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Diminution of experimental autoimmune uveoretinitis (EAU) in mice depleted of NK cells Nobuyoshi Kitaichi,* Satoshi Kotake,† Taiki Morohashi,‡ Kazunori Onoe´,‡ Shigeaki Ohno,† and Andrew W. Taylor* *Schepens Eye Research Institute and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; and †Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine, and ‡Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan

Abstract: To evaluate the potential role of NK1.1 (CD161c) cells in autoimmune uveoretinitis, we treated experimental autoimmune uveoretinitis (EAU)-susceptible mice with anti-CD161c antibodies (PK136) to deplete natural killer (NK) cells. Injection of anti-CD161c antibodies deleted NK cells from the peripheral blood of EAU-susceptible mice. The T cell proliferative response against the ocular autoantigen K2 was not suppressed in mice treated with anti-CD161c antibody when compared with T cells from control mice. Although mice treated with anti-CD161c developed EAU, the clinical severity on days 17 and 19 after induction of EAU was significantly mild in anti-CD161ctreated mice compared with control mice. In addition, the histopathological severity of EAU was significantly milder in mice treated with anti-CD161c antibodies than controls 21 days after induction of EAU. Our results indicate that the severity of EAU is augmented by NK1.1ⴙ NK cells. J. Leukoc. Biol. 72: 1117–1121; 2002. Key Words: NK1.1 䡠 anti-CD161c 䡠 mouse

INTRODUCTION Experimental autoimmune uveoretinitis (EAU) is an animal model for several human endogenous uveoretinitis diseases [1, 2]. It is a T cell-mediated inflammatory disease that is induced by immunizing specific strains of mice with ocular autoantigens. The disease model is limited to strains of mice of the H-2r, H-2k, and H-2b haplotypes [3–5]. As EAU is dependent on the activation of autoreactive CD4⫹ T cells [6], strains of mice that produce a low T helper cell type 1 (Th1) or high Th2 response are resistant to the induction of EAU [7]. Thus, accessory factors from other immune cells that promote autoreactive Th1 cell activation may also promote EAU. Several studies demonstrate a role for CD161c⫹ (NK 1.1) cells in regulating autoimmunity [8]. For Th1 cell-mediated autoimmune diseases, diabetes and experimental autoimmune encephalomyelitis (EAE), it is clear that CD161c⫹ CD3⫹ natural killer (NK) T cells suppress the induction of autoimmune diseases [9]. This is possibly a result of their ability to mediate immune deviation, thus preventing the activation of

autoreactive Th1 cells. Moreover, NK T cells are central to the induction of anterior chamber-associated immune deviation, an active, immunosuppressive mechanism of the immune-privileged eye [10]. In contrast, the literature reports a paradox for the role of CD161c⫹ CD3- NK cells. It is well-characterized that microbial products activate interferon-␥ production by NK cells and promote the induction of Th1 cells. However, the depletion of NK cells with anit-CD161c antibody causes an increase in the severity of EAE [11] and colitis [12]. Also, NK cells are needed to induce a remission in EAE [11]. Therefore, it appears that NK cells have a role in suppressing the induction of autoimmunity; however, nothing is known about NK cell activity in ocular autoimmune disease. There are only a few reports describing NK cell activity in the eye [13–16], and these reports describe the suppression of NK cell functions in the normal, ocular microenvironment. Based on these reports and the effects of depleting NK cells in other autoimmune disease models, it is reasonable to think that NK cells may have a role in suppressing the induction of ocular autoimmunity with little involvement in the intraocular inflammation. To examine the influence of NK cells on EAU, we depleted EAU-susceptible mice of NK cells by intravenous (i.v.) injections of anti-CD161c antibody. Unexpectedly, we found that anti-CD161c treatment resulted in the diminution of EAU in the mice but not in suppressing activation of autoreactive T cells.

MATERIALS AND METHODS Reagents The K2 peptide (ADKDVVVLTSSRTGGV, MW⫽1603.78) corresponds to the amino acid sequence 201–216 of bovine interphotoreceptor retinoid-binding protein (IRBP) and is the immunodominate retinal autoantigen of EAU in H-2k mice [4]. K2 was synthesized and purified by high-pressure liquid chromatography [Invitrogen (ResGen) Corp., Huntsville, AL]. Whole IRBP protein was provided to us as a kind gift from Dr. Igal Gery (National Eye Institute, NIH, Bethesda, MD). Incomplete and complete Freund’s adjuvant (CFA) and desiccated Mycobacterium tuberculosis H37RA were purchased from Difco (De-

Correspondence: Andrew W. Taylor, Ph.D., Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114. E-mail: awtaylor@vision. eri.harvard.edu Received June 10, 2002; revised July 26, 2002; accepted August 27, 2002.

