mice) were measured by sandwich ELISA, as previously reported (25). Ab (M5) treatment ..... Calder, C. J., L. B. Nicholson, and A. D. Dick. 2005. A selective role ...
The Journal of Immunology
Osteopontin Aggravates Experimental Autoimmune Uveoretinitis in Mice1 Mizuki Kitamura,*† Kazuya Iwabuchi,* Nobuyoshi Kitaichi,† Shigeyuki Kon,‡ Hirokuni Kitamei,† Kenichi Namba,† Kazuhiko Yoshida,† David T. Denhardt,§ Susan R. Rittling,¶ Shigeaki Ohno,† Toshimitsu Uede,‡ and Kazunori Onoe´2* Human endogenous uveitis is a common sight-threatening intraocular inflammatory disease and has been studied extensively using a murine model of experimental autoimmune uveoretinitis (EAU). It is possibly mediated by Th1 immune responses. In the present study, we investigated the role of osteopontin (OPN), a protein with pleiotropic functions that contributes to the development of Th1 cell-mediated immunity. Accompanying EAU progression, OPN was elevated in wild-type (WT) mice that had been immunized with human interphotoreceptor retinoid-binding protein (hIRBP) peptide 1–20. OPN-deficient (OPNⴚ/ⴚ) mice showed milder EAU progression in clinical and histopathological scores compared with those of WT mice. The T cells from hIRBPimmunized OPNⴚ/ⴚ mice exhibited reduced Ag-specific proliferation and proinflammatory cytokine (TNF-␣ and IFN-␥) production compared with those of WT T cells. When hIRBP-immunized WT mice were administered M5 Ab reacting to SLAYGLR sequence, a cryptic binding site to integrins within OPN, EAU development was significantly ameliorated. T cells from hIRBPimmunized WT mice showed significantly reduced proliferative responses and proinflammatory cytokine production upon stimulation with hIRBP peptide in the presence of M5 Ab in the culture. Our present results demonstrate that OPN may represent a novel therapeutic target to control uveoretinitis. The Journal of Immunology, 2007, 178: 6567– 6572.
E
xperimental autoimmune uveoretinitis (EAU)3 is an animal model of human endogenous uveitis, such as sympathetic ophthalmia, birdshot retinochoroidopathy, Vogt-Koyanagi-Harada’s disease, and Behc¸et’s disease (1). EAU is an organ-specific, T cell-mediated autoimmune disease that can be induced by immunization with retinal Ag, e.g., human interphotoreceptor retinoid-binding protein or by the adoptive transfer of retinal Ag-specific T lymphocytes (2– 4). EAU is a Th1-dominant response. It has been demonstrated that the augmentation of the Th2 response and T regulatory cytokine production or down-regulation of the Th1 responses prevents inflammatory responses and protects against the EAU development (5–7).
*Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; †Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine, Sapporo, Japan; ‡Division of Molecular Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; § Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854; and ¶The Forsyth Institute, The Fenway, Boston, MA 02115 Received for publication October 19, 2006. Accepted for publication February 22, 2007. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by a grant for research on sensory and communicative disorders from the Ministry of Health, Labor, and Welfare, Japan; by Grantsin-Aid for Scientific Research (S) and (C) from Japan Society for the Promotion of Science; and a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. 2 Address correspondence and reprint requests to Dr. Kazunori Onoe´, Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan. E-mail address: kazunori@igm. hokudai.ac.jp 3 Abbreviations used in this paper: EAU, experimental autoimmune uveoretinitis; hIRBP, human interphotoreceptor retinoid-binding protein; OPN, osteopontin; PTX, pertussis toxin; RGD, arginine-glycine-aspartic acid; WT, wild type.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org
