Intraocular injection of tamoxifen-loaded nanoparticles: a new treatment of experimental autoimmune uveoretinitis

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Intraocular injection of tamoxifen-loaded nanoparticles: a new treatment of experimental autoimmune uveoretinitis Yvonne de Kozak1, Karine Andrieux2, Henri Villarroya1, Christophe Klein3, Brigitte Thillaye-Goldenberg1, Marie-Christine Naud1, Elisabeth Garcia2 and Patrick Couvreur2 1 2 3

INSERM U598, Paris, France UMR CNRS 8612, Universite´ Paris-Sud XI, Chaˆtenay-Malabry, France IFR 58, Paris, France

In this study, we tested the efficiency of an intravitreal injection of tamoxifen, a non-steroidal estrogen receptor modulator, in retinal soluble antigen (S-Ag)-induced experimental autoimmune uveoretinitis (EAU). To increase the bioavailability of tamoxifen, we incorporated tamoxifen into polyethylene glycol (PEG)-coated nanoparticles (NP-PEG-TAM). The localization of the nanoparticles within the eye was investigated using fluorescent-labeled PEG-coated nanoparticles after injection into the vitreous cavity of rats with EAU. Some nanoparticles were distributed extracellularly throughout the ocular tissues, others were concentrated in resident ocular cells and in infiltrating macrophages. Whereas the injection of free tamoxifen did not alter the course of EAU, injection of NP-PEG-TAM performed 1–2 days before the expected onset of the disease in controls resulted in significant inhibition of EAU. NP-PEG-TAM injection significantly reduced EAU compared to injection of NP-PEG-TAM with 17b-estradiol (E2), suggesting that tamoxifen is acting as a partial antagonist to E2. Diminished infiltration by MHC class II+ inflammatory cells and low expression of TNF-a, IL1b, and RANTES mRNA were noted in eyes of NP-PEG-TAM-treated rats. Intravitreal injection of NP-PEG-TAM decreased S-Ag lymphocyte proliferation, IFN-c production by inguinal lymph node cells, and specific delayed-type hypersensitivity indicative of a reduced Th1-type response. It increased the anti-S-Ag IgG1 isotype indicating an antibody class switch to Th2 response. These data suggest that NP-PEG-TAM inhibition of EAU could result from a form of immune deviation. Tamoxifen-loaded nanoparticles may represent a new option for the treatment of experimental uveitis. Key words: Experimental autoimmune uveoretinitis / Nanoparticles / Tamoxifen / Treatment / Drug delivery

1 Introduction Experimental autoimmune uveoretinitis (EAU) is a wellestablished model of organ-specific endogenous uveitis [1]. It is a CD4+ T lymphocyte-mediated disease in which macrophages play an important role as effector cells of the intraocular inflammation [2, 3] and photoreceptor cell damage [1, 3]. The breakdown of the blood-ocular [DOI 10.1002/eji.200425022] Abbreviations: EAU: Experimental autoimmune uveoretinitis S-Ag: Retinal soluble antigen PEG: Polyethylene glycol NP-PEG: PEG-coated nanoparticles NP-PEG-F: Fluorescent PEG-coated nanoparticles NP-PEG-TAM: Tamoxifenloaded PEG-coated nanoparticles RPE: Retinal pigment epithelium E2: 17b-Estradiol DTH: Delayed-type hypersensitivity GFAP: Glial fibrillary acidic protein f 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Received Revised Accepted

25/2/04 3/9/04 15/9/04

barriers that occurs before the clinical onset of EAU [4] leads to a substantial inflammatory cell infiltration of both anterior and posterior segments of the eye, followed by an irreversible destruction of the retinal photoreceptors. To induce EAU, laboratory animals are immunized with retinal autoantigens such as retinal soluble antigen (S-Ag) [1]. The inflammation resembles certain noninfectious visually disabling intraocular pathologies which are observed in humans [5, 6]. This experimental model has been used to evaluate the effect of immunomodulatory molecules [5, 6] and immunomodulatory cytokines [1, 7–9]. Some of these agents are currently used in clinical practice (e.g. corticosteroids, cyclosporine) [5, 6]; however, these therapies are frequently complicated by systemic side effects. www.eji.de

