October 2005
Volume 46, Issue 10
Free
Immunology and Microbiology  |   October 2005
CCR5-Deficient Mice Develop Experimental Autoimmune Uveoretinitis in the Context of a Deviant Effector Response
Author Affiliations
  • Aya Takeuchi
    From the Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan; and the
  • Yoshihiko Usui
    From the Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan; and the
  • Masaru Takeuchi
    From the Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan; and the
  • Takaaki Hattori
    From the Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan; and the
  • Takeshi Kezuka
    From the Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan; and the
  • Jun Suzuki
    From the Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan; and the
  • Yoko Okunuki
    From the Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan; and the
  • Takuya Iwasaki
    From the Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan; and the
  • Makoto Haino
    Department of Molecular Preventive Medicine and Solution Oriented Research for Science and Technology (SORST), School of Medicine, The University of Tokyo, Tokyo, Japan.
  • Kouji Matsushima
    Department of Molecular Preventive Medicine and Solution Oriented Research for Science and Technology (SORST), School of Medicine, The University of Tokyo, Tokyo, Japan.
  • Masahiko Usui
    From the Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan; and the
Investigative Ophthalmology & Visual Science October 2005, Vol.46, 3753-3760. doi:10.1167/iovs.04-1429
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Aya Takeuchi, Yoshihiko Usui, Masaru Takeuchi, Takaaki Hattori, Takeshi Kezuka, Jun Suzuki, Yoko Okunuki, Takuya Iwasaki, Makoto Haino, Kouji Matsushima, Masahiko Usui; CCR5-Deficient Mice Develop Experimental Autoimmune Uveoretinitis in the Context of a Deviant Effector Response. Invest. Ophthalmol. Vis. Sci. 2005;46(10):3753-3760. doi: 10.1167/iovs.04-1429.

      Download citation file:


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

      ×
  • Supplements
Abstract

purpose. Experimental autoimmune uveoretinitis (EAU) is an organ-specific, Th1-cell–mediated disease that targets the neural retina. CCR5 is a chemokine receptor expressed on Th1 cells that promotes their migration. In CCR5-deficient mice, we examined the role of CCR5 in the development of EAU induced by immunization with interphotoreceptor retinoid-binding protein (IRBP) peptide.

methods. Wild-type or CCR5-deficient B6 mice were immunized with human IRBP peptide 1-20 (hIRBP-p), and the severity of EAU was assessed clinically and histologically. Splenocytes and cells of regional lymph nodes near the eye were collected and their proliferation and production of IL-6, IL-10, IFN-γ, and CCL2 (MCP-1) in response to hIRBP-p stimulation were measured. Moreover, the intraocular levels of these cytokines were analyzed.

results. Immunization with hIRBP-p induced EAU in CCR5-deficient mice with a severity comparable to that in wild-type mice. Histologically, T-cell infiltration of the eye was reduced, but granulocyte infiltration was augmented in CCR5-deficient mice. Although splenic T cells from CCR5-deficient mice produced IFN-γ but not IL-10 on stimulation by hIRBP-p, T cells from the regional lymph nodes failed to produce both cytokines. IL-6 production in the eye and IL-6 and CCL2 production by splenic T cells were predominantly augmented in CCR5-deficient mice.

conclusions. The development of EAU is not prevented in CCR5-deficient mice. Although T-cell infiltration into the eye is apparently reduced in CCR5-deficient mice, the defect is compensated for by granulocyte infiltration, supposedly mediated by augmented intraocular production of IL-6.