Osteopontin (OPN), also known as early T lymphocyte activation 1 (Eta-1), is a secreted phosphoglycoprotein that contains the arginine-glycine-aspartic acid (RGD) integrin-binding sequence. The RGD sequence is also found in many extracellular matrix proteins (8). OPN has been shown to mediate adhesion and migration of a number of different cell types. It has been reported that the integrins ␣v1, ␣v3, ␣v5, ␣51, ␣81, and ␣v6 bind to OPN through RGD sequence and ␣41, ␣91 bind to non-RGD sites of OPN (9). CD44 is known as one of the OPN receptors, and the interaction of OPN with CD44 regulates macrophage migration and activation (10, 11). OPN is produced by a variety of inflammatory cells, including T cells, macrophages, NK cells, and NKT cells (12, 13); induces IL-12 and IFN-␥ production; and inhibits IL-10 expression (14). Thus, OPN is considered to act as a cytokine that contributes to the development of Th1-mediated immunity and diseases. Xu et al. (15) reported that CD44 was up-regulated in the eyes of EAU mice and the disease severity was reduced by administration of antiCD44 mAb. Of interest, OPN-positive cells were observed in the ganglion cell layer of retinas in C57BL/6J mice (16). Recent studies with OPN-deficient (OPN⫺/⫺) mice have demonstrated that OPN deficiency inhibits disease progression in some experimental autoimmune models, such as experimental autoimmune encephalomyelitis (17, 18) and anti-type II collagen Abinduced arthritis (19). Furthermore, an Ab (M5) specific for SLAYGLR sequence, which is newly exposed within OPN by thrombin cleavage, significantly suppressed a murine model of rheumatoid arthritis (20). In the present study, using a murine EAU model, we examined the role of OPN in this organ-specific autoimmune disease. We demonstrate in this study that OPN⫺/⫺ mice develop significantly milder EAU in comparison with that in wild-type (WT) controls. Furthermore, we will show that EAU is ameliorated by a single administration of M5 Ab.
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Materials and Methods Experimental animals Six- to 8-wk-old female C57BL/6 (H-2b) (WT) mice were obtained from Japan SLC. OPN⫺/⫺ mice (21) were backcrossed 11 times to C57BL/6 mice at Institute for Genetic Medicine at Hokkaido University. These animals were maintained in a specific pathogen-free condition in the animal facility of Laboratory of Animal Experiment for Disease Model, Institute for Genetic Medicine at Hokkaido University. All studies were conducted in compliance with the Association for Research in Vision and Ophthalmology.
Reagents Human interphotoreceptor retinoid-binding protein (hIRBP) peptide sequence 1–20 (GPTHLFQPSLVLDMAKVLLD) was purchased from Sigma-Genosys. Purified Bordetella pertussis toxin (PTX) was from Sigma-Aldrich, and CFA and Mycobacterium tuberculosis strain H37Ra were from Difco.
Immunization To analyze the cell proliferative response, hIRBP 1–20 (100 g) was emulsified in CFA (1:1 v/v), and a total of 50 l of the emulsion was injected s.c. (four mice/group). To induce EAU, hIRBP 1–20 (200 g) was emulsified in CFA (1:1 v/v) containing 2.5 mg/ml M. tuberculosis. A total of 200 l of the emulsion was injected s.c. Concurrent with immunization, 0.1 g of PTX in 100 l of PBS was injected i.p. as an additional adjuvant (22).
Evaluation of EAU Clinical assessment by funduscopic examination of the retinal inflammation was conducted every 3 or 4 days from day 7 after immunization (23) (10 mice/group). The severity of EAU was graded 0 – 4, as described previously (24). Briefly, the clinical scoring was based on vessel dilatation, the number of vessel white focal lesions, vessel white linear lesions, hemorrhages, and the extent of retinal detachment. For the histological assessment of EAU, eyes were enucleated on day 21 after immunization (10 mice/group). Mice were deeply anesthetized with ether and then transcardially perfused with 4% paraformaldehyde. Eyes were immediately removed and postfixed in the same fixative at 4°C for 4 h and transferred into 10% phosphate-buffered formaldehyde until processing. Fixed samples were embedded in paraffin, and 5-m sagittal sections were cut near the optic nerve head and stained with H&E. The severity of EAU in each eye was scored on a scale of 0 – 4, as described previously (1). In brief, focal nongranulomatous, monocytic infiltrations in the choroids, ciliary body, and retina were scored as 0.5. Retinal perivascular infiltration and monocytic infiltration in the vitreous were scored as 1. Granuloma formation in the uvea and retina, the presence of occluded retinal vasculitis, along with photoreceptor folds, serous detachment, and loss of photoreceptor were scored as 2. In addition, the formation of Dalen-Fuchs nodules (granuloma at the level of the retinal pigmented epithelium) and the development of subretinal neovascularization were scored as 3 and 4, according to the number and the size of the lesions. The EAU severity was double-blindly scored by two ophthalmologists.