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An inhibitory effect of tamoxifen treatment (a nonsteroidal estrogen receptor modulator) has been reported on different experimental models of autoimmune diseases [10–14]. These observations have prompted us to test the effect of this molecule in the EAU model of intraocular inflammation. To avoid side effects observed when tamoxifen is used in high doses and/or for long periods of time [15, 16], we encapsulated tamoxifen into nanoparticles before administration to rats with EAU [17, 18]. Indeed, encapsulation of biologically active molecules may increase their bioavailability and may induce a sustained release, thus avoiding repeated intraocular injections. Additionally, the use of nanotechnologies is expected to decrease the doses of drug administered, thus reducing side effects. Poly[methoxy poly(ethylene glycol) cyanoacrylate-co-hexadecyl cyanoacrylate] (PEG-PHDCA 1:4) was chosen as polymer material for nanoparticle preparation because it has previously been shown that this type of nanoparticle is biodegradable, biocompatible, and in general well tolerated. The efficacy of this approach was first investigated by the study of the ocular biodistribution of fluorescent polyethylene glycolcoated nanoparticles (NP-PEG-F) and then by the evaluation of the pharmacological efficacy of intravitreal administration of tamoxifen loaded onto PEG-nanoparticles (NP-PEG-TAM), in rats with EAU.

2 Results 2.1 NP-PEG-F is found distributed within ocular tissues after intravitreal injection In nonimmunized rats (Fig. 1a–c), from 8 to 24 h postinjection, free nanoparticles were seen to disperse from the vitreous body and to accumulate along the internal limiting membrane of the retina (Fig. 1b) and in the ciliary body (Fig. 1a). After 24 h, evidence of phagocytosis of NP-PEG-F was observed in astrocytes along the internal limiting membrane of the retina (Fig. 1c). Then, the nanoparticles diffused through the retina to reach the retinal pigment epithelium (RPE) by 3 days. Nanoparticles that had been internalized by ED2+ resident tissue macrophages were detected in the choroid (Fig. 1b) and in the ciliary body (Fig. 1a). Phagocytosis of nanoparticles by a small number of ED1+ macrophages, present mainly in the anterior segment of the eye, was also observed from 8 h (not shown). This observation suggested that the nanoparticles might have a modest pro-inflammatory effect. In rats with EAU (Fig. 1d–l), substantial numbers of free nanoparticles were seen from 8 h after intravitreal injection dispersing throughout the anterior and posterior segments of the eye. Free nanoparticles infiltrated the f 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Fig. 1. Ocular biodistribution of NP-PEG-F 24 h after intravitreal injection of nonimmunized controls (panels a–c) and 8 h (panels g–i) and 24 h (panels d–f, j–l) after injection on the day of clinical onset of EAU. Colocalization of NP-PEG-F (yellow) with numerous ED2+ resident macrophages cells in iris/ciliary body (panel a), in the choroid (panel b, arrows) and in GFAP+ astrocytes of the retina (panel c, arrowhead). Eight hours after injection of NP-PEG-F, numerous free nanoparticles (green) infiltrating the retina between the end feet (panels g–i) of the GFAP+ (red) Mu¨ller glial cells (panel g). At 24 h after injection, NP-PEG-F accumulate in cells of the trabecular meshwork (panel d, arrow), in the epithelium of the ciliary body (panel f, arrow), and in the RPE (panels j–l, arrows in k, l). Phagocytosis of NP-PEG-F by inflammatory cells is shown by the colocalization with ED1+ macrophages, ED2+ resident macrophages, and OX42+ macrophages/microglia markers (yellow) in the anterior segment tissues (panels d–f) and in the posterior segment mainly in the subretinal space (panels j–l). Abbreviations used within panels: c, cornea; t, trabeculum; i, iris; ah, aqueous humor, cb, ciliary body; r, retina, ch, choroid; srs, subretinal space. Original magnification (panels a–c) 500. Scale bars in confocal microscope digital images: panels d, k, 50 lm; panels e, f, g–i, l, 15 lm; panel j, 145 lm; panel j (insert, 5 lm.

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retina by migrating in between the end feet of the activated glial fibrillary acidic protein (GFAP)+ Mu¨ller glial cells (Fig. 1g–i). At 24 h, nanoparticles crossed the edematous retina, accumulating in the subretinal space and within RPE (Fig. 1j–l). No free nanoparticles were detected in the choroid (Fig. 1j, k). Large numbers of free nanoparticles accumulated in the anterior chamber of the eye (Fig. 1d–f). Phagocytosis of NP-PEG-F by resident ocular cells, including the corneal endothelial cells (not shown), the ciliary epithelial cells (Fig. 1f), and cells of the trabecular meshwork (Fig. 1d), was obvious. Evidence of phagocytosis of NP-PEG-F was detected in numerous ED1+ and OX42+ inflammatory cells and ED2+ resident cells in the iris and ciliary body (Fig. 1e, f), in the subretinal space (Fig. 1j), and in the photoreceptor cell layer (Fig. 1k, l). At days 3 and 9 post-injection, accumulation of nanoparticles in the RPE, trabecular meshwork, and ED1+ and OX42+ cells were still detectable in ocular tissues (not shown). To detect a possible systemic spread of NP-PEG-F, NPPEG-F were injected into the vitreous body, 1 day before expected clinical onset of EAU. Three days later, nanoparticles were detected in cervical lymph nodes as free nanoparticles or phagocytosed in ED1+, ED2+ macrophages. Rare nanoparticles were detected in the spleen and the liver, but not in inguinal lymph nodes (not shown).