Experimental autoimmune uveoretinitis (EAU) is an organ-specific, T-cell–mediated disease that targets the neural retina. 1 EAU is induced in susceptible animals by immunization with uveitogenic retinal antigens emulsified in complete Freund’s adjuvant (CFA) or by adoptive transfer of retina-specific syngeneic CD4+ T cells. 2 3 Murine EAU closely resembles several human noninfectious uveitic diseases, and the murine EAU serves as an ideal model for human posterior uveitis, a major sight-threatening disease. 4 In genetically unmanipulated animals, EAU is a T-helper type 1 (Th1)–mediated disease, because uveitogenic T cells show a Th1-like cytokine profile (high IFN-γ) and susceptible rodent strains typically mount a Th1-dominant response to the uveitogenic antigen. 5 6 7 The balance between Th1 and Th2 immune responses plays an important role in determining the outcome of an uveitogenic challenge. 
The recruitment of leukocytes is a critical feature of ocular inflammation in EAU. Chemokines and their G-protein-coupled surface membrane receptors mediate innate and adaptive immunity through mechanisms of selection and recruitment of cells to sites of inflammation and disease. 8 A recent study has reported the expression of CCL3/macrophage-inflammatory protein-1α (MIP-1α), CCL2/monocyte chemoattractant protein–1 (CCL2), and CCL5/regulated on activation of normal T-cell-expressed and secreted (RANTES) in the choroids and retina during the course of EAU, and CCL3 is particularly expressed concomitant with the Th1 cytokine IFN-γ. 9 In addition, a study in patients with uveitis has shown that some of the chemokines are involved in leukocyte recruitment into the eye and that CXCL8/IL-8, CXCL10/interferon-inducible 10-kDa protein (IP-10), CCL2, CCL5, and CCL3 increase significantly in the aqueous humor during the active stages of anterior uveitis. 10  
It is also known that chemokines, through interaction with their corresponding receptors that are differentially expressed on Th1 and Th2 cells, modulate the Th1 versus Th2 balance, thereby regulating the inflammatory response. 11 12 For example, the chemokine receptors CXCR3 and CCR5 are associated with a Th1 phenotype, whereas CCR3, CCR4, and CCR8 are found in the Th2 phenotype. 13 14 15 16 CCR5 is the receptor for CCL3, CCL4, and CCL5, which is expressed on activated and memory (CD45RO+) T cells but not on naïve T cells. 17 18 19 Accumulating evidence suggests that CCR5 is involved in a series of T-cell–mediated autoimmune diseases that are in favor of Th1 immune responses. For example, elevated expression of CCR5 can be found in the infiltrating T cells, macrophages, activated microglia, and astrocytes in patients with multiple sclerosis. 20 Moreover, in patients with rheumatoid arthritis, 80% of the T cells in the synovial fluid compared with only 15% in the blood are CCR5 positive. 21 We have recently reported that CCR2, CCR5, and CXCR3 are overexpressed in the posterior segment of the eyes after IRBP immunization. 22 In the present study, we examined whether CCR5 deficiency prevents the development of EAU induced by immunization with IRBP peptide. Contrary to our expectation, the development of EAU was not prevented in CCR5-deficient mice, and the severity was comparable to that in wild-type mice. Although T-cell infiltration of the eye was in part impaired in CCR5-deficient mice, the defect was compensated for by augmented granulocyte infiltration. 
Materials and Methods
Mice
Six- to eight-week old female C57BL/6 mice were obtained from Japan CLEA Inc. (Shizuoka, Japan). C57BL/6 CCR5-deficient mice (CCR5 KO) were kindly provided by Makoto Haino and Kouji Matsushima (Department of Molecular Preventive Medicine and Solution Oriented Research for Science and Technology (SORST), School of Medicine, The University of Tokyo). All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures were performed with animals under pentobarbital sodium or ketamine-xylazine anesthesia. 
Reagents
Human IRBP peptide 1-20 (hIRBP-p) was synthesized (model 432A Peptide Synthesizer; Applied Biosystems, Inc., Foster City, CA), with Fmoc chemistry. Purified Bordetella pertussis toxin (PTX) was from Sigma-Aldrich (St. Louis, MO). CFA and Mycobacterium tuberculosis strain H37Ra were from Difco (Detroit, MI). 
Induction and Scoring of EAU
C57BL/6 mice were immunized subcutaneously with 0.2 mL of emulsion containing 200 μg of hIRBP-p in CFA (1:1 wt/vol) containing 5 mg/mL Mycobacterium tuberculosis H37Ra. Concurrent with immunization, 1 μg PTX was injected intraperitoneally. Funduscopic examinations were performed on days 7, 14, 21, and 28 after immunization. Tropicamide and phenylephrine saline (0.5%) were applied to the eyes of mice, and the fundi of the eyes were examined. Three ophthalmologists performed the clinical assessments in a masked fashion. The presence of vascular dilatation, vascular white focal lesions, vascular white linear lesions, retinal hemorrhage, and retinal detachment were determined. According to the severity of these findings, the EAU clinical scores were graded on a scale of 0 to 4, as described by Thurau et al. 23 Eyes were then collected, and ocular inflammation was assessed histologically. Eyes were fixed in Bouin’s solution and embedded in paraffin. Six-micrometer-thick sections were prepared and stained with hematoxylin and eosin. The severity of EAU was assessed in each eye and scored on a scale of 0 to 4 in half-point increments, according to a semiquantitative system described previously. 3 Briefly, the minimal criterion for scoring an eye as positive by histopathology was inflammatory cell infiltration of the ciliary body, choroids, or retina. Progressively higher grades were assigned for the presence of discrete lesions in the tissue, such as vasculitis, granuloma formation, retinal folding and/or detachment, and photoreceptor damage. The grading system takes into account lesion type, size, and number. 
Immunohistochemical Studies
Twenty-micrometer-thick cryostat tissue sections were prepared from the eyes of wild-type or CCR5-deficient mice on day 21 after immunization. The sections were fixed in acetone for 30 minutes at room temperature (RT) and incubated in 2% bovine serum albumin (BSA) diluted in PBS (PBS-BSA) for 15 minutes, to block nonspecific staining before addition of primary and secondary antibodies. FITC-conjugated anti-CD3 Ab (clone 145-2C11; eBioscience, San Diego, CA), anti-Ly-6G (Gr-1) Ab (clone RB6-8C5; BD-PharMingen, San Diego, CA), or the respective isotype-matched control antibodies were applied to the sections, and incubated for 12 hours. The sections were then washed and incubated with biotin-conjugated goat anti-rat IgG and streptavidin Alexa 594 (red; Molecular Probes, Eugene, OR). No significant staining was observed with isotope-matched control IgG. All staining procedures were performed at RT, and each step was followed by three thorough washings in PBS for 5 minutes each. Finally, the sections were covered with mounting medium (Vector, Burlingame, CA) and analyzed with a confocal microscope equipped with an image-analysis system. At least three different sections were examined per double-staining experiment. Five different fields of the posterior segments were analyzed in each specimen. 
Lymphocyte Proliferation