Measurement of plasma OPN level For the measurement of OPN concentration in plasma of EAU mice, mice were deeply anesthetized with ether, and then blood was collected transcardially before immunization and on days 3, 7, 10, 14, 21, and 28 after immunization. All blood samples were collected under EDTA to avoid being fragmented in vitro by thrombin, centrifuged to remove cells and debris, and stored at ⫺80°C until used. Plasma levels of OPN (totally 24 mice) were measured by sandwich ELISA, as previously reported (25).
Ab (M5) treatment A purified IgG fraction of rabbit serum immunized with synthetic peptide (VDVPNGRGDSLAYGLRS), referred to as M5 Ab (20), was used in this study. M5 Ab (400 g/mouse) was i.v. administered concurrent with immunization (n ⫽ 10). The control group was i.v. administered 400 g of normal rabbit IgG (Jackson ImmunoResearch Laboratories) (n ⫽ 10).
Determination of immunological responses We performed three types of T cell proliferation assay. In the first type of experiment, 10 days after immunization, primed lymphocytes obtained from draining lymph nodes were suspended at 5 ⫻ 105 cells per 0.2 ml of serum-free medium (RPMI 1640 medium, 10 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 50 g/ml gentamicin sulfate, supplemented with 0.1% BSA, and ITS ⫹ 1 liquid medium supple-
FIGURE 1. Plasma OPN levels in EAU mice. C57BL/6 mice (n ⫽ 24) were immunized with hIRBP 1–20 (200 g) emulsified in CFA (1:1 v/v) containing 2.5 mg/ml M. tuberculosis. A total of 200 l of the emulsion was injected s.c. Concurrent with immunization, 0.1 g of PTX in 100 l of PBS was injected i.p. as an additional adjuvant. Blood was collected transcardially before immunization and on days 3, 7, 10, 14, 21, and 28 after immunization. All blood samples were collected under EDTA, centrifuged to remove cells and debris, and stored at ⫺80°C until used. Plasma levels of OPN were measured by sandwich ELISA.
ment (2 g/ml insulin from bovine pancreas, 1.1 g/ml iron-free transferrin, 1 ng/ml sodium selenite, 100 g/ml BSA, and 1 g/ml linoleic acid)) (Sigma-Aldrich). Triplicate 0.2-ml cultures in 96-well flat-bottom plates were stimulated with the hIRBP peptide at the indicated concentration. In the second type of experiment, CD4⫹ T cell-enriched fractions were prepared by passing the dispersed cells from the draining lymph nodes of hIRBP-immunized WT or OPN⫺/⫺ mice over mouse T cell CD4 subset column kit (R&D Systems). The collected T cells (4 ⫻ 105/well) were cultured with 30 Gy-irradiated splenocytes as APC (1 ⫻ 105/well) from untreated WT or OPN⫺/⫺ mice in 0.2 ml of the serum-free medium. Triplicate 0.2-ml cultures in 96-well flat-bottom plates were stimulated with the hIRBP peptide at the indicated concentration. In the third type of experiment, to investigate the effect of the M5 Ab in vitro, M5 (50 g/ml) was added in each well. The cultures (first, second, and third) were incubated for 64 h at 37°C in 5% CO2 in air, pulsed with 1 Ci of [3H]thymidine (PerkinElmer) per well during the last 16 h of incubation, and then harvested. [3H]Thymidine incorporation was quantitated with a direct beta counter (Packard Instrument) in the first and third experiments, and with a liquid scintillation counter (Beckman Coulter) in the second experiment. The data were presented as the mean cpm minus the background (medium alone; ⌬cpm), as described previously (26). Cytokines produced in the culture supernatant were measured by an ELISA kit (OptEIA; BD Pharmingen). Briefly, flat-bottom 96-well plates were coated with anti-IFN-␥, anti-TNF-␣, anti-IL-4, or anti-IL-10 capture Ab at 4°C overnight. The plates were blocked with PBS with 10% FBS for 1 h and incubated for another 2 h with samples or standards at room temperature. Then, detection Ab and streptavidin-HRP conjugate were added and incubated for 1 h at room temperature. Finally, the plates were developed using tetramethylbenzidine substrate.