2.2 Intravitreal injection of NP-PEG-TAM is an effective treatment of EAU 2.2.1 Clinical observation The injection of NP-PEG-TAM performed on day 13 after immunization, i.e. 1–2 days before the expected onset of EAU in controls, led to a significant delay (p=0.006) of disease onset in treated rats (mean onset time 16.0.2 days; n=10 rats) compared to control eyes (14.40.2 days; n=12 rats). The disease severity was significantly inhibited (p=0.007) by the treatment up to 17 days after immunization, indicating that local intraocular therapy was very effective (Fig. 2A). NP-PEG-injected rats presented a higher ocular inflammation than rats injected with pyrogen-free sterile water, but this difference never reached significance, suggesting that the contribution of NP-PEG to EAU was low. The inhibitory effect of NP-PEG-TAM treatment in female rats was similar to the one obtained in male rats (data not shown).

f 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Fig. 2. Effect on clinical EAU of intravitreal injection of (A) NPPEG-TAM (10 ll containing 0.25 lg of tamoxifen) or (B) tamoxifen (10 ll/eye containing 0.20 lg of tamoxifen) performed on day 13 post-immunization. (A) Data are mean scores  SEM representing two separate experiments (n=8 rats in treated group, n=7 rats in control group). (B) Data are mean  SEM representative of two separate experiments (n=8 rats in treated group, n=9 rats in control group). Each point represents one rat (average of both eyes).

2.2.2 Histological and immunohistochemical analysis NP-PEG-TAM-treated rats presented low-grade EAU compared to lesions observed in control rats (mean EAU severity grade: 0.80.2, n=8 rats and 2.50.4, n=7 rats, respectively; p=0.0003). Rats injected with NP-PEG (Fig. 3A) showed a severe posterior uveitis with destruction of photoreceptor cell layer and part of the bipolar cells with inflammatory cell infiltration in the retina. Rats injected with NP-PEG-TAM (Fig. 3B) showed low level of inflammation and preservation of the photoreceptor cell layer, target of the immune reaction. Intravitreal injection of NP-PEG-TAM (Fig. 4A) reduced the level of OX6 (MHC class II) expression in OX62+ and OX42+ cells (dendritic cells, macrophages, and microglia) in ocular tissues compared to high expression of OX6 observed in control NP-PEG-injected rats (Fig. 4B).

2.3 Intravitreal injection of free tamoxifen had no effect on EAU Injection of free tamoxifen performed 13 days after immunization had no effect (p=0.1) on the time of disease onset (mean disease onset time: 15.30.3 days in tamoxifen-treated rats versus 14.60.2 days in control). There was no effect on the severity of the

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Fig. 3. Histopathological features of EAU in rats that received an intravitreal injection of NP-PEG-TAM (10 ll containing 0.25 lg of tamoxifen) on day 13 after immunization compared to controls that received NP-PEG. (A) Controls presented an acute inflammatory cell infiltration in all layers of the retina, around retinal capillaries (arrow) with fibrin exsudate (white arrowhead), and a complete destruction of the photoreceptor cell layer and part of the bipolar cell layer (dark arrowhead). (B) Rats treated with one injection of NPPEG-TAM had few inflammatory cells in the retina and the subretinal space, and a preservation of the photoreceptor cell layer (asterisk) with partial loss of outer segments (arrow). Abbreviations: srs, subretinal space; r, retina; c, choroid; v, vitreous. Original magnification: 500.

clinical intraocular inflammation (Fig. 2B). At the time of maximal severity in controls, mean clinical EAU severity score was 3.560.3 versus 3.70.1 in controls (p=0.6). Histologically, free tamoxifen had no inhibitory effect on EAU (mean severity grade in treated rats: 2.40.2 versus 2.50.0 in controls; p=0.8).

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Fig. 4. Immunohistochemical expression of MHC class II (OX6) in inflammatory cell infiltration (A) in rats treated with intravitreal injection of NP-PEG-TAM and (B) in controls that received NP-PEG. Expression of MHC class II was determined by immunostaining with mouse anti-rat OX6 (green) in macrophages/microglia (OX42, red) and dendritic cells (OX62, red). In treated rats (panels a–d), inflammatory cells infiltrated the ocular tissues and expressed OX6 (yellow) with low intensity. In contrast, in all control ocular tissues (panels e–h), high numbers of inflammatory cells show intense colocalization of OX42 and OX62 markers with OX6 (yellow). Abbreviations: ah, aqueous humor; cb, ciliary body; r, retina; v, vitreous. Scale bars: 30 lm; confocal microscope digital images.