Spleens and regional lymph nodes near the eye (cervical and submandibular lymph nodes) were collected on day 21 after immunization and pooled within groups. Each group consisted of five mice. The suspended cells were cultured in triplicate at 5 × 105 cells per 0.2 mL of culture medium in the presence of various concentrations of hIRBP-p. The cultures were incubated for 72 hours at 37°C in 5% CO2 in air, pulsed with [3H]thymidine (1.0 μCi/10 μL/well) during the last 18 hours of incubation, and harvested onto glass filters with an automated cell harvester (Tomtec, Orange, CT). Radioactivity was assessed by liquid scintillation spectrometry, and the amount expressed as counts per million (cpm). 
Cytokine and Chemokine Production Assay
Spleen cells and lymph node cells, prepared as described earlier, were cultured in 96-well, round-bottomed plates at a concentration of 5 × 105 cells/well in 0.2 mL of culture medium, either alone or with hIRBP-p. Supernatants were collected after 72 hours for detection of IL-6, IL-10, TNFα, IFN-γ, and CCL2 using cytometric bead array immunoassay as previously described. 24 Briefly, particles (polystyrene beads, 7.5 m; Bangs Laboratories, Carmel, IN) were dyed in five different fluorescence intensities. The proprietary dye has an emission wavelength of approximately 650 nm (FL-3). Each particle is coupled by covalent linkage based on thiol-maleimide chemistry to an antibody (BD-PharMingen) against IL-6, IL-10, TNFα, IFN-γ, or CCL2, and represents a discrete population unique in FL-3 intensity. The Ab particles serve as a capture for a given cytokine in the immunoassay panel and can be detected simultaneously in a mixture. The captured cytokines are detected by a direct immunoassay involving five different antibodies coupled to phycoerythrin (PE), which emits at approximately 585 nm (FL-2). Two-color flow cytometric analysis was performed (FACSCalibur flow cytometer; BD Biosciences, Franklin Lakes, NJ). 
Intraocular levels of these cytokines were also measured by cytometric bead array immunoassay, as previously reported. 25 Briefly, samples were prepared from six eyes of three wild-type or three CCR5-deficient mice on days 14 and 28 after immunization. A rounded surgical blade was used to incise each whole eye at the equator, and intraocular contents were scraped into a 1.5-mL tube (Eppendorf, Fremont, CA) containing 100 μL PBS on ice. Each sample of intraocular content was homogenized in an ultrasonic disrupter (UD201; Tomy, Tokyo, Japan) and centrifuged at 15,000 rpm for 30 minutes at 4°C. Supernatants were collected, and the levels of IL-6, IL-10, TNFα, IFN-γ, and CCL2 in the supernatant of each sample were immediately assayed. 
Statistical Analyses and Reproducibility
Experiments were repeated at least twice and usually three times. Response patterns were highly reproducible. Statistical analyses for parametric data (proliferation, cytokine, and chemokine production) were performed by independent t-test. Nonparametric data (EAU scores) were analyzed by Mann-Whitney test. P < 0.05 was considered significant. 
Results
EAU in CCR5-Deficient Mice
To investigate whether CCR5 is involved in the development of EAU, CCR5-deficient mice were immunized with hIRBP-p, as described in the Materials and Methods section. Clinical and histopathological observation of EAU was performed on day 21 after immunization. The results of 2 separate experiments are summarized in Figures 1A and 1B . Contrary to our expectation, all except one CCR5-deficient mouse developed EAU clinically, as in wild-type mice (Fig. 1A) . The average clinical score was 2.5 ± 0.4 in CCR5-deficient mice, which was slightly higher than the score of 2.1 ± 0.4 in wild-type mice, although there was no significant difference. In addition, histologic examination confirmed the development of EAU in CCR5-deficient mice induced by immunization with hIRBP-p (Fig. 1B) . The disease was histologically observed in 80% of CCR5-deficient mice, which was higher than the 60% in wild-type, although the average EAU histologic score in CCR5-deficient mice (1.8 ± 1.1) was not significantly higher than that of wild-type mice (1.5 ± 1.4). We also examined the time course of the clinical severity of EAU from 7 to 28 days after immunization with hIRBP-p. As shown in Figure 1C , the average clinical scores in CCR5-deficient mice were slightly higher than the scores in wild-type mice at all tested time points, although there were no significant differences. 
Figure 2shows the representative micrograph of sections of eyes with EAU induced in wild-type or CCR5-deficient mice. Massive infiltration of inflammatory cells in multiple regions of the vitreous cavity and retina, retinal vasculitis, and retinal folds were observed, which appeared similar in both wild-type and CCR5-deficient mice (Figs. 2A 2B) . However, at higher magnification, it became evident that the inflammatory cells infiltrating the eyes of CCR5-deficient mice contained an excessive proportion of granulocytes to mononuclear cells—apparently more than that in wild-type mice (Fig. 2C 2D)
Cells Infiltrating the Eyes with EAU in CCR5-Deficient Mice
Using indirect immunofluorescence, we confirmed the histopathologic observation that T-cells infiltrating the eye decreased, whereas granulocytes increased in EAU in CCR5-deficient mice compared with wild-type mice. Figure 3presents the representative results of the experiments. 
The cells infiltrating the vitreous clearly contained a smaller proportion of CD3+ T cells and a greater proportion of Gr-1+ granulocytes in CCR5-deficient mice with EAU compared with wild-type mice with EAU. 
Cellular Responses of CCR5-Deficient Mice
Because T-cell infiltration of the eye with EAU was reduced in CCR5-deficient mice compared with wild-type mice and was compensated for by granulocyte infiltration, it is possible that, in CCR5-deficient mice, the IRBP-specific T cells failed to be activated by immunization with hIRBP-p, and the T cells are activated but do not migrate to the eye. Antigen-presenting cells such as dendritic cells capture antigen at the immunized site and migrate to the draining lymph nodes and the spleen where the antigen-specific T cells are activated and migrate through lymphatics and lymph nodes to tissues containing the antigen. Therefore, we obtained splenocytes and cells from the regional lymph nodes near the eye (the cervical and submandibular lymph nodes) of CCR5-deficient mice on day 21 after immunization with hIRBP-p and examined their proliferation responses to hIRBP-p. Representative results are displayed in Figure 4 . Both splenocytes and lymph node cells of CCR5-deficient mice proliferated by stimulation with hIRBP-p. Furthermore, although the proliferation responses of lymph node cells to hIRBP-p were similar in wild-type and CCR5-deficient mice, splenocytes of CCR5-deficient mice exhibited markedly more intensive proliferation responses to hIRBP-p than did splenocytes of wild-type mice. These data indicate that circulating hIRBP-p-specific T cells can be sufficiently activated by the specific antigen through antigen-presenting cells in the spleen, even in the absence of CCR5. 
Ag-Specific Cytokine Production by Splenocytes and Lymph Node Cells of CCR5-Deficient Mice
