCP-1 expression in the retina with EAU (Fig. 1B)
Dual Staining for MOMA2 and CCR2
Cryostat sections from animals were stained using antibodies to CCR2 (MC21) and MOMA2. CCR2 expression was clear on a substantial number of infiltrating cells throughout the retina and within the vitreous (Figs. 2A 2B) . The percentage of infiltrating cells within the vitreous and retina expressing CCR2 was 36.18 ± 6.04 (SD). Dual fluorescent staining of sections showed that the majority (88.18% ± 6.52 SD) of infiltrating MOMA2 positive cells were also CCR2 positive (Figs. 2C 2D)
Development of EAU in Mice Lacking CCR2
EAU was induced in both WT and CCR2−/− mice and disease was graded in terms of inflammatory infiltrate and structural damage at day 26 pi. Mice homozygous for the CCR2 deletion showed no difference in disease severity in terms of either infiltrate or structural damage at day 26 pi compared to WT mice (Fig. 3A)
When the infiltrate was examined after immunohistochemical staining for the macrophage marker F4/80, the percentage of macrophages in the infiltrate was not significantly changed when CCR2−/− mice were compared with WT mice at day 26 pi (Fig. 3B) . Similar results were obtained if antibodies to MOMA2 or CD11b were used (data not shown). 
Effect of CCR2 Deletion on Trafficking of Adoptively Transferred Bone Marrow Monocytes to the Inflamed Retina
Bone marrow monocytes from either C57BL/6 WT or CCR2−/− mice that had been labeled with CDFA-SE were injected via the tail vein into WT mice with EAU day 25 pi and eyes were examined by SLO 48 hours later. When WT and CCR2−/− monocytes were compared there was no significant difference in the percentage of labeled cells rolling (Fig. 4A) . However there was a slight but significant increase (P < 0.05) in the velocity of the CCR2−/− monocytes compared to WT and in the length of time they rolled (Figs. 4B 4C)
There appeared to be a decrease in the numbers of CCR2−/− monocytes infiltrating the retina, as determined by confocal microscopy, compared to WT but this was not statistically significant (Fig. 4D) . Confocal images were also examined for extent of Evan’s blue leakage, which indicates breakdown of the BRB, and it was confirmed that disease was comparable in the recipient animals included (data not shown). 
Effect of CCR2 Blockade with Antibody and Antagonist on Rolling of Monocytes on Inflamed Retinal Endothelium
As bone marrow monocytes from CCR2−/− mice might have a modified phenotype due to adjustment to long-term absence of CCR2, we also examined the rolling of labeled WT monocytes on inflamed retinal endothelium in response to blockade of CCR2 with either CCR2 neutralizing antibody (MC21) or CCR2 antagonist (JE 9-76). SLO readings were recorded for 15 minutes before IV injection of either antibody or antagonist and then for a further 15 minutes. This method gives a very accurate determination of the effect of the antibody or antagonist as the control readings are from the same mouse as the test results. 29 Figure 5Ashows that although treatment with the positive control antibody to CD62L significantly decreased the percentage of labeled monocytes that rolled (P < 0.05), treatment with either antibody or antagonist to CCR2 had no significant effect on rolling. Similarly, treatment with CCR2 antibody or antagonist had no effect on the velocity of the rolling cells (Fig. 5B)
Discussion
The increase in expression of mRNA for MCP-1 in the retina as EAU develops is consistent with our earlier immunohistochemistry data 30 and the association of CCR2 with infiltrating monocytes. This, combined with evidence that CCR2 on adoptively transferred monocytes increases in parallel with the ability of the cells to infiltrate the retina in EAU, 20 led us to expect that lack of CCR2 would hinder monocyte recruitment and potentially reduce the severity of EAU. However, this was not the case. Only the slight but significant increase in the rolling velocity of labeled CCR2−/− monocytes in EAU indicated that CCR2 might be involved. This resulted in the CCR2−/− monocytes rolling for longer than WT, but did not lead to a statistically significant reduction in the number of CCR2−/− monocytes infiltrating the retina. CCR2 antibody or antagonist had no effect on either the percentage of WT monocytes rolling or their velocity, and differences in monocyte subsets present 10 may explain this difference between WT and CCR2−/− mice. 
Some of the experimental data underlining the importance of CCR2 in monocyte recruitment in inflammation comes from models of atherosclerosis. 9 31 32 However, in these situations monocyte adhesion is arteriolar rather than venular and there is evidence that monocyte recruitment at arterioles is regulated more by MCP-1 than at venules. 33 We have shown that in EAU, recruitment of leukocytes is via post-capillary venules 34 and that MIP-1α, a CCR5 ligand, is important for leukocyte recruitment in this situation. 35 Additional chemokines and chemoattractants are upregulated in the retina during EAU 30 36 37 and may be involved. Other site-specific differences are also likely to have a substantial effect on which chemokine/chemokine receptor pairs take precedence, such as the increased shear stress at the retinal vasculature compared to more peripheral sites. 20  
The stage of the inflammatory response also influences which chemokine receptors predominantly regulate monocyte recruitment. We have confined our investigations to recipients with well-developed EAU and it may be that monocyte recruitment is more dependent on CCR2 earlier in EAU development. However, even if this is the case, this recruitment is not fundamental to EAU as no difference in disease severity is detected in CCR2−/− compared to WT mice. 
As the retina is an extension of the CNS, it is appropriate to compare the role of CCR2 in EAU with that in EAE. Development of EAE in CCR2−/− mice is delayed but appears to be critically dependent on the mouse strain used. 38 CCR2−/− mice were 100% susceptible to EAE if they were on C57BL/6 x J129 or BalbC strains, but less susceptible on the C57BL/6 background. 8 Age of the mice and differences in methods of disease induction, including antigen dose and route of immunization, were also thought to have contributed to increased resistance to EAE in CCR2−/− mice in earlier studies. 
We have shown that CCR2 does not have a primary role in the recruitment of monocytes to the inflammatory site across the BRB in well-developed EAU in C57BL/6 mice. Hence, drugs targeting CCR2 are unlikely to be helpful in the treatment of human endogenous posterior intraocular inflammation. Strong MCP-1 expression in the retina and CCR2 expression by recruited monocytes suggests a role in monocyte retention and activation although as CCR2 deletion did not lead to reduction in disease severity, these actions are likely to be compensated for by other chemokine/ chemokine receptor pairs. 
 