Statistical analysis Data are presented as mean ⫾ SD in clinical and histopathological scoring, and as mean ⫾ SEM in analyses of cell proliferation and cytokine production. Statistical analysis of EAU scoring was performed using the nonparametric Mann-Whitney U test. Analyses of cell proliferation and cytokine production were performed using two-tailed Student’s t test. Values of p ⬍ 0.05 were considered statistically significant.
Results Plasma OPN levels are increased by the induction of EAU To examine whether plasma OPN levels change during the course of EAU progression, C57BL/6 (WT) mice were immunized with hIRBP 1–20 emulsified in CFA containing 2.5 mg/ml M. tuberculosis. Blood samples were collected before immunization and on days 3, 7, 10, 14, 21, and 28 after immunization, and the plasma levels of OPN were measured by ELISA. It will be seen in Fig. 1 that plasma OPN levels increased after immunization with a peak
The Journal of Immunology
FIGURE 2. Clinical score of EAU in OPN⫺/⫺ mice. EAU was induced in WT (F) and OPN⫺/⫺ (E) mice. Funduscopic examination was conducted every 3 or 4 days from day 7 after immunization. The results are presented as mean clinical score for all eyes of each group of mice (10 mice per group) ⫾ SD. Significance was determined using the Mann-Whitney U test (ⴱⴱ, p ⬍ 0.01).
at day 14. The concentration of OPN was significantly higher in EAU mice (from days 3 to 28) than that of before immunization. OPN⫺/⫺ mice show reduced EAU scores We then examined the role of OPN in development of EAU using OPN⫺/⫺ mice. OPN⫺/⫺ and WT mice were immunized with hIRBP 1–20 emulsified in CFA containing 2.5 mg/ml M. tuberculosis. From day 7 after immunization, we conducted clinical assessment every 3 or 4 days. In WT control mice, clinically diagnosable EAU began developing at ⬃day 10 and reached a peak at day 21 after immunization (Fig. 2). In OPN⫺/⫺ mice, the clinical score of EAU followed a similar time course to that in control mice. However, the EAU onset was delayed in OPN⫺/⫺ mice and the clinical severity in the OPN⫺/⫺ mice was significantly milder than that of control mice. Then, histopathological examinations were performed in these two groups of mice. Eyes were removed from OPN⫺/⫺ and WT EAU mice 21 days after hIRBP immunization. Representative histopathology of the eyes from WT control mice and OPN⫺/⫺ mice is shown in Fig. 3, A and B, respectively. In WT eyes, infiltration of inflammatory cells, granuloma formation, and photoreceptor cell damage in retina were seen, whereas slight changes were seen in OPN⫺/⫺ eyes. The histological scores of retinal sections were significantly lower in OPN⫺/⫺ mice (average scores: 0.68 ⫾ 0.41)
FIGURE 3. Ocular histopathology of WT and OPN⫺/⫺ mice. A, EAU in WT mice immunized with hIRBP. Inflammatory cells are present in the retina, vitreous, and choroid with retinal folding and granulomatous lesions. B, EAU in OPN⫺/⫺ mice immunized with hIRBP. Almost normal retinal architecture is seen.
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FIGURE 4. Histopathological score of EAU in OPN⫺/⫺ mice. EAU was induced in WT (F) and OPN⫺/⫺ (E) mice. On day 21, the eyes were enucleated and scored by examining histopathologically. The results are presented as the histopathological score of each eye, and the mean EAU score of each group is indicated by a bar. Significance was determined by the Mann-Whitney U test (p ⬍ 0.05).
than those in the control mice (1.33 ⫾ 0.91) (Fig. 4). These results demonstrate that OPN plays a role in EAU progression. OPN deficiency results in reduced T cell proliferative responses to hIRBP To analyze proliferative and cytokine responses of T cells, draining lymph nodes were collected from WT and OPN⫺/⫺ mice 10 days after immunization. These cells were stimulated with hIRBP peptide Ag at various concentrations for 64 h. The Ag-specific proliferation was seen in a dose-dependent manner of hIRBP added in both culture groups. Of note, the cells from OPN⫺/⫺ mice showed considerably reduced proliferative responses compared with those in WT cells (Fig. 5A).
FIGURE 5. Cell proliferative response and cytokine production of lymph node cells from OPN⫺/⫺ mice. A, [3H]Thymidine incorporation by primed lymphocytes. Lymphocytes were obtained from draining lymph nodes of WT (F) and OPN⫺/⫺ (E) mice 10 days after immunization and incubated with indicated dose of hIRBP peptide for 64 h and [3H]thymidine for the last 16 h. B, IFN-␥ produced in the culture supernatant. C, TNF-␣ produced in the culture supernatant. The results are presented as mean ⫾ SEM. Significance was determined using two-tailed Student’s t test (ⴱⴱ, p ⬍ 0.01). Data are representative of two separate experiments with the same results.
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FIGURE 6. Cell proliferative response of CD4⫹ T lymph node cells from OPN⫺/⫺ mice. Lymphocytes were obtained from draining lymph nodes of WT or OPN⫺/⫺ mice 10 days after immunization. CD4⫹ T cellenriched fractions were prepared by passing the dispersed cells over mouse T cell CD4 subset column kit. The collected cells (4 ⫻ 105/well) were incubated with 30 Gy-irradiated splenocytes (1 ⫻ 105/well) as APC from WT or OPN⫺/⫺ mice with indicated dose of hIRBP peptide for 64 h and [3H]thymidine for the last 16 h. The results are presented as mean ⫾ SEM. Significance was determined using two-tailed Student’s t test (ⴱⴱ, p ⬍ 0.01: F vs E; ††, p ⬍ 0.01, †, p ⬍ 0.05: f vs E; ‡, p ⬍ 0.05: 䡺 vs E; §§, p ⬍ 0.01, §, p ⬍ 0.05: F vs f; ¶, p ⬍ 0.05: F vs 䡺).
Then, the supernatants were collected from cultures of the cell proliferation assay, and concentrations of proinflammatory cytokines, IFN-␥, TNF-␣, IL-4, and IL-10, were quantitated by ELISA. The production levels of IFN-␥ and TNF-␣ were reduced in supernatants from OPN⫺/⫺ cell cultures compared with those of WT cell cultures (Fig. 5, B and C). No IL-4 and IL-10 were detected in both OPN⫺/⫺ and WT culture supernatants (data not shown). Next, we examined the proliferative responses of purified T cells from OPN⫺/⫺ and WT mice in response to OPN⫺/⫺ or WT APC plus hIRBP peptide to elucidate whether OPN deficiency in T cells or APC is responsible for the low proliferative responses of OPN⫺/⫺ lymph node cells. When CD4⫹ T cells were stimulated with hIRBP peptide Ag at various concentrations for 64 h, the Ag-specific proliferation was seen in a dose-dependent manner of hIRBP added in all culture groups. Of note, the cells from OPN⫺/⫺ mice, not only CD4⫹ T cells, but also APC, generated considerably reduced proliferative responses compared with those from WT cells (Fig. 6). The coculture of OPN⫺/⫺ CD4⫹ T cells and OPN⫺/⫺ APC showed the lowest responses.
OPN IN EAU
FIGURE 7. Clinical score of EAU in mice treated with M5 Ab. EAU was induced in C57BL/6 mice. These mice were treated with M5 Ab (E) or isotype control Ab (F), as described in Materials and Methods. Funduscopic examination was conducted every 3 or 4 days from day 7 after immunization. The results are presented as the mean clinical score for all eyes of each group of mice (10 mice per group) ⫾ SD. Significance was determined using the Mann-Whitney U test (ⴱ, p ⬍ 0.05).
filtration of inflammatory cells, granuloma formation, and photoreceptor cell damage in retina were seen as in previous experiments (Figs. 3 and 4), whereas only slight changes were seen in M5-treated eyes. The histological scores of retinal sections were significantly lower in M5-treated mice (average scores: 0.20 ⫾ 0.33) than those in the control mice (1.30 ⫾ 0.83) (Fig. 8). These results demonstrate that OPN plays a role in EAU progression and the severity of EAU is attenuated by the treatment with M5 Ab. M5 Ab suppresses cell proliferative responses and cytokine production in vitro To examine the direct effect of M5 Ab in T cell proliferative responses in vitro, T cells were collected from draining lymph nodes of WT mice 10 days after immunization with hIRBP 1–20. These cells were stimulated with hIRBP in the presence of M5 Ab or control IgG. Ag-specific proliferation was seen in a dose-dependent manner of hIRBP in both groups, as already shown in Fig. 5A (Fig. 9). Notably, M5-treated cells showed significantly reduced proliferative responses compared with those in control Ig-treated cells (Fig. 9A). Then, the supernatants were collected from these
Administration of M5 Ab reduced EAU scores To investigate whether OPN serves as a potential target for an intervention of the disease process, WT mice were treated with M5 Ab or control IgG concurrent with immunization with hIRBP 1–20. From day 7 after immunization, we conducted clinical assessment every 3 or 4 days. M5-treated and control mice followed a similar time course to those shown in Fig. 2 (Fig. 7). However, it should be noted in the M5-treated group that EAU reached a peak at day 31 after immunization. This finding stands in marked contrast to that in the control group, which showed the peak at day 24 (Fig. 7). In addition, the clinical severity of EAU in the M5treated mice was significantly milder than that of control mice. The mean maximum clinical score was significantly lower in M5treated group (average scores: 0.75 ⫾ 1.1) than that in the control (1.67 ⫾ 0.91). The incidence of the retinal disease was also significantly decreased in M5-treated group (35%) compared with control group (94%). Then, histopathological examinations were performed in these two groups of mice. Eyes were removed from M5-treated and control mice 21 days after hIRBP immunization. In control eyes, in-
FIGURE 8. Histopathological score of EAU in M5-treated mice. EAU was induced in C57BL/6 mice. These mice were treated with M5 Ab (E) or control IgG as described in Materials and Methods. On day 21, the eyes were enucleated and scored by examining histopathologically. The results are presented as the histopathological score of each eye (M5-treated group, 22 eyes; control group, 20 eyes), and the mean EAU score of each group is indicated by a bar. Significance was determined by the Mann-Whitney U test (p ⬍ 0.01).
The Journal of Immunology
FIGURE 9. Cell proliferative response and cytokine production of hIRBP-immunized and M5-treated lymphocytes. A, [3H]Thymidine incorporation by primed lymphocytes. Lymphocytes were obtained from draining lymph nodes of C57BL/6 mice (n ⫽ 8) 10 days after immunization. Lymphocytes were treated with M5 Ab (E) or isotype control Ab (F) and incubated with indicated dose of hIRBP peptide for 64 h and [3H]thymidine for the last 16 h. B, IFN-␥ produced in the culture supernatant. C, TNF-␣ produced in the culture supernatant. The results are presented as mean ⫾ SEM. Significance was determined using two-tailed Student’s t test (ⴱⴱ, p ⬍ 0.01; ⴱ, p ⬍ 0.05). Data are representative of two separate experiments with the same results.
cultures of the cell proliferation assay, and concentrations of proinflammatory cytokines, IFN-␥ and TNF-␣, in the culture supernatant were quantitated by ELISA. The production levels of IFN-␥ and TNF-␣ were significantly reduced in the supernatants from M5treated cells compared with those in control cells (Fig. 9, B and C).
Discussion Recently, it has been reported that OPN is expressed in various inflammatory diseases, such as pulmonary sarcoidosis (27, 28), rheumatoid arthritis (29), and multiple sclerosis (17, 30). In models of multiple sclerosis, the proinflammatory effect of OPN appeared to be mediated by CD44, a ligand of OPN (17, 31). To date, however, the precise role of OPN has been unclear in these models of rheumatoid arthritis and multiple sclerosis. In the present study, we investigated the influence of OPN in EAU. When the plasma OPN levels were quantitated in EAU mice during the disease progression, the level increased gradually and showed a peak at day 14 after immunization. Thereafter, the concentration of OPN decreased, whereas the EAU score continued increasing and showed a peak 21 days after immunization. From these findings, we postulated that OPN was involved in the afferent phase of the EAU development. Next, we analyzed development of EAU in OPN⫺/⫺ mice and compared the disease manifestation with that developed in WT mice. We found that clinical EAU development was significantly ameliorated in OPN⫺/⫺ mice. Thus, OPN⫺/⫺ mice displayed reduced mean maximal clinical scores and delayed disease onset days compared with those in WT mice. Consistent results were obtained when histopathological scores were compared between OPN⫺/⫺ mice and WT mice developing EAU. On ex vivo hIRBP peptide restimulation of lymphocytes, we could show that the proliferative responses and IFN-␥ and TNF-␣ productions were reduced in OPN⫺/⫺ mice compared with those in WT mice. These findings suggested that Th1-mediated responses were suppressed in OPN⫺/⫺ mice. Moreover, we examined whether OPN deficiency in T cells or APC was responsible for the low proliferative responses by
6571 analyzing T cell proliferative responses in reciprocal combinations of WT and OPN⫺/⫺ cells. We found that either T cells or APC from OPN⫺/⫺ mice were responsible for the low responses. EAU is induced by Th1 cells, which produce the Th1-specific cytokines (32). The elevated Th1 response seems to be related to high susceptibility to ocular autoimmunity in EAU (33), although conflicting results with respect to EAU development have been reported in different cytokine or cytokine receptor-deficient mice (34, 35). Nonetheless, Hoey et al. (36) reported that IFN-␥-stimulated macrophage activation and NO production led to destruction of the retina in EAU. It was also demonstrated that TNF-␣ provoked inflammatory responses (37). TNF p55 receptor-deficient mice were resistant to EAU (38). These previous studies (32) appeared to be consistent with our present observations. IL-6 is involved in T cell proliferation through induction of IL-2R (39) and thought to be more active in this respect than TNF-␣ (40). IL-6 levels have been found to be increased in the aqueous humor in EAU, which appears to antagonize TGF- (41, 42). We administered M5 Ab to EAU mice to pursue whether OPN could be a therapeutic target. We could clearly show that EAU was ameliorated by a single treatment with the M5 Ab. Moreover, when the M5 Ab was added in the culture of hIRBP-primed lymph node cells, IFN-␥ and TNF-␣ productions as well as T cell proliferative responses were significantly suppressed. These findings demonstrate that OPN plays a role in not only afferent phase of EAU development, but also the efferent phase in which primed T cells respond to the immunogen, hIRBP peptide. Furthermore, it has been reported that OPN initiates migration of macrophages and dendritic cells to site of inflammation (10, 43, 44). Thus, M5 Ab may also diminish the migration of macrophages and dendritic cells to the inflammatory site. It has been reported that M5 Ab binds to the cryptic epitope, SLAYGLR, exposed by thrombin cleavage of murine OPN. SVVYGLR, the corresponding sequence in human OPN (45), directly binds to ␣91 that interacts with VCAM-1 in extravasation of neutrophils at sites of acute inflammation (46). By this means, the SVVYGLR sequence appears to promote adhesion and migration of leukocytes to the lesion. On the basis of these previous reports on human OPN, we consider that murine SLAYGLR functions similarly and contributes to migration of leukocytes, including macrophages and neutrophils to the inflammatory lesion. Hikita et al. (47) recently reported that OPN was involved in EAU development. We demonstrated two new findings concerning involvement of OPN in EAU in the present study, i.e., a correlation between the plasma OPN level and the disease progression, and the preventive effect of M5 Ab on EAU. Recently, it has been reported that anti-TNF-␣ Ab, infliximab, and soluble p75 rTNFR, etanercept, are efficacious treatment of various autoimmune diseases, such as rheumatoid arthritis and refractory intraocular inflammation. However, the treatment with antiTNF-␣ Ab sometimes brings about litany of adverse effects, especially serious infections (48, 49). M5, an OPN-neutralizing Ab, has been reported to be efficacious in a murine model of rheumatoid arthritis (20, 50). Notably, M5 showed no influence on susceptibility to Candida albicans infection (20, 50). In addition to these previous studies, our present results suggest that Ab treatment is applicable to treatment of human uveoretinitis without detrimental side effects when a neutralizing Ab for human OPN becomes available. At any rate, it seems important to elucidate more precisely in vivo influences of anti-OPN Ab administration in various disease models. In conclusion, our findings highlight the importance of studies that correlate OPN expression with clinical signs in human uveoretinitis patients, and propose that OPN may represent a new therapeutic target to ameliorate uveoretinitis. Further studies should be conducted to elucidate precisely how OPN functions in EAU.
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Disclosures The authors have no financial conflict of interest.
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