Splenocytes and cells from the regional lymph nodes near the eye (cervical and submandibular lymph nodes) were collected on day 21 after immunization with hIRBP-p, pooled within each group, and stimulated in culture with hIRBP-p. Supernatants were assayed for IFN-γ, IL-10, IL-6, and CCL2 by flow cytometry, as described in the Materials and Methods section. Representative results are displayed in Figure 5 . Compatible with the notion that EAU is mediated by Th1 cells, splenocytes from CCR5-deficient mice produced IFN-γ but not IL-10, and the results were comparable to those in wild-type mice. However, the lymph node cells from CCR5-deficient mice failed to produce IFN-γ or detectable levels of IL-10, when stimulated with hIRBP-p. These data suggest that although hIRBP-p-specific T cells in CCR5-deficient mice were activated and differentiated into Th1 cells in the spleen by immunization with hIRBP-p, in the same manner as in wild-type mice, their migration to the regional lymph nodes near the eye was impaired. In contrast, it is noteworthy that splenocytes of CCR5-deficient mice produced markedly more IL-6 and CCL2, which are chemoattractant factors for granulocytes, than did the splenocytes of wild-type mice. 
Cytokine Production in the Eye of CCR5-Deficient Mice
We observed augmented IL-6 and CCL2 production by splenocytes in CCR5-deficient mice with induced EAU and secretion of these cytokines in the intraocular environment is critical for recruitment of granulocytes into the eye. In addition, if recruitment of Th1 cells into the eye were impaired in CCR5-deficient mice, the level of IFN-γ in the eyes with EAU would be lower in CCR5-deficient mice than in wild-type mice. Therefore, we collected eyes from wild-type and CCR5-deficient mice on days 14 and 28 after immunization with hIRBP-p and measured the intraocular levels of IFN-γ, TNFα, IL-10, IL-6, and CCL2. Although intraocular TNFα and IL-10 were undetectable, IFN-γ, IL-6, and MCP-1 were measured as shown in Figure 6 . Intraocular IFN-γ was generally low. The level was apparently higher in wild-type mice than in CCD5-deficient mice as expected, but there was no statistically significant difference. Intraocular IL-6 and CCL2 were higher on day 14 than on day 28 in both wild-type and CCR5-deficient mice, and the intraocular IL-6 level was significantly higher in CCR5-deficient mice than in wild-type mice on day 14. 
Discussion
The findings in the present study indicate that on immunization with hIRBP-p, CCR5-deficient mice develop EAU with a degree of severity comparable to that in wild-type mice. However, T-cell infiltration in the eye with EAU is apparently reduced in CCR5-deficient mice and is compensated for by augmented granulocyte infiltration, presumably mediated by increased production of IL-6 in the eye and also increased IL-6 and CCL2 production by the splenic T cells. 
Our previous findings of a high expression of CCR5 in the posterior segment of the eyes with EAU induced by IRBP immunization in addition to the expression of CCR2 and CXCR3 22 led us to hypothesize that CCR5 plays a role in the recruitment of effector T cells into the eye in EAU, which is essential for the development of EAU induced by hIRBP-p immunization. However, our present data do not support the hypothesis. It is possible that the discrepancy may have been caused by the relationship between chemokines and their receptors, which are not mutually exclusive. It is well known that the chemokine system plays redundant roles, and there is considerable overlap in ligand-receptor specificity and responses. 26 One particular chemokine can bind several receptors and vice versa. 27 CCR5 can bind CCL2, -3, -4, and -5, but none is selective. 28 In addition to binding CCR5, these chemokines also bind other receptors—for example, CCL3 also binds CCR1 and -4; CCL4 binds CCR1 and -8; CCL5 binds CCR1, -3, and -4; and CCL2 binds -2 and -11. 28 29 Because of the high degree of redundancy, it is possible that CCR5 deficiency may be functionally replaced by other chemokines and their receptors. In addition, our result is consistent with the reports of other autoimmune disease models. CCR5-deficient mice are fully susceptible to myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), P0 peptide–induced experimental autoimmune neuritis (EAN), and experimental autoimmune gastritis (EAG) induced by day 3 neonatal thymectomy, 30 31 32 although other reports indicate that CCR5 antagonist inhibits collagen-induced arthritis by modulating T cell migration, 33 and that CCR5-deficient mice are protected from dextran sodium sulfate-mediated colitis. 34  
Although EAU developed in CCR5-deficient mice, recruitment of effector T cells into the eye was apparently impaired, as shown in Figure 3 . This impaired recruitment is also suggested by the finding that T cells from the spleen of CCR5-deficient mice immunized with hIRBP-p produced IFN-γ when stimulated with the immunized antigen, but cells from the regional lymph nodes near the eye failed to produce IFN-γ (Fig. 5) . The intraocular IFN-γ level in CCR5-deficient mice was not significantly lower than that in wild-type mice, probably because intraocular IFN-γ was low. Some of samples were below the detection limit, and the individual difference was large, as shown by the high standard deviation. 
The proliferation response of splenic T cells to hIRBP-p was markedly more intensive in CCR5-deficient mice than in wild-type mice. This phenomenon may be in part because of the presence of some hIRBP-p-specific T cells that were activated and expanded in the spleen but failed to migrate. Furthermore, T cells from the regional lymph nodes in CCR5-deficient mice exhibited proliferation responses to hIRBP-p comparable to those of wild-type mice, but could produce either IFN-γ or IL-10, suggesting that activated T cells which cannot be classified into Th1 or Th2 cells may exist in CCR5-deficient mice on immunization with hIRBP-p. 
The present study provides evidence that granulocyte infiltration into the inflamed site is promoted in CCR5-deficient mice to compensate for the impaired migration of effector T cells. IL-6, particularly, activates granulocytes and macrophages in the context of acute phase response, and CCL2 exhibits chemoattractant properties for granulocytes in addition to macrophages, memory T cells, and natural killer (NK) cells. 35 Therefore, augmented IL-6 production in the eye and enhanced IL-6 and CCL2 production by splenic T cells may reflect the characteristics of EAU in CCR5-deficient mice. Although we cannot find an appropriate answer for the mechanism involved in the promoted IL-6 and CCL2 production, CCL2 that binds only CCR2 would be one of the best chemokines capable of compensating for the deficiency of CCR5, since it is also a chemoattractant factor in effector Th1 cells. 36 In addition, CCL2 production by splenic T cells was measured on day 21 after immunization, whereas that in the eye was measured on days 14 and 28. It is possible that the cytokine levels in each site are altered throughout the course of EAU, which may be the reason that augmented CCL2 production in the eye of CCR5-deficient mice was not observed in this study. 
In conclusion, CCR5 deficiency does not prevent the development of EAU induced by IRBP-peptide immunization. Although the effector T cell migration is partially impaired, the disadvantage is compensated for by augmented granulocyte infiltration. 
 