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Fig. 5. Tamoxifen is acting as a partial antagonist to E2. Effect of intravitreal injection performed on day 13 post-immunization of 10 ll/eye of NP-PEG-TAM loaded with 0.25 lg of tamoxifen associated with 2 lg of E2 (n=4 rats), or 10 ll of NP-PEG associated with 2 ll of E2 (n=4 rats) on clinical EAU compared to injection of NP-PEG-TAM (n=3 rats) and NPPEG (n=6 rats). Data are mean scores  SEM. Each point represents one rat (average of both eyes).

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Fig. 6. Effect of NP-PEG-TAM treatment on cytokine mRNA expression in rat eyes. mRNA expression was evaluated by semiquantitative PCR. Total mRNA was isolated from the whole eye at day 13 after immunization and reversetranscribed into cDNA. The PCR fragments were analyzed by agarose gel electrophoresis. Results obtained in two rats in each group are shown and are representative of results found in four rats per group. Agarose gel electrophoresis of the PCR products is presented below in (1): TNF-a, 295 bp; IL-1b, 264 pb; RANTES, 211 bp; TGF-b2, 315 bp; in (2): bactin, 245 bp.

2.4 Tamoxifen is acting as a partial antagonist to the estrogen receptors

2.6 Effect of NP-PEG-TAM treatment on the immune response to S-Ag

The injection of NP-PEG with 2 lg of 17b-estradiol (E2) significantly increased EAU scores compared to rats injected with NP-PEG alone at day 15, the time of maximal severity of EAU (p=0.02). This suggests that E2 could be responsible for an increase of Th1 immunity. NP-PEG-TAM injection significantly reduced EAU scores (day 15: p=0.05) compared to injection of NP-PEG-TAM with 2 lg of E2, suggesting that tamoxifen is acting as a partial antagonist to E2 (Fig. 5).

2.6.1 Delayed-type hypersensitivity Rats treated with NP-PEG-TAM (Fig. 7) showed significant inhibition (p=0.009) of ear swelling at 48 h after SAg challenge (0.70.04 mm thickness) compared to controls injected with NP-PEG (0.90.06 mm thickness), who showed ear swelling response typical of delayed-

2.5 Analysis of cytokine mRNA expression in the eye Expression of cytokine mRNA was investigated by semiquantitative reverse transcription (RT)-PCR analysis of eyes from four rats in each group taken at days 16–17. Results in two controls showing EAU grade 3.5–4 and two treated animals presenting no sign of EAU are shown in Fig. 6. When compared to controls, NP-PEG-TAM treatment reduced TNF-a (p=0.03), IL-1b (p=0.02), and RANTES (p=0.02) mRNA expression and increased TGFb2 mRNA levels, but the difference to controls was not significant.

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Fig. 7. DTH to S-Ag in treated and control rats. Rats were challenged with 10 lg of S-Ag in one ear and with saline in the other ear, 17 days after immunization. Specific ear swelling was calculated at 48 h after S-Ag challenge, as the difference in thickness (mm) between the two ears. Each point represents one rat. www.eji.de

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type hypersensitivity (DTH). Tamoxifen-treated rats and rats treated with NP-PEG-TAM + E2 showed ear swelling (1.10.04 mm and 0.950.03 mm thickness, respectively) that was not different from controls injected with sterile water and NP-PEG + E2 (0.90.07 mm and 1.050.06 mm thickness, respectively; p=0.1 and 0.3).

2.6.2 Lymphocyte proliferation Lymphocyte proliferation test (Fig. 8A) showed that NPPEG-TAM treatment induced a decrease of lymphocyte proliferation in the presence of 50, 5, and 0.5 lg/ml S-Ag compared to controls (p=0.03, 0.03, and 0.05, respectively). In contrast, the lymphocyte proliferation in response to Con A was identical in treated and control rats (Fig. 8A).

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2.6.3 Cytokine production In the supernatants of lymph node cells stimulated with S-Ag (50 lg/ml), 34% inhibition of IFN-c production in the presence of S-Ag was found in treated rats compared to controls, indicating a decrease in type-1 response. No difference in IFN-c production in the presence of Con A was found between treated rats and controls (Fig. 8B). SAg-induced IL-2 and IL-10 production by cells from treated rats was not different from that of controls. IL-4 was not detected in any group of rats (not shown). No cytokine could be detected in the sera (not shown).

2.6.4 Serum antibody isotypