Aggravates Experimental Autoimmune Uveoretinitis in Mice Mizuki Kitamura, Kazuya Iwabuchi, Nobuyoshi Kitaichi, Shigeyuki Kon, Hirokuni Kitamei, Kenichi Namba, This information is current as Kazuhiko Yoshida, David T. Denhardt, Susan R. Rittling, of September 28, 2021. Shigeaki Ohno, Toshimitsu Uede and Kazunori Onoé J Immunol 2007; 178:6567-6572; ; doi: 10.4049/jimmunol.178.10.6567

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 immu- nized with human interphotoreceptor retinoid-binding protein (hIRBP) peptide 1–20. OPN-deficient (OPN؊/؊) mice showed milder EAU progression in clinical and histopathological scores compared with those of WT mice. The T cells from hIRBP- Downloaded from -immunized OPN؊/؊ mice exhibited reduced Ag-specific proliferation and proinflammatory (TNF-␣ and IFN-␥) produc tion 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 hIRBP- immunized WT mice showed significantly reduced proliferative responses and proinflammatory cytokine production upon stim- ulation with hIRBP peptide in the presence of M5 Ab in the culture. Our present results demonstrate that OPN may represent http://www.jimmunol.org/ a novel therapeutic target to control uveoretinitis. The Journal of Immunology, 2007, 178: 6567–6572.

xperimental autoimmune uveoretinitis (EAU)3 is an an- Osteopontin (OPN), also known as early T activa- imal model of human endogenous uveitis, such as sym- tion 1 (Eta-1), is a secreted phosphoglycoprotein that contains the E pathetic ophthalmia, birdshot retinochoroidopathy, arginine-glycine-aspartic acid (RGD) integrin-binding sequence. Vogt-Koyanagi-Harada’s disease, and Behc¸et’s disease (1). The RGD sequence is also found in many extracellular matrix EAU is an organ-specific, -mediated autoimmune disease proteins (8). OPN has been shown to mediate adhesion and mi- that can be induced by immunization with retinal Ag, e.g., hu- gration of a number of different cell types. It has been reported that man interphotoreceptor retinoid-binding protein or by the adop- ␣ ␤ ␣ ␤ ␣ ␤ ␣ ␤ ␣ ␤ ␣ ␤ by guest on September 28, 2021 the integrins v 1, v 3, v 5, 5 1, 8 1, and v 6 bind to OPN tive transfer of retinal Ag-specific T (2–4). EAU ␣ ␤ ␣ ␤ through RGD sequence and 4 1, 9 1 bind to non-RGD sites of is a Th1-dominant response. It has been demonstrated that the OPN (9). CD44 is known as one of the OPN receptors, and the augmentation of the Th2 response and T regulatory cytokine interaction of OPN with CD44 regulates migration production or down-regulation of the Th1 responses prevents and activation (10, 11). inflammatory responses and protects against the EAU develop- OPN is produced by a variety of inflammatory cells, including ment (5–7). T cells, , 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 *Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; †Department of Ophthalmology and Visual Sciences, Hokkaido Uni- development of Th1-mediated immunity and diseases. Xu et al. versity Graduate School of Medicine, Sapporo, Japan; ‡Division of Molecular Im- (15) reported that CD44 was up-regulated in the eyes of EAU mice munology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; §Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ and the disease severity was reduced by administration of anti- 08854; and ¶The Forsyth Institute, The Fenway, Boston, MA 02115 CD44 mAb. Of interest, OPN-positive cells were observed in the Received for publication October 19, 2006. Accepted for publication February ganglion cell layer of retinas in C57BL/6J mice (16). 22, 2007. Recent studies with OPN-deficient (OPNϪ/Ϫ) mice have dem- The costs of publication of this article were defrayed in part by the payment of page onstrated that OPN deficiency inhibits disease progression in some charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. experimental autoimmune models, such as experimental autoim- 1 This work was supported in part by a grant for research on sensory and communi- mune encephalomyelitis (17, 18) and anti-type II collagen Ab- cative disorders from the Ministry of Health, Labor, and Welfare, Japan; by Grants- induced arthritis (19). Furthermore, an Ab (M5) specific for in-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 Min- SLAYGLR sequence, which is newly exposed within OPN by istry of Education, Culture, Sports, Science, and Technology, Japan. thrombin cleavage, significantly suppressed a murine model of 2 Address correspondence and reprint requests to Dr. Kazunori Onoe´, Division of rheumatoid arthritis (20). Immunobiology, Institute for Genetic Medicine, Hokkaido University, Kita-15, In the present study, using a murine EAU model, we examined Nishi-7, Kita-ku, Sapporo 060-0815, Japan. E-mail address: kazunori@igm. hokudai.ac.jp the role of OPN in this organ-specific autoimmune disease. We Ϫ/Ϫ 3 Abbreviations used in this paper: EAU, experimental autoimmune uveoretinitis; demonstrate in this study that OPN mice develop significantly hIRBP, human interphotoreceptor retinoid-binding protein; OPN, osteopontin; milder EAU in comparison with that in wild-type (WT) controls. PTX, pertussis toxin; RGD, arginine-glycine-aspartic acid; WT, wild type. Furthermore, we will show that EAU is ameliorated by a single Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 administration of M5 Ab. www.jimmunol.org 6568 OPN IN EAU

Materials and Methods Experimental animals Six- to 8-wk-old female C57BL/6 (H-2b) (WT) mice were obtained from Japan SLC. OPNϪ/Ϫ 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 Bordetella pertussis Sigma-Genosys. Purified toxin (PTX) was from FIGURE 1. Plasma OPN levels in EAU mice. C57BL/6 mice (n ϭ 24) Sigma-Aldrich, and CFA and Mycobacterium tuberculosis strain were immunized with hIRBP 1–20 (200 ␮g) emulsified in CFA (1:1 v/v) H37Ra were from Difco. containing 2.5 mg/ml M. tuberculosis. A total of 200 ␮l of the emulsion Immunization 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 To analyze the cell proliferative response, hIRBP 1–20 (100 ␮g) was emul- transcardially before immunization and on days 3, 7, 10, 14, 21, and 28 sified 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 emul- after immunization. All blood samples were collected under EDTA, cen- Downloaded from Ϫ sified in CFA (1:1 v/v) containing 2.5 mg/ml M. tuberculosis. A total of trifuged to remove cells and debris, and stored at 80°C until used. Plasma 200 ␮l of the emulsion was injected s.c. Concurrent with immunization, 0.1 levels of OPN were measured by sandwich ELISA. ␮g of PTX in 100 ␮l of PBS was injected i.p. as an additional adjuvant (22). Evaluation of EAU ment (2 ␮g/ml insulin from bovine pancreas, 1.1 ␮g/ml iron-free Clinical assessment by funduscopic examination of the retinal inflamma- transferrin, 1 ng/ml sodium selenite, 100 ␮g/ml BSA, and 1 ␮g/ml linoleic tion was conducted every 3 or 4 days from day 7 after immunization (23) acid)) (Sigma-Aldrich). Triplicate 0.2-ml cultures in 96-well flat-bottom http://www.jimmunol.org/ (10 mice/group). The severity of EAU was graded 0–4, as described pre- plates were stimulated with the hIRBP peptide at the indicated concentra- viously (24). Briefly, the clinical scoring was based on vessel dilatation, the tion. In the second type of experiment, CD4ϩ T cell-enriched fractions number of vessel white focal lesions, vessel white linear lesions, hemor- were prepared by passing the dispersed cells from the draining lymph rhages, and the extent of retinal detachment. For the histological assess- nodes of hIRBP-immunized WT or OPNϪ/Ϫ mice over mouse T cell CD4 ment of EAU, eyes were enucleated on day 21 after immunization (10 subset column (R&D Systems). The collected T cells (4 ϫ 105/well) mice/group). Mice were deeply anesthetized with ether and then transcar- were cultured with 30 Gy-irradiated splenocytes as APC (1 ϫ 105/well) dially perfused with 4% paraformaldehyde. Eyes were immediately re- from untreated WT or OPNϪ/Ϫ mice in 0.2 ml of the serum-free medium. moved and postfixed in the same fixative at 4°C for 4 h and transferred into Triplicate 0.2-ml cultures in 96-well flat-bottom plates were stimulated 10% phosphate-buffered formaldehyde until processing. Fixed samples with the hIRBP peptide at the indicated concentration. In the third type of were embedded in paraffin, and 5-␮m sagittal sections were cut near the experiment, to investigate the effect of the M5 Ab in vitro, M5 (50 ␮g/ml) by guest on September 28, 2021 optic nerve head and stained with H&E. The severity of EAU in each eye was added in each well. The cultures (first, second, and third) were incu- was scored on a scale of 0–4, as described previously (1). In brief, focal ␮ 3 bated for 64 h at 37°C in 5% CO2 in air, pulsed with 1 Ci of [ H]thy- nongranulomatous, monocytic infiltrations in the choroids, ciliary body, midine (PerkinElmer) per well during the last 16 h of incubation, and then and retina were scored as 0.5. Retinal perivascular infiltration and mono- harvested. [3H]Thymidine incorporation was quantitated with a direct beta cytic infiltration in the vitreous were scored as 1. Granuloma formation in counter (Packard Instrument) in the first and third experiments, and with a the uvea and retina, the presence of occluded retinal vasculitis, along with liquid scintillation counter (Beckman Coulter) in the second experiment. photoreceptor folds, serous detachment, and loss of photoreceptor were The data were presented as the mean cpm minus the background (medium scored as 2. In addition, the formation of Dalen-Fuchs nodules (granuloma alone; ⌬cpm), as described previously (26). at the level of the retinal pigmented epithelium) and the development of produced in the culture supernatant were measured by an subretinal neovascularization were scored as 3 and 4, according to the ELISA kit (OptEIA; BD Pharmingen). Briefly, flat-bottom 96-well plates number and the size of the lesions. The EAU severity was double-blindly were coated with anti-IFN-␥, anti-TNF-␣, anti-IL-4, or anti-IL-10 capture scored by two ophthalmologists. Ab at 4°C overnight. The plates were blocked with PBS with 10% FBS for Measurement of plasma OPN level 1 h and incubated for another 2 h with samples or standards at room tem- perature. Then, detection Ab and streptavidin-HRP conjugate were added For the measurement of OPN concentration in plasma of EAU mice, mice and incubated for1hatroom temperature. Finally, the plates were devel- were deeply anesthetized with ether, and then blood was collected tran- oped using tetramethylbenzidine substrate. scardially before immunization and on days 3, 7, 10, 14, 21, and 28 after immunization. All blood samples were collected under EDTA to avoid Statistical analysis being fragmented in vitro by thrombin, centrifuged to remove cells and Ϯ Ϫ Data are presented as mean SD in clinical and histopathological scoring, debris, and stored at 80°C until used. Plasma levels of OPN (totally 24 and as mean Ϯ SEM in analyses of cell proliferation and cytokine pro- mice) were measured by sandwich ELISA, as previously reported (25). duction. Statistical analysis of EAU scoring was performed using the non- Ab (M5) treatment parametric Mann-Whitney U test. Analyses of cell proliferation and cyto- kine production were performed using two-tailed Student’s t test. Values of A purified IgG fraction of rabbit serum immunized with synthetic peptide p Ͻ 0.05 were considered statistically significant. (VDVPNGRGDSLAYGLRS), referred to as M5 Ab (20), was used in this study. M5 Ab (400 ␮g/mouse) was i.v. administered concurrent with im- Results ϭ ␮ munization (n 10). The control group was i.v. administered 400 gof Plasma OPN levels are increased by the induction of EAU normal rabbit IgG (Jackson ImmunoResearch Laboratories) (n ϭ 10). To examine whether plasma OPN levels change during the course Determination of immunological responses of EAU progression, C57BL/6 (WT) mice were immunized with We performed three types of T cell proliferation assay. In the first type of hIRBP 1–20 emulsified in CFA containing 2.5 mg/ml M. tuber- experiment, 10 days after immunization, primed lymphocytes obtained culosis. Blood samples were collected before immunization and on from draining lymph nodes were suspended at 5 ϫ 105 cells per 0.2 ml of serum-free medium (RPMI 1640 medium, 10 mM HEPES, 0.1 mM non- days 3, 7, 10, 14, 21, and 28 after immunization, and the plasma essential amino acids, 1 mM sodium pyruvate, 50 ␮g/ml gentamicin sul- levels of OPN were measured by ELISA. It will be seen in Fig. 1 fate, supplemented with 0.1% BSA, and ITS ϩ 1 liquid medium supple- that plasma OPN levels increased after immunization with a peak The Journal of Immunology 6569

FIGURE 2. Clinical score of EAU in OPNϪ/Ϫ mice. EAU was induced in WT (F) and OPNϪ/Ϫ (E) mice. Funduscopic examination was con- ducted 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 FIGURE 4. Histopathological score of EAU in OPNϪ/Ϫ mice. EAU per group) Ϯ SD. Significance was determined using the Mann-Whitney U was induced in WT (F) and OPNϪ/Ϫ (E) mice. On day 21, the eyes were p Ͻ 0.01). enucleated and scored by examining histopathologically. The results are ,ءء) test Downloaded from 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 at day 14. The concentration of OPN was significantly higher in the Mann-Whitney U test (p Ͻ 0.05). EAU mice (from days 3 to 28) than that of before immunization.

OPNϪ/Ϫ mice show reduced EAU scores than those in the control mice (1.33 Ϯ 0.91) (Fig. 4). These results

demonstrate that OPN plays a role in EAU progression. http://www.jimmunol.org/ We then examined the role of OPN in development of EAU using Ϫ Ϫ Ϫ Ϫ OPN / mice. OPN / and WT mice were immunized with OPN deficiency results in reduced T cell proliferative responses hIRBP 1–20 emulsified in CFA containing 2.5 mg/ml M. tuber- to hIRBP culosis. From day 7 after immunization, we conducted clinical To analyze proliferative and cytokine responses of T cells, drain- assessment every 3 or 4 days. In WT control mice, clinically di- Ϫ Ϫ ing lymph nodes were collected from WT and OPN / mice 10 agnosable EAU began developing at ϳday 10 and reached a peak Ϫ Ϫ days after immunization. These cells were stimulated with hIRBP at day 21 after immunization (Fig. 2). In OPN / mice, the clinical peptide Ag at various concentrations for 64 h. The Ag-specific score of EAU followed a similar time course to that in control Ϫ/Ϫ proliferation was seen in a dose-dependent manner of hIRBP mice. However, the EAU onset was delayed in OPN mice Ϫ/Ϫ by guest on September 28, 2021 Ϫ Ϫ added in both culture groups. Of note, the cells from OPN mice and the clinical severity in the OPN / mice was significantly showed considerably reduced proliferative responses compared milder than that of control mice. with those in WT cells (Fig. 5A). Then, histopathological examinations were performed in these two groups of mice. Eyes were removed from OPNϪ/Ϫ and WT EAU mice 21 days after hIRBP immunization. Representative his- topathology of the eyes from WT control mice and OPNϪ/Ϫ 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 OPNϪ/Ϫ eyes. The histological scores of retinal sections were significantly lower in OPNϪ/Ϫ mice (average scores: 0.68 Ϯ 0.41)

FIGURE 5. Cell proliferative response and cytokine production of lymph node cells from OPNϪ/Ϫ mice. A,[3H]Thymidine incorporation by primed lymphocytes. Lymphocytes were obtained from draining lymph nodes of WT (F) and OPNϪ/Ϫ (E) mice 10 days after immunization and incubated with indicated dose of hIRBP peptide for 64 h and [3H]thymi- FIGURE 3. Ocular histopathology of WT and OPNϪ/Ϫ mice. A, EAU in dine for the last 16 h. B, IFN-␥ produced in the culture supernatant. C, WT mice immunized with hIRBP. Inflammatory cells are present in the TNF-␣ produced in the culture supernatant. The results are presented as retina, vitreous, and choroid with retinal folding and granulomatous le- mean Ϯ SEM. Significance was determined using two-tailed Student’s t p Ͻ 0.01). Data are representative of two separate experiments ,ءء) sions. B, EAU in OPNϪ/Ϫ mice immunized with hIRBP. Almost normal test retinal architecture is seen. with the same results. 6570 OPN IN EAU

ϩ FIGURE 6. Cell proliferative response of CD4 T lymph node cells FIGURE 7. Clinical score of EAU in mice treated with M5 Ab. EAU Ϫ/Ϫ from OPN mice. Lymphocytes were obtained from draining lymph was induced in C57BL/6 mice. These mice were treated with M5 Ab (E) Ϫ/Ϫ ϩ nodes of WT or OPN mice 10 days after immunization. CD4 T cell- or isotype control Ab (F), as described in Materials and Methods. Fun- enriched fractions were prepared by passing the dispersed cells over mouse duscopic examination was conducted every 3 or 4 days from day 7 after 5 T cell CD4 subset column kit. The collected cells (4 ϫ 10 /well) were immunization. The results are presented as the mean clinical score for all 5 incubated with 30 Gy-irradiated splenocytes (1 ϫ 10 /well) as APC from eyes of each group of mice (10 mice per group) Ϯ SD. Significance was Ͻ ء WT or OPNϪ/Ϫ mice with indicated dose of hIRBP peptide for 64 h and

determined using the Mann-Whitney U test ( , p 0.05). Downloaded from [3H]thymidine for the last 16 h. The results are presented as mean Ϯ SEM. p Ͻ ,ءء) Significance was determined using two-tailed Student’s t test 0.01: F vs E; ††, p Ͻ 0.01, †, p Ͻ 0.05: f vs E;‡,p Ͻ 0.05: Ⅺ vs E; filtration of inflammatory cells, granuloma formation, and photo- Ͻ Ͻ F f Ͻ F Ⅺ §§, p 0.01, §, p 0.05: vs ; ¶, p 0.05: vs ). receptor 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

Then, the supernatants were collected from cultures of the cell http://www.jimmunol.org/ significantly lower in M5-treated mice (average scores: 0.20 Ϯ proliferation assay, and concentrations of proinflammatory cyto- 0.33) than those in the control mice (1.30 Ϯ 0.83) (Fig. 8). These kines, IFN-␥, TNF-␣, IL-4, and IL-10, were quantitated by ELISA. results demonstrate that OPN plays a role in EAU progression and The production levels of IFN-␥ and TNF-␣ were reduced in su- Ϫ Ϫ the severity of EAU is attenuated by the treatment with M5 Ab. pernatants 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 M5 Ab suppresses cell proliferative responses and cytokine Ϫ/Ϫ both OPN and WT culture supernatants (data not shown). production in vitro Next, we examined the proliferative responses of purified T cells Ϫ Ϫ Ϫ Ϫ To examine the direct effect of M5 Ab in T cell proliferative re- from OPN / and WT mice in response to OPN / or WT APC

sponses in vitro, T cells were collected from draining lymph nodes by guest on September 28, 2021 plus hIRBP peptide to elucidate whether OPN deficiency in T cells of WT mice 10 days after immunization with hIRBP 1–20. These or APC is responsible for the low proliferative responses of Ϫ Ϫ ϩ cells were stimulated with hIRBP in the presence of M5 Ab or OPN / lymph node cells. When CD4 T cells were stimulated control IgG. Ag-specific proliferation was seen in a dose-depen- with hIRBP peptide Ag at various concentrations for 64 h, the dent manner of hIRBP in both groups, as already shown in Fig. 5A Ag-specific proliferation was seen in a dose-dependent manner of Ϫ Ϫ (Fig. 9). Notably, M5-treated cells showed significantly reduced hIRBP added in all culture groups. Of note, the cells from OPN / ϩ proliferative responses compared with those in control Ig-treated mice, not only CD4 T cells, but also APC, generated consider- cells (Fig. 9A). Then, the supernatants were collected from these ably reduced proliferative responses compared with those from WT cells (Fig. 6). The coculture of OPNϪ/Ϫ CD4ϩ T cells and OPNϪ/Ϫ APC showed the lowest responses.

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 as- sessment 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 M5- treated mice was significantly milder than that of control mice. The mean maximum clinical score was significantly lower in M5- treated group (average scores: 0.75 Ϯ 1.1) than that in the control 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) (1.67 Ϯ 0.91). The incidence of the retinal disease was also sig- or control IgG as described in Materials and Methods. On day 21, the eyes nificantly decreased in M5-treated group (35%) compared with were enucleated and scored by examining histopathologically. The results control group (94%). are presented as the histopathological score of each eye (M5-treated group, Then, histopathological examinations were performed in these 22 eyes; control group, 20 eyes), and the mean EAU score of each group two groups of mice. Eyes were removed from M5-treated and con- is indicated by a bar. Significance was determined by the Mann-Whitney U trol mice 21 days after hIRBP immunization. In control eyes, in- test (p Ͻ 0.01). The Journal of Immunology 6571

analyzing T cell proliferative responses in reciprocal combinations of WT and OPNϪ/Ϫ cells. We found that either T cells or APC from OPNϪ/Ϫ mice were responsible for the low responses. EAU is induced by Th1 cells, which produce the Th1-specific cy- tokines (32). The elevated Th1 response seems to be related to high susceptibility to ocular autoimmunity in EAU (33), although conflict- ing results with respect to EAU development have been reported in different cytokine or -deficient mice (34, 35). None- theless, 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 re- sponses (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 ␣ FIGURE 9. Cell proliferative response and cytokine production of than TNF- (40). IL-6 levels have been found to be increased in the hIRBP-immunized and M5-treated lymphocytes. A,[3H]Thymidine incor- aqueous humor in EAU, which appears to antagonize TGF-␤ (41, 42). poration by primed lymphocytes. Lymphocytes were obtained from drain- We administered M5 Ab to EAU mice to pursue whether OPN Downloaded from ing lymph nodes of C57BL/6 mice (n ϭ 8) 10 days after immunization. could be a therapeutic target. We could clearly show that EAU was Lymphocytes were treated with M5 Ab (E) or isotype control Ab (F) and ameliorated by a single treatment with the M5 Ab. Moreover, when 3 incubated with indicated dose of hIRBP peptide for 64 h and [ H]thymi- the M5 Ab was added in the culture of hIRBP-primed lymph node dine for the last 16 h. B, IFN-␥ produced in the culture supernatant. C, cells, IFN-␥ and TNF-␣ productions as well as T cell proliferative TNF-␣ produced in the culture supernatant. The results are presented as mean Ϯ SEM. Significance was determined using two-tailed Student’s t responses were significantly suppressed. These findings demonstrate

/p Ͻ 0.05). Data are representative of two separate that OPN plays a role in not only afferent phase of EAU development, http://www.jimmunol.org ,ء ;p Ͻ 0.01 ,ءء) test experiments with the same results. 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 cultures of the cell proliferation assay, and concentrations of proin- inflammation (10, 43, 44). Thus, M5 Ab may also diminish the mi- flammatory cytokines, IFN-␥ and TNF-␣, in the culture supernatant gration of macrophages and dendritic cells to the inflammatory site. were quantitated by ELISA. The production levels of IFN-␥ and It has been reported that M5 Ab binds to the cryptic epitope, TNF-␣ were significantly reduced in the supernatants from M5- SLAYGLR, exposed by thrombin cleavage of murine OPN. treated cells compared with those in control cells (Fig. 9, B and C). SVVYGLR, the corresponding sequence in human OPN (45), di- ␣ ␤ by guest on September 28, 2021 rectly binds to 9 1 that interacts with VCAM-1 in extravasation of Discussion neutrophils at sites of acute inflammation (46). By this means, the Recently, it has been reported that OPN is expressed in various SVVYGLR sequence appears to promote adhesion and migration of inflammatory diseases, such as pulmonary sarcoidosis (27, 28), leukocytes to the lesion. On the basis of these previous reports on rheumatoid arthritis (29), and multiple sclerosis (17, 30). In models human OPN, we consider that murine SLAYGLR functions similarly of multiple sclerosis, the proinflammatory effect of OPN appeared and contributes to migration of leukocytes, including macrophages to be mediated by CD44, a ligand of OPN (17, 31). To date, how- and neutrophils to the inflammatory lesion. Hikita et al. (47) recently ever, the precise role of OPN has been unclear in these models of reported that OPN was involved in EAU development. We demon- rheumatoid arthritis and multiple sclerosis. strated two new findings concerning involvement of OPN in EAU in In the present study, we investigated the influence of OPN in the present study, i.e., a correlation between the plasma OPN level and EAU. When the plasma OPN levels were quantitated in EAU mice the disease progression, and the preventive effect of M5 Ab on EAU. during the disease progression, the level increased gradually and Recently, it has been reported that anti-TNF-␣ Ab, infliximab, showed a peak at day 14 after immunization. Thereafter, the con- and soluble p75 rTNFR, etanercept, are efficacious treatment of centration of OPN decreased, whereas the EAU score continued various autoimmune diseases, such as rheumatoid arthritis and refrac- increasing and showed a peak 21 days after immunization. From tory intraocular inflammation. However, the treatment with anti- these findings, we postulated that OPN was involved in the afferent TNF-␣ Ab sometimes brings about litany of adverse effects, espe- phase of the EAU development. Ϫ Ϫ cially serious infections (48, 49). M5, an OPN-neutralizing Ab, has Next, we analyzed development of EAU in OPN / mice and compared the disease manifestation with that developed in WT been reported to be efficacious in a murine model of rheumatoid ar- mice. We found that clinical EAU development was significantly thritis (20, 50). Notably, M5 showed no influence on susceptibility to ameliorated in OPNϪ/Ϫ mice. Thus, OPNϪ/Ϫ mice displayed re- Candida albicans infection (20, 50). In addition to these previous duced mean maximal clinical scores and delayed disease onset studies, our present results suggest that Ab treatment is applicable to days compared with those in WT mice. Consistent results were treatment of human uveoretinitis without detrimental side effects obtained when histopathological scores were compared between when a neutralizing Ab for human OPN becomes available. At any OPNϪ/Ϫ mice and WT mice developing EAU. On ex vivo hIRBP rate, it seems important to elucidate more precisely in vivo influences peptide restimulation of lymphocytes, we could show that the pro- of anti-OPN Ab administration in various disease models. liferative responses and IFN-␥ and TNF-␣ productions were re- In conclusion, our findings highlight the importance of studies duced in OPNϪ/Ϫ mice compared with those in WT mice. These that correlate OPN expression with clinical signs in human uveo- findings suggested that Th1-mediated responses were suppressed in retinitis patients, and propose that OPN may represent a new ther- OPNϪ/Ϫ mice. Moreover, we examined whether OPN deficiency in T apeutic target to ameliorate uveoretinitis. Further studies should be cells or APC was responsible for the low proliferative responses by conducted to elucidate precisely how OPN functions in EAU. 6572 OPN IN EAU

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