Table 1.
 
Oligonucleotide Primer Sequences Used for Standard and Real-time PCR
Table 1.
 
Oligonucleotide Primer Sequences Used for Standard and Real-time PCR
Sequence
Mouse B-actin (standard PCR) Forward: 5′-GTG GGG CGC CCC AGG CAC CA-3′
Reverse: 5′-GCT CGG TGG TGG TGA AGC-3′
Mouse MCP-1 (standard PCR) Forward: 5′-CAC TCA CCT GCT GCT ACT CAT TCA C-3′
Reverse: 5′-GGA TTC ACA GAG AGG GAA AAA TGG-3′
Mouse MCP-1 (real-time PCR) Forward: 5′-CAT CCA CGT GTT GGC TCA-3′
Reverse: 5′-GAT CAT CTT GCT GGT GAA TGA GT-3′
Figure 1.
 
Expression of mRNA for MCP-1 in retina of mice with EAU. (A) Agarose gel electrophoresis of RT-PCR products β-actin (Aa) and MCP-1 (Ab). Lanes 1 to 3: perfused retina from non-immunized mice; lanes 4 to 6: perfused retina from mice with EAU d23 pi; lane 7: ladder. Retinas from the same animal were pooled. (B) Q-PCR results showing crossing point (Cp) for MCP-1 as a ratio to GAPDH Cp for retinas from mice at different times pi. Retinas from each mouse pooled (3 or 4 mice at each time point). Error bars indicate SEM. *P < 0.05.
Figure 1.
 
Expression of mRNA for MCP-1 in retina of mice with EAU. (A) Agarose gel electrophoresis of RT-PCR products β-actin (Aa) and MCP-1 (Ab). Lanes 1 to 3: perfused retina from non-immunized mice; lanes 4 to 6: perfused retina from mice with EAU d23 pi; lane 7: ladder. Retinas from the same animal were pooled. (B) Q-PCR results showing crossing point (Cp) for MCP-1 as a ratio to GAPDH Cp for retinas from mice at different times pi. Retinas from each mouse pooled (3 or 4 mice at each time point). Error bars indicate SEM. *P < 0.05.
Figure 2.
 
CCR2 staining of infiltrating cells in EAU retina. (A) Immunohistochemical staining of a cryostat section from an eye with EAU, for CCR2 (MC21 antibody) and (B) for control a, same area using rat IgG2b. Black arrows: positively stained cells. (C) Immunofluorescent dual staining of similar sections with antibody to CCR2 (Texas red) and to MOMA2 (FITC) or (D) control b with no primary antibodies included. Yellow staining: areas stained dually (also indicated by white arrows). EAU d28 pi; layers identified: V, vitreous; GL, ganglion layer; INL, inner nuclear layer. Scale bar, 20 μm.
Figure 2.
 
CCR2 staining of infiltrating cells in EAU retina. (A) Immunohistochemical staining of a cryostat section from an eye with EAU, for CCR2 (MC21 antibody) and (B) for control a, same area using rat IgG2b. Black arrows: positively stained cells. (C) Immunofluorescent dual staining of similar sections with antibody to CCR2 (Texas red) and to MOMA2 (FITC) or (D) control b with no primary antibodies included. Yellow staining: areas stained dually (also indicated by white arrows). EAU d28 pi; layers identified: V, vitreous; GL, ganglion layer; INL, inner nuclear layer. Scale bar, 20 μm.
Figure 3.
 
EAU in WT and CCR2−/− mice at day 26 pi. (A) EAU score as determined by grading inflammatory infiltrate and structural damage. Circles: infiltrative score; triangles: structural score. Open symbols: WT mice; filled symbols: CCR2−/− mice. Bar shows the mean. n = 12 eyes (6 mice) per group. Each point represents the average grade of at least three sections. (B) Percentage of cells in the inflammatory infiltrate stained positively for F4/80. Error bars indicate SEM. n = 3 mice with 3 sections per point. No statistical difference between WT and CCR2−/− groups for either (A) or (B).
Figure 3.
 
EAU in WT and CCR2−/− mice at day 26 pi. (A) EAU score as determined by grading inflammatory infiltrate and structural damage. Circles: infiltrative score; triangles: structural score. Open symbols: WT mice; filled symbols: CCR2−/− mice. Bar shows the mean. n = 12 eyes (6 mice) per group. Each point represents the average grade of at least three sections. (B) Percentage of cells in the inflammatory infiltrate stained positively for F4/80. Error bars indicate SEM. n = 3 mice with 3 sections per point. No statistical difference between WT and CCR2−/− groups for either (A) or (B).
Figure 4.
 
The effect of CCR2 deficiency on trafficking of labeled monocytes on inflamed retinal endothelium 48 hours after adoptive transfer. Bone marrow monocytes from CCR2−/− and WT mice compared for rolling efficiency, expressed as rolling cells as a percentage of all labeled rolling and free-floating cells in a vessel (A); rolling velocity, 40 cells per group measured (B); length of time the cells rolled (C); and infiltration of cells into the retina counted in retinal wholemounts (D). Error bars indicate SEM. (n = 6) retinas per group. *P < 0.05.
Figure 4.
 
The effect of CCR2 deficiency on trafficking of labeled monocytes on inflamed retinal endothelium 48 hours after adoptive transfer. Bone marrow monocytes from CCR2−/− and WT mice compared for rolling efficiency, expressed as rolling cells as a percentage of all labeled rolling and free-floating cells in a vessel (A); rolling velocity, 40 cells per group measured (B); length of time the cells rolled (C); and infiltration of cells into the retina counted in retinal wholemounts (D). Error bars indicate SEM. (n = 6) retinas per group. *P < 0.05.
Figure 5.
 
The effect of CCR2 blockade on rolling of labeled monocytes on inflamed retinal endothelium 48 hours after adoptive transfer. Rolling efficiency of labeled WT monocytes, rolling cells as a percentage of all labeled rolling and free-floating cells in a vessel, was compared before and after the addition of antibody against CCR2 (MC21), antagonist to CCR2 (JE9-76), antibody to CD62L or isotype (iso) control (A). SLO images were recorded for 15 minutes before and after the addition. Velocites of labeled rolling cells were compared after the additions (B). Error bars indicate SEM. n = 3 mice per group. *P < 0.05.
Figure 5.
 
The effect of CCR2 blockade on rolling of labeled monocytes on inflamed retinal endothelium 48 hours after adoptive transfer. Rolling efficiency of labeled WT monocytes, rolling cells as a percentage of all labeled rolling and free-floating cells in a vessel, was compared before and after the addition of antibody against CCR2 (MC21), antagonist to CCR2 (JE9-76), antibody to CD62L or isotype (iso) control (A). SLO images were recorded for 15 minutes before and after the addition. Velocites of labeled rolling cells were compared after the additions (B). Error bars indicate SEM. n = 3 mice per group. *P < 0.05.
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Figure 1.
 
Expression of mRNA for MCP-1 in retina of mice with EAU. (A) Agarose gel electrophoresis of RT-PCR products β-actin (Aa) and MCP-1 (Ab). Lanes 1 to 3: perfused retina from non-immunized mice; lanes 4 to 6: perfused retina from mice with EAU d23 pi; lane 7: ladder. Retinas from the same animal were pooled. (B) Q-PCR results showing crossing point (Cp) for MCP-1 as a ratio to GAPDH Cp for retinas from mice at different times pi. Retinas from each mouse pooled (3 or 4 mice at each time point). Error bars indicate SEM. *P < 0.05.
Figure 1.
 
Expression of mRNA for MCP-1 in retina of mice with EAU. (A) Agarose gel electrophoresis of RT-PCR products β-actin (Aa) and MCP-1 (Ab). Lanes 1 to 3: perfused retina from non-immunized mice; lanes 4 to 6: perfused retina from mice with EAU d23 pi; lane 7: ladder. Retinas from the same animal were pooled. (B) Q-PCR results showing crossing point (Cp) for MCP-1 as a ratio to GAPDH Cp for retinas from mice at different times pi. Retinas from each mouse pooled (3 or 4 mice at each time point). Error bars indicate SEM. *P < 0.05.
Figure 2.
 
CCR2 staining of infiltrating cells in EAU retina. (A) Immunohistochemical staining of a cryostat section from an eye with EAU, for CCR2 (MC21 antibody) and (B) for control a, same area using rat IgG2b. Black arrows: positively stained cells. (C) Immunofluorescent dual staining of similar sections with antibody to CCR2 (Texas red) and to MOMA2 (FITC) or (D) control b with no primary antibodies included. Yellow staining: areas stained dually (also indicated by white arrows). EAU d28 pi; layers identified: V, vitreous; GL, ganglion layer; INL, inner nuclear layer. Scale bar, 20 μm.
Figure 2.
 
CCR2 staining of infiltrating cells in EAU retina. (A) Immunohistochemical staining of a cryostat section from an eye with EAU, for CCR2 (MC21 antibody) and (B) for control a, same area using rat IgG2b. Black arrows: positively stained cells. (C) Immunofluorescent dual staining of similar sections with antibody to CCR2 (Texas red) and to MOMA2 (FITC) or (D) control b with no primary antibodies included. Yellow staining: areas stained dually (also indicated by white arrows). EAU d28 pi; layers identified: V, vitreous; GL, ganglion layer; INL, inner nuclear layer. Scale bar, 20 μm.
Figure 3.
 
EAU in WT and CCR2−/− mice at day 26 pi. (A) EAU score as determined by grading inflammatory infiltrate and structural damage. Circles: infiltrative score; triangles: structural score. Open symbols: WT mice; filled symbols: CCR2−/− mice. Bar shows the mean. n = 12 eyes (6 mice) per group. Each point represents the average grade of at least three sections. (B) Percentage of cells in the inflammatory infiltrate stained positively for F4/80. Error bars indicate SEM. n = 3 mice with 3 sections per point. No statistical difference between WT and CCR2−/− groups for either (A) or (B).
Figure 3.
 
EAU in WT and CCR2−/− mice at day 26 pi. (A) EAU score as determined by grading inflammatory infiltrate and structural damage. Circles: infiltrative score; triangles: structural score. Open symbols: WT mice; filled symbols: CCR2−/− mice. Bar shows the mean. n = 12 eyes (6 mice) per group. Each point represents the average grade of at least three sections. (B) Percentage of cells in the inflammatory infiltrate stained positively for F4/80. Error bars indicate SEM. n = 3 mice with 3 sections per point. No statistical difference between WT and CCR2−/− groups for either (A) or (B).
Figure 4.
 
The effect of CCR2 deficiency on trafficking of labeled monocytes on inflamed retinal endothelium 48 hours after adoptive transfer. Bone marrow monocytes from CCR2−/− and WT mice compared for rolling efficiency, expressed as rolling cells as a percentage of all labeled rolling and free-floating cells in a vessel (A); rolling velocity, 40 cells per group measured (B); length of time the cells rolled (C); and infiltration of cells into the retina counted in retinal wholemounts (D). Error bars indicate SEM. (n = 6) retinas per group. *P < 0.05.
Figure 4.
 
The effect of CCR2 deficiency on trafficking of labeled monocytes on inflamed retinal endothelium 48 hours after adoptive transfer. Bone marrow monocytes from CCR2−/− and WT mice compared for rolling efficiency, expressed as rolling cells as a percentage of all labeled rolling and free-floating cells in a vessel (A); rolling velocity, 40 cells per group measured (B); length of time the cells rolled (C); and infiltration of cells into the retina counted in retinal wholemounts (D). Error bars indicate SEM. (n = 6) retinas per group. *P < 0.05.
Figure 5.
 
The effect of CCR2 blockade on rolling of labeled monocytes on inflamed retinal endothelium 48 hours after adoptive transfer. Rolling efficiency of labeled WT monocytes, rolling cells as a percentage of all labeled rolling and free-floating cells in a vessel, was compared before and after the addition of antibody against CCR2 (MC21), antagonist to CCR2 (JE9-76), antibody to CD62L or isotype (iso) control (A). SLO images were recorded for 15 minutes before and after the addition. Velocites of labeled rolling cells were compared after the additions (B). Error bars indicate SEM. n = 3 mice per group. *P < 0.05.
Figure 5.
 
The effect of CCR2 blockade on rolling of labeled monocytes on inflamed retinal endothelium 48 hours after adoptive transfer. Rolling efficiency of labeled WT monocytes, rolling cells as a percentage of all labeled rolling and free-floating cells in a vessel, was compared before and after the addition of antibody against CCR2 (MC21), antagonist to CCR2 (JE9-76), antibody to CD62L or isotype (iso) control (A). SLO images were recorded for 15 minutes before and after the addition. Velocites of labeled rolling cells were compared after the additions (B). Error bars indicate SEM. n = 3 mice per group. *P < 0.05.
Table 1.
 
Oligonucleotide Primer Sequences Used for Standard and Real-time PCR
Table 1.
 
Oligonucleotide Primer Sequences Used for Standard and Real-time PCR
Sequence
Mouse B-actin (standard PCR) Forward: 5′-GTG GGG CGC CCC AGG CAC CA-3′
Reverse: 5′-GCT CGG TGG TGG TGA AGC-3′
Mouse MCP-1 (standard PCR) Forward: 5′-CAC TCA CCT GCT GCT ACT CAT TCA C-3′
Reverse: 5′-GGA TTC ACA GAG AGG GAA AAA TGG-3′
Mouse MCP-1 (real-time PCR) Forward: 5′-CAT CCA CGT GTT GGC TCA-3′
Reverse: 5′-GAT CAT CTT GCT GGT GAA TGA GT-3′
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