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troit, MI). Inactive Bordetella pertussis suspension was purchased from Wako Pure Chemical Industries (Osaka, Japan). Anti-CD161c antibodies [mouse immunoglobulin G (IgG)2a] were purified from culture supernatant of PK136 monoclonal cells (American Type Culture Collection, Manassas, VA). AntiCD244.2 (2B4)-fluorescein isothiocyanate (FITC) was purchased from BDPharMingen (San Diego, CA).

Mice and induction of EAU Female B10.BR (mice 6 – 8 weeks old) were purchased from Shizuoka Laboratory Animal Corporation (Hamamatsu, Japan) and Jackson Laboratories (Bar Harbor, ME). All mice were provided food and water and kept on a 12-h light-dark cycle. All experiments were conducted with the approval and supervision of the Institutional Animal Care and Use Committee. To induce EAU, mice were immunized in the footpad and the base of the tail with 100 ␮l 60 nmol K2 peptide or with 100 ␮g IRBP emulsified in CFA (1:1 v/v) plus an intraperitoneal injection of 50 ␮l B. pertussis suspension (1010 bacteria per mouse) as described previously [4, 17]. Double-blind clinical assessment by funduscopic examinations of the retinal inflammation was done every 2 days from 7 to 19 days after the immunization. The severity of the retinal inflammation was graded double-blind on a five-point scale as described previously [18, 19]. The clinical scoring was based on vessel dilatation, number of vessel white focal lesions, and the extent of retinal vessel exudate, hemorrhage, and detachment. On day 21 after immunization, the eyes were enucleated and fixed in 4% phosphate-buffered glutaraldehyde for 1 h and transferred into 10% phosphate-buffered formaldehyde. Fixed tissues were stained with hematoxylin and eosin, and the histological severity was graded double-blind on a scale of 0 – 4 as reported [20]. The histology score differs from the clinical score, as the histology score was based on the number of focal and linear lesions along with the physical features of vasculitis, retinal hemorrhage, detachment, and atrophy.

Anti-CD161c treatment To delete CD161c⫹ cells, B10.BR mice were injected into their tail vein with 500 ␮g anti-CD161c monoclonal antibody (mAb; PK136) as previously reported [11] before the mice were immunized on the same day with K2 peptide. The anti-CD161c antibody was purified from the supernatant of PK136 cell cultures. The supernatant was passed through a GammaBind Plus Sepharose column from Pharmacia (Peapack, NJ), and the Ig was eluted from the column, concentrated, and filtered sterilized. Control mice were treated with 500 ␮g ChromPure mouse IgG (Jackson ImmunoResearch, West Grove, PA) or phosphate-buffered saline.

TABLE 1. Histopathology Score of Retinas on Day 19 of EAU Induced by K2 or IRBP in Mice Treated with Anti-CD161c Antibody

Control IgG Anti-CD161

K2 peptide

IRBP

1.08 ⫾ 1.04a 0.88 ⫾ 0.94

3.2 ⫾ 0.4b 0.9 ⫾ 0.8b

a Mean histopathological score ⫾ SD of five samples per 10 retinas. b Significant difference P ⬍ 0.0001 (Mann-Whitney U test).

a nylon wool column (95% CD3⫹ cells). The T cells (4x105 cells/well) were cultured with 30 Gy-irradiated syngeneic splenic antigen-presenting cells (APC) and peptides in a 96-well flat-bottomed plate. The cells were incubated for 48 h, and 50 ␮Ci 3H-thymidine was added to each well. The cultures were further incubated for 24 h. Evaluation of T cell proliferation was determined by 3 H-thymidine incorporation, and the data are presented as the mean counts per minute (CPM) minus the background (medium alone; CPM) as described elsewhere [21–23].

Immunofluorescence analysis The effectiveness of anti-CD161c antibody treatment to deplete NK cells was determined by flow cytometric analysis of spleen cells 7 days after the mice were injected with anti-CD161c. The spleen cells were stained with FITCconjugated 2B4 antibody (anti-CD244.2). The cells were incubated with the antibody for 30 min on ice and then analyzed on a Coulter Epics XL flow cytometer. The results were generated by gating on the 2B4-positive cells defined by comparing the results of mice not treated with anti-CD161c antibody with the FITC isotype control.

Statistical analysis Statistical analysis was performed using Kruscal-Wallis and Mann-Whitney U tests for nonparametric comparisons of treated and untreated groups. Calculations were performed using the Statview statistical software application (Abacus Concepts, Berkeley, CA).

T cell proliferation assay Cells were collected from the popliteal lymph nodes of mice 10 days after immunization. T cells were enriched by passing the lymph node cells through

Fig. 1. Mice treated with anti-CD161c have diminished EAU. Serial observations of EAU severity were scored on mice injected with anti-CD161c antibody (500 ␮g) or control IgG (500 ␮g). The EAU was induced by immunizing the mice with K2 peptide in CFA. Results are reported as the mean score for all eyes of each group of mice (10 mice per group). *Significance was determined using Kruscal-Wallis nonparametric analysis.

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Fig. 2. The anti-CD161c treatment suppresses the severity of cellular infiltration into the retina of EAU mice. The severity of EAU induced by IRBP between anti-CD161c-treated and controls was scored by examining histological sections of EAU mice described in Material and Methods. The results are presented as the maximum histopathology score for each eye. The maximum histopathology score was determined from examining five sections of each eye. Significant (P⬍0.0001) changes in EAU severity were determined by MannWhitney U nonparametric analysis.

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Fig. 3. Mice treated with anti-CD161c antibody are depleted of NK cells. A flow cytometry analysis was conducted on the spleen cells of mice treated 7 days prior with anti-CD161c antibody. The spleen cells were stained with FITC-conjugated 2B4 (anti-CD244.2). Presented are the histograms of flow cytometry analysis of stained spleen cells from untreated mice (A), from mice treated with anti-CD161c (B), and FITC-isotype IgG-stained spleen cells of normal, untreated mice (C). The percentage stated is the percent of the total number of spleen cells that are 2B4⫹ NK cells.

RESULTS Injections of anti-CD161c diminishes EAU To analyze the functional role of CD161c (NK1.1)-positive cells in EAU, we injected EAU-susceptible B10.BR mice with anti-CD161c monoclonal PK136, which can deplete NK1.1positive cells in vivo [11, 12, 24]. Susceptibility for EAU was induced by immunizing the mice with K2 peptide, an autologous autoantigen peptide of human IRBP [4, 17]. We visually examined the severity of inflammation in the retinas of the anti-CD161c-treated EAU mice from day 7 to day 19 after K2 immunization. The average clinical severity of EAU in the anti-CD161c-treated mice was significantly mild compared with that of control mice on days 17 (P⫽.0004) and 19 (P⬍.0001; Fig. 1). However, when we compared the histology of EAU retinal tissues 21 days after K2 immunization with anti-CD161c-treated and untreated mice, we found no significant difference in the level of cellular infiltration (Table 1). As immunization with K2 induces EAU with mild cellular infiltration of the retina [4], we induced EAU with whole bovine IRBP protein to enhance the clonal expansion of activated EAU-mediating T cells in vivo. The maximum level of retinitis in EAU in mice immunized with whole IRBP occurs 21 days after immunization. The histopathology scores of mouse retinas 21 days after whole IRBP immunization were significantly different between the mice treated with irrelevant IgG control (3.2⫾0.4) and mice injected with anti-CD161c mAb (0.9⫾0.8; Fig. 2 and Table 1). The severity of the disease was significantly (P⬍0.0001) milder in the anti-CD161c-treated mice.

with anti-CD161c antibody are significantly depleted in the NK cells. This is consistent with previous reports demonstrating a similar in vivo depletion pattern of cells by anti-CD161c injections [11, 12, 24].

Depletion of NK cells does not suppress induction of K2-reactive T cells As EAU is a T cell-mediated disease, we examined whether anti-CD161c depletion of NK cells and thus the suppression in

Treatment of B10.BR mice with anti-CD161c depletes NK cells To determine whether the i.v. injections of anti-CD161c depleted NK cells in the mice, we stained spleen cells with 2B4 antibody (anti-CD244.2), an antibody that does not compete for binding with anti-CD161c on NK1.1⫹ NK cells. The stained cells were analyzed by flow cytometry. Normally, the spleen cells of B10.BR mice are 4.3% 2B4⫹ NK cells (Fig. 3A). Injection of anti-CD161c depleted the 2B4⫹ cell population 7 days after antibody injection (Fig. 3B). Therefore, mice treated

Fig. 4. Anti-CD161c treatment does not change T cell response to ocularautoantigen K2. Mice treated with anti-CD161c antibody (500 ␮g) or control IgG (500 ␮g) were immunized with K2 and CFA in a manner similar to the procedures used to induce EAU. T cells were isolated from draining lymph nodes 10 days after immunization and placed into cultures of K2-pulsed APC. Proliferation was assayed by scintillation counting of 3H-thymidine incorporation by the proliferating T cells. The results are presented as average difference in the CPM over background (⌬CPM) of three independent experiments.

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the severity of autoimmune retinitis resulted from a reduction in the activation of K2-specific T cells. We obtained T cells from K2-immunized mice treated with anti-CD161 mAb or an irrelevant IgG control 10 days after K2 immunization. There was no significant difference in K2 antigen-stimulated proliferation between T cells from anti-CD161c- and IgG-treated mice (Fig. 4). Therefore, these results indicate that NK cells are not required for the induction of ocular-autoantigen-reactive T cells.

DISCUSSION We demonstrated that anti-CD161c treatment of EAU-susceptible B10.BR mice diminished the severity of EAU. Also, the depletion of NK cells in the mice did not prevent the induction of ocular-autoreactive T cells. An initial evaluation of the results suggests that the severity of autoimmune disease in the eye is dependent on functional NK cells. As the anti-CD161c treatment does not inhibit the induction of autoreactive T cells in the EAU mice, it suggests that the NK1.1⫹ cells must influence later phases of EAU. The target tissues in several animal models of autoimmune disease contain NK cells [25–30]. In the diabetes and in encephalomyelitis autoimmune disease models, isolated NK cells have been found to destroy pancreatic ␤-cells and neurons, respectively, in vitro [31, 32]. This suggests that NK cells can contribute to the pathology of autoimmunity in the target tissue. Also, the cellular damage caused by the NK cells would release more immune-targeted autoantigens. Although retinas of mice with EAU have yet to be examined for NK cells, our deletion of NK cells has removed an important effector cell participating in the pathology of autoimmune uveoretinitis. Therefore, the NK cells could participate directly in the retinal pathology, or they could support the activation of autoimmuneeffector T cells [33] in the retina or regional lymph nodes. In addition, there is also the possibility that as we deleted the NK cells, we simultaneously activated NK T cells [34]. As there is an association of NKT cell activation with protection from autoimmune diseases in mice [35, 36], it is likely that the observed diminution of EAU is caused by the combined loss of a mediator of the disease (deletion of NK cells) with the activation of immune protection (activated NK T cells) [34]. Another reason for the unexpected results is that the eye is normally a tissue site of extreme regional immunity [37–39]. Aqueous humor, the fluid filling the ocular-anterior chamber, inhibits NK cell killing of major histocompatibility complex I-negative cells [15]. This inhibitory activity of aqueous humor is mediated by the constitutively present macrophage migration-inhibitory factor (MIF) [15]. We previously reported that MIF increases in sera of patients with uveitis [40 – 42]. Therefore, the constitutive presence of MIF in the eye suggests that the ocular microenvironment has the potential to regulate the functionality of NK cells in a manner different from other tissue sites. Other than suppressed cytotoxic activity, very little else is known about other NK cell functions regulated by MIF in the eye. Our results suggest that NK cell activity in an autoimmune-susceptible eye would be proinflammatory. 1120

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Finally, our results could be because an autoimmune disease of the eye is different from autoimmune diseases in other tissues. Zhang et al. [11] reported that the severity of EAE was enhanced by NK1.1⫹ cell depletion by anti-CD161c antibody in C57BL/6 mice. They also showed that the enhanced severity of EAE was associated with enhanced T cell proliferation in response to pathogenic peptide and that it was NK cells normally suppressing the activation of autoreactive T cells. In our present study, we found no significant change in T cell proliferation between anti-CD161c-injected mice and control mice groups. Also, the severity of EAU was milder in mice injected with anti-CD161 mAb than those injected with control Ig. The cause of the diminution in EAU severity cannot be a result of any changes in lymphocyte activation to the autoantigens among all groups in the present study. Our results demonstrate that NK1.1⫹ cells are uniquely involved in the inflammatory response of EAU. Therefore, not only T cells but also NK cells are involved in mediating the severity of autoimmune uveoretinitis. The diminution of EAU severity by anti-CD161c antibody treatment is the opposite of our expectations, based on reports demonstrating that antiCD161c antibody treatment enhances the severity of other autoimmune diseases [11, 12].

ACKNOWLEDGMENTS This work was supported by a grant-in-aid from the Japan Eye Bank Association; the Ministry of Health, Labor and Welfare of Japan; and by PHS grants EY10752 and EY07145 of the National Eye Institute (Bethesda, MD). Authors are grateful to Dr. Joan Stein-Streilein (Schepens Eye Research Institute, Boston, MA) for her helpful discussions.

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