Figure 1.
 
Clinical and histopathologic evaluation of EAU in CCR5-deficient mice. (A) Comparison of the funduscopic EAU severity between wild-type and CCR5-deficient mice on day 21 after immunization with hIRBP-p (200 μg per mouse). Vascular dilatation, vascular white focal lesions, vascular white linear lesions, retinal hemorrhage, and retinal detachment were examined. According to the severity of these findings, the EAU clinical scores were assigned on a scale of 0 to 4 and are indicated on the ordinate. Each data point represents one mouse (average of both eyes). The data are compiled from two separate experiments. The horizontal lines denote the average EAU score of the group. (B) Comparison of the histologic EAU severity between wild-type and CCR5-deficient mice. Eyes of wild-type and CCR5-deficient mice immunized with hIRBP-p were harvested after clinical evaluation on day 21. The EAU histologic scores were graded on a scale of 0 to 4 in half-point increments. Each point represents one mouse (average of both eyes). The horizontal lines denote the average EAU score of the group. (C) Time course of the clinical severity of EAU in wild-type (•) and CCR5-deficient mice (▪). Funduscopic examination of EAU was performed on days 7, 14, 21, and 28 after immunized with hIRBP-p. Each group consisted of five mice.
Figure 1.
 
Clinical and histopathologic evaluation of EAU in CCR5-deficient mice. (A) Comparison of the funduscopic EAU severity between wild-type and CCR5-deficient mice on day 21 after immunization with hIRBP-p (200 μg per mouse). Vascular dilatation, vascular white focal lesions, vascular white linear lesions, retinal hemorrhage, and retinal detachment were examined. According to the severity of these findings, the EAU clinical scores were assigned on a scale of 0 to 4 and are indicated on the ordinate. Each data point represents one mouse (average of both eyes). The data are compiled from two separate experiments. The horizontal lines denote the average EAU score of the group. (B) Comparison of the histologic EAU severity between wild-type and CCR5-deficient mice. Eyes of wild-type and CCR5-deficient mice immunized with hIRBP-p were harvested after clinical evaluation on day 21. The EAU histologic scores were graded on a scale of 0 to 4 in half-point increments. Each point represents one mouse (average of both eyes). The horizontal lines denote the average EAU score of the group. (C) Time course of the clinical severity of EAU in wild-type (•) and CCR5-deficient mice (▪). Funduscopic examination of EAU was performed on days 7, 14, 21, and 28 after immunized with hIRBP-p. Each group consisted of five mice.
Figure 2.
 
Histopathology of EAU in wild-type and CCR5-deficient mice. Retina of an hIRBP-p-immunized wild-type (A) and a CCR5-deficient (B) mouse. Hematoxylin and eosin staining. Note the massive cell infiltration in the vitreous and retina, retinal vasculitis, and retinal folds. Infiltrating cells in the vitreous of a wild-type (C) and a CCR5-deficient (D) mouse. Note that the infiltrating cells in the vitreous of CCR5-deficient mice contain an excessive proportion of granulocytes compared with that of wild-type mice. Magnification: (A, B) ×200; (C, D) ×400.
Figure 2.
 
Histopathology of EAU in wild-type and CCR5-deficient mice. Retina of an hIRBP-p-immunized wild-type (A) and a CCR5-deficient (B) mouse. Hematoxylin and eosin staining. Note the massive cell infiltration in the vitreous and retina, retinal vasculitis, and retinal folds. Infiltrating cells in the vitreous of a wild-type (C) and a CCR5-deficient (D) mouse. Note that the infiltrating cells in the vitreous of CCR5-deficient mice contain an excessive proportion of granulocytes compared with that of wild-type mice. Magnification: (A, B) ×200; (C, D) ×400.
Figure 3.
 
Cells infiltrating eyes with EAU in wild-type and CCR5-deficient mice. To evaluate T cells and granulocytes infiltrating the eye with EAU in wild-type and CCR5-deficient mice, eye sections prepared on day 21 after immunization were stained with anti-CD3 and/or anti-Gr-1 Ab, and the posterior segments are shown in confocal micrographs. CD3+ T cells (green) infiltrating the eye of a wild-type (A) and a CCR5-deficient (B) mouse. Gr-1+ granulocytes (red) infiltrating the eye of a wild-type (C) and a CCR5-deficient (D) mouse. CD3 (green) and Gr-1 (red) double-stained sections of a wild-type (E) and a CCR5-deficient (F) mouse. Note a smaller proportion of CD3+ T cells but a larger proportion of Gr-1+ granulocytes in the CCR5-deficient mouse compared with the number of those cells in the wild-type mouse.
Figure 3.
 
Cells infiltrating eyes with EAU in wild-type and CCR5-deficient mice. To evaluate T cells and granulocytes infiltrating the eye with EAU in wild-type and CCR5-deficient mice, eye sections prepared on day 21 after immunization were stained with anti-CD3 and/or anti-Gr-1 Ab, and the posterior segments are shown in confocal micrographs. CD3+ T cells (green) infiltrating the eye of a wild-type (A) and a CCR5-deficient (B) mouse. Gr-1+ granulocytes (red) infiltrating the eye of a wild-type (C) and a CCR5-deficient (D) mouse. CD3 (green) and Gr-1 (red) double-stained sections of a wild-type (E) and a CCR5-deficient (F) mouse. Note a smaller proportion of CD3+ T cells but a larger proportion of Gr-1+ granulocytes in the CCR5-deficient mouse compared with the number of those cells in the wild-type mouse.
Figure 4.
 
IRBP-specific proliferation responses of splenocytes and lymph node cells from wild-type and CCR5-deficient mice. Splenocytes and cells from the cervical and submandibular lymph node were collected on day 21 after immunization and were pooled within each group. Triplicate cultures were stimulated with the indicated concentrations of hIRBP-p. The cultures were incubated for 54 hours and pulsed with [3H]thymidine for the last 18 hours. Proliferation responses of splenocytes (A) and lymph node cells (B) from wild-type (•) and CCR5-deficient (▪) mice are shown. *P < 0.05.
Figure 4.
 
IRBP-specific proliferation responses of splenocytes and lymph node cells from wild-type and CCR5-deficient mice. Splenocytes and cells from the cervical and submandibular lymph node were collected on day 21 after immunization and were pooled within each group. Triplicate cultures were stimulated with the indicated concentrations of hIRBP-p. The cultures were incubated for 54 hours and pulsed with [3H]thymidine for the last 18 hours. Proliferation responses of splenocytes (A) and lymph node cells (B) from wild-type (•) and CCR5-deficient (▪) mice are shown. *P < 0.05.
Figure 5.
 
IRBP-specific lymphokine production by splenocytes and lymph node cells from wild-type and CCR5-deficient mice. Splenocytes and cells from the cervical and submandibular lymph node were collected on day 21 after immunization and pooled within each group. The cultures were stimulated with the indicated concentrations of hIRBP-p. Supernatants were collected at 72 hours from cultures of wild-type (•) and CCR5-deficient (▪) mice, and IFN-γ (A, B), IL-10 (C, D), IL-6 (E, F), and MCP-1 (G, H) levels were measured by cytometric bead array immunoassay. *P < 0.05.
Figure 5.
 
IRBP-specific lymphokine production by splenocytes and lymph node cells from wild-type and CCR5-deficient mice. Splenocytes and cells from the cervical and submandibular lymph node were collected on day 21 after immunization and pooled within each group. The cultures were stimulated with the indicated concentrations of hIRBP-p. Supernatants were collected at 72 hours from cultures of wild-type (•) and CCR5-deficient (▪) mice, and IFN-γ (A, B), IL-10 (C, D), IL-6 (E, F), and MCP-1 (G, H) levels were measured by cytometric bead array immunoassay. *P < 0.05.
Figure 6.
 
Intraocular cytokines in CCR5-deficient mice by immunization with hIRBP-p. Intraocular samples were prepared from six eyes of three wild-type and three CCR5-deficient mice on days 14 and 28 after immunization. Intraocular cytokines and chemokines in each sample were measured by cytometric bead array immunoassay. Mean ± SD levels of IFN-γ (A), IL-6 (B) and MCP-1 (C) in six eyes of wild-type (□) or CCR5-deficient (▪) mice are shown. *P < 0.05.
Figure 6.
 
Intraocular cytokines in CCR5-deficient mice by immunization with hIRBP-p. Intraocular samples were prepared from six eyes of three wild-type and three CCR5-deficient mice on days 14 and 28 after immunization. Intraocular cytokines and chemokines in each sample were measured by cytometric bead array immunoassay. Mean ± SD levels of IFN-γ (A), IL-6 (B) and MCP-1 (C) in six eyes of wild-type (□) or CCR5-deficient (▪) mice are shown. *P < 0.05.
CaspiRR, ChanCC, WiggertB, ChaderGJ. The mouse as a model of experimental autoimmune uveoretinitis (EAU). Curr Eye Res. 1990;9(suppl)169–174.
CaspiRR, RobergeFG, McAllisterCG, et al. T cell lines mediating experimental autoimmune uveoretinitis (EAU) in the rat. J Immunol. 1986;136:928–933. [PubMed]
CaspiRR, RobergeFG, ChanCC, et al. A new model of autoimmune disease: experimental autoimmune uveoretinitis induced in mice with two different retinal antigens. J Immunol. 1988;140:1490–1495. [PubMed]
ForresterJV, LiversidgeJ, DuaHS, DickA, HarperF, McMenaminPG. Experimental autoimmune uveoretinitis: a model system for immunointervention: a review. Curr Eye Res. 1992;11(suppl)33–40. [CrossRef] [PubMed]
CaspiRR, SunB, AgarwalRK, et al. T cell mechanisms in experimental autoimmune uveoretinitis: susceptibility is a function of the cytokine response profile. Eye. 1997;11:209–212. [CrossRef] [PubMed]
XuH, RizzoLV, SilverPB, CaspiRR. Uveitogenicity is associated with a Th1-like lymphokine profile: cytokine-dependent modulation of early and committed effector T cells in experimental autoimmune uveitis. Cell Immunol. 1997;178:69–78. [CrossRef] [PubMed]
PrendergastRA, IliffCE, CoskuncanNM, et al. T cell traffic and the inflammatory response in experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci. 1998;39:754–762. [PubMed]
LusterAD. Chemokines–chemotactic cytokines that mediate inflammation. N Engl J Med. 1998;338:436–445. [CrossRef] [PubMed]
CraneIJ, McKillop-SmithS, WallaceCA, LamontGR, ForresterJV. Expression of the chemokines MIP-1alpha, CCL2, and RANTES in experimental autoimmune uveitis. Invest Ophthalmol Vis Sci. 2001;42:1547–1552. [PubMed]
VermaMJ, LloydA, RagerH, StrieterR, KunkelS, TaubD, WakefieldD. Chemokines in acute anterior uveitis. Curr Eye Res. 1997;16:1202–1208. [CrossRef] [PubMed]
O’GarraA, McEvoyLM, ZlotnikA. T-cell subsets: chemokine receptors guide the way. Curr Biol. 1998;8:R646–R649. [CrossRef] [PubMed]
ZlotnikA, YoshieO. Chemokines: a new classification system and their role in immunity. Immunity. 2000;12:121–127. [CrossRef] [PubMed]
SallustoF, LenigD, MackayCR, LanzavecchiaA. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J Exp Med. 1998;187:875–883. [CrossRef] [PubMed]
BonecchiR, BianchiG, BordignonPP, et al. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J Exp Med. 1998;187:129–134. [CrossRef] [PubMed]
ImaiT, NagiraM, TakagiS, et al. Selective recruitment of CCR4-bearing Th2 cells toward antigen-presenting cells by the CC chemokines thymus and activation-regulated chemokine and macrophage-derived chemokine. Int Immunol. 1999;11:81–88. [CrossRef] [PubMed]
LoetscherP, UguccioniM, BordoliL, et al. CCR5 is characteristic of Th1 lymphocytes. Nature. 1998;391:344–345. [PubMed]
BleulCC, WuL, HoxieJA, et al. The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc Natl Acad Sci USA. 1997;94:1925–1930. [CrossRef] [PubMed]
SchallTJ, BaconK, ToyKJ, GoeddelDV. Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. Nature. 1990;347:669–671. [CrossRef] [PubMed]
TaubDD, ConlonK, LloydAR, OppenheimJJ, KelvinDJ. Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-1 alpha and MIP-1 beta. Science. 1993;260:355–358. [CrossRef] [PubMed]
SimpsonJ, RezaieP, NewcombeJ, CuznerML, MaleD, WoodroofeMN. Expression of the beta-chemokine receptors CCR2, CCR3 and CCR5 in multiple sclerosis central nervous system tissue. J Neuroimmunol. 2000;108:192–200. [CrossRef] [PubMed]
QinS, RottmanJB, MyersP, et al. The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J Clin Invest. 1998;101:746–754. [CrossRef] [PubMed]
KeinoH, TakeuchiM, KezukaT, et al. Chemokine and chemokine receptor expression during experimental autoimmune uveoretinitis in mice. Graefes Arch Clin Exp Ophthalmol. 2003;241:111–115. [CrossRef] [PubMed]
ThurauSR, ChanCC, NussenblattRB, CaspiRR. Oral tolerance in a murine model of relapsing experimental autoimmune uveoretinitis (EAU): induction of protective tolerance in primed animals. Clin Exp Immunol. 1997;109:370–376. [CrossRef] [PubMed]
CookEB, StahlJL, LoweL, et al. Simultaneous measurement of six cytokines in a single sample of human tears using microparticle-based flow cytometry: allergics vs. non-allergics. J Immunol Methods. 2001;254:109–118. [CrossRef] [PubMed]
OkadaAA, KeinoH, SuzukiJ, SakaiJ, UsuiM, MizuguchiJ. Kinetics of intraocular cytokines in the suppression of experimental autoimmune uveoretinitis by type I IFN. Int Immunol. 1998;10:1917–1922. [CrossRef] [PubMed]
ThelenM. Dancing to the tune of chemokines. Nature Immunology. 2001;2:129–134. [CrossRef] [PubMed]
BajettoA, BonaviaR, BarberoS, FlorioT, SchettiniG. Chemokines and their receptors in the central nervous system. Front Neuroendocrinol. 2001;22:147–184. [CrossRef] [PubMed]
MurphyPM, BaggioliniM, CharoIF, et al. International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev. 2000;52:145–176. [PubMed]
RossiD, ZlotnikA. The biology of chemokines and their receptors. Ann Rev Immunol. 2000;18:217–242. [CrossRef]
TranEH, KuzielWA, OwensT. Induction of experimental autoimmune encephalomyelitis in C57BL/6 mice deficient in either the chemokine macrophage inflammatory protein-1alpha or its CCR5 receptor. Eur J Immunol. 2000;30:1410–1415. [CrossRef] [PubMed]
DuanRS, ChenZ, BaoL, et al. CCR5 deficiency does not prevent P0 peptide 180–199 immunized mice from experimental autoimmune neuritis. Neurobiol Dis. 2004;16:630–637. [CrossRef] [PubMed]
FieldJ, MarshallAC, HertzogJP, et al. Chemokine receptor CCR5 is not required for development of experimental autoimmune gastritis. Clin Immunol. 2003;109:238–247. [CrossRef] [PubMed]
YangYF, MukaiT, GaoP, et al. A non-peptide CCR5 antagonist inhibits collagen-induced arthritis by modulating T cell migration without affecting anti-collagen T cell responses. Eur J Immunol. 2002;32:2124–2132. [CrossRef] [PubMed]
AndresPG, BeckPL, MizoguchiE, et al. Mice with a selective deletion of the CC chemokine receptors 5 or 2 are protected from dextran sodium sulfate-mediated colitis: lack of CC chemokine receptor 5 expression results in a NK1.1+ lymphocyte-associated Th2-type immune response in the intestine. J Immunol. 2000;164:6303–6312. [CrossRef] [PubMed]
TuaillonN, Shen deF, BergerRB, LuB, RollinsBJ, ChanCC. CCL2 expression in endotoxin-induced uveitis. Invest Ophthalmol Vis Sci. 2002;43:1493–1498. [PubMed]
BoringL, GoslingJ, ChensueSW, et al. Impaired monocyte migration and reduced type 1 (Th1) cytokine responses in C-C chemokine receptor 2 knockout mice. J Clin Invest. 1997;100:2552–2561. [CrossRef] [PubMed]
Figure 1.
 
Clinical and histopathologic evaluation of EAU in CCR5-deficient mice. (A) Comparison of the funduscopic EAU severity between wild-type and CCR5-deficient mice on day 21 after immunization with hIRBP-p (200 μg per mouse). Vascular dilatation, vascular white focal lesions, vascular white linear lesions, retinal hemorrhage, and retinal detachment were examined. According to the severity of these findings, the EAU clinical scores were assigned on a scale of 0 to 4 and are indicated on the ordinate. Each data point represents one mouse (average of both eyes). The data are compiled from two separate experiments. The horizontal lines denote the average EAU score of the group. (B) Comparison of the histologic EAU severity between wild-type and CCR5-deficient mice. Eyes of wild-type and CCR5-deficient mice immunized with hIRBP-p were harvested after clinical evaluation on day 21. The EAU histologic scores were graded on a scale of 0 to 4 in half-point increments. Each point represents one mouse (average of both eyes). The horizontal lines denote the average EAU score of the group. (C) Time course of the clinical severity of EAU in wild-type (•) and CCR5-deficient mice (▪). Funduscopic examination of EAU was performed on days 7, 14, 21, and 28 after immunized with hIRBP-p. Each group consisted of five mice.
Figure 1.
 
Clinical and histopathologic evaluation of EAU in CCR5-deficient mice. (A) Comparison of the funduscopic EAU severity between wild-type and CCR5-deficient mice on day 21 after immunization with hIRBP-p (200 μg per mouse). Vascular dilatation, vascular white focal lesions, vascular white linear lesions, retinal hemorrhage, and retinal detachment were examined. According to the severity of these findings, the EAU clinical scores were assigned on a scale of 0 to 4 and are indicated on the ordinate. Each data point represents one mouse (average of both eyes). The data are compiled from two separate experiments. The horizontal lines denote the average EAU score of the group. (B) Comparison of the histologic EAU severity between wild-type and CCR5-deficient mice. Eyes of wild-type and CCR5-deficient mice immunized with hIRBP-p were harvested after clinical evaluation on day 21. The EAU histologic scores were graded on a scale of 0 to 4 in half-point increments. Each point represents one mouse (average of both eyes). The horizontal lines denote the average EAU score of the group. (C) Time course of the clinical severity of EAU in wild-type (•) and CCR5-deficient mice (▪). Funduscopic examination of EAU was performed on days 7, 14, 21, and 28 after immunized with hIRBP-p. Each group consisted of five mice.
Figure 2.
 
Histopathology of EAU in wild-type and CCR5-deficient mice. Retina of an hIRBP-p-immunized wild-type (A) and a CCR5-deficient (B) mouse. Hematoxylin and eosin staining. Note the massive cell infiltration in the vitreous and retina, retinal vasculitis, and retinal folds. Infiltrating cells in the vitreous of a wild-type (C) and a CCR5-deficient (D) mouse. Note that the infiltrating cells in the vitreous of CCR5-deficient mice contain an excessive proportion of granulocytes compared with that of wild-type mice. Magnification: (A, B) ×200; (C, D) ×400.
Figure 2.
 
Histopathology of EAU in wild-type and CCR5-deficient mice. Retina of an hIRBP-p-immunized wild-type (A) and a CCR5-deficient (B) mouse. Hematoxylin and eosin staining. Note the massive cell infiltration in the vitreous and retina, retinal vasculitis, and retinal folds. Infiltrating cells in the vitreous of a wild-type (C) and a CCR5-deficient (D) mouse. Note that the infiltrating cells in the vitreous of CCR5-deficient mice contain an excessive proportion of granulocytes compared with that of wild-type mice. Magnification: (A, B) ×200; (C, D) ×400.
Figure 3.
 
Cells infiltrating eyes with EAU in wild-type and CCR5-deficient mice. To evaluate T cells and granulocytes infiltrating the eye with EAU in wild-type and CCR5-deficient mice, eye sections prepared on day 21 after immunization were stained with anti-CD3 and/or anti-Gr-1 Ab, and the posterior segments are shown in confocal micrographs. CD3+ T cells (green) infiltrating the eye of a wild-type (A) and a CCR5-deficient (B) mouse. Gr-1+ granulocytes (red) infiltrating the eye of a wild-type (C) and a CCR5-deficient (D) mouse. CD3 (green) and Gr-1 (red) double-stained sections of a wild-type (E) and a CCR5-deficient (F) mouse. Note a smaller proportion of CD3+ T cells but a larger proportion of Gr-1+ granulocytes in the CCR5-deficient mouse compared with the number of those cells in the wild-type mouse.
Figure 3.
 
Cells infiltrating eyes with EAU in wild-type and CCR5-deficient mice. To evaluate T cells and granulocytes infiltrating the eye with EAU in wild-type and CCR5-deficient mice, eye sections prepared on day 21 after immunization were stained with anti-CD3 and/or anti-Gr-1 Ab, and the posterior segments are shown in confocal micrographs. CD3+ T cells (green) infiltrating the eye of a wild-type (A) and a CCR5-deficient (B) mouse. Gr-1+ granulocytes (red) infiltrating the eye of a wild-type (C) and a CCR5-deficient (D) mouse. CD3 (green) and Gr-1 (red) double-stained sections of a wild-type (E) and a CCR5-deficient (F) mouse. Note a smaller proportion of CD3+ T cells but a larger proportion of Gr-1+ granulocytes in the CCR5-deficient mouse compared with the number of those cells in the wild-type mouse.
Figure 4.
 
IRBP-specific proliferation responses of splenocytes and lymph node cells from wild-type and CCR5-deficient mice. Splenocytes and cells from the cervical and submandibular lymph node were collected on day 21 after immunization and were pooled within each group. Triplicate cultures were stimulated with the indicated concentrations of hIRBP-p. The cultures were incubated for 54 hours and pulsed with [3H]thymidine for the last 18 hours. Proliferation responses of splenocytes (A) and lymph node cells (B) from wild-type (•) and CCR5-deficient (▪) mice are shown. *P < 0.05.
Figure 4.
 
IRBP-specific proliferation responses of splenocytes and lymph node cells from wild-type and CCR5-deficient mice. Splenocytes and cells from the cervical and submandibular lymph node were collected on day 21 after immunization and were pooled within each group. Triplicate cultures were stimulated with the indicated concentrations of hIRBP-p. The cultures were incubated for 54 hours and pulsed with [3H]thymidine for the last 18 hours. Proliferation responses of splenocytes (A) and lymph node cells (B) from wild-type (•) and CCR5-deficient (▪) mice are shown. *P < 0.05.
Figure 5.
 
IRBP-specific lymphokine production by splenocytes and lymph node cells from wild-type and CCR5-deficient mice. Splenocytes and cells from the cervical and submandibular lymph node were collected on day 21 after immunization and pooled within each group. The cultures were stimulated with the indicated concentrations of hIRBP-p. Supernatants were collected at 72 hours from cultures of wild-type (•) and CCR5-deficient (▪) mice, and IFN-γ (A, B), IL-10 (C, D), IL-6 (E, F), and MCP-1 (G, H) levels were measured by cytometric bead array immunoassay. *P < 0.05.
Figure 5.
 
IRBP-specific lymphokine production by splenocytes and lymph node cells from wild-type and CCR5-deficient mice. Splenocytes and cells from the cervical and submandibular lymph node were collected on day 21 after immunization and pooled within each group. The cultures were stimulated with the indicated concentrations of hIRBP-p. Supernatants were collected at 72 hours from cultures of wild-type (•) and CCR5-deficient (▪) mice, and IFN-γ (A, B), IL-10 (C, D), IL-6 (E, F), and MCP-1 (G, H) levels were measured by cytometric bead array immunoassay. *P < 0.05.
Figure 6.
 
Intraocular cytokines in CCR5-deficient mice by immunization with hIRBP-p. Intraocular samples were prepared from six eyes of three wild-type and three CCR5-deficient mice on days 14 and 28 after immunization. Intraocular cytokines and chemokines in each sample were measured by cytometric bead array immunoassay. Mean ± SD levels of IFN-γ (A), IL-6 (B) and MCP-1 (C) in six eyes of wild-type (□) or CCR5-deficient (▪) mice are shown. *P < 0.05.
Figure 6.
 
Intraocular cytokines in CCR5-deficient mice by immunization with hIRBP-p. Intraocular samples were prepared from six eyes of three wild-type and three CCR5-deficient mice on days 14 and 28 after immunization. Intraocular cytokines and chemokines in each sample were measured by cytometric bead array immunoassay. Mean ± SD levels of IFN-γ (A), IL-6 (B) and MCP-1 (C) in six eyes of wild-type (□) or CCR5-deficient (▪) mice are shown. *P < 0.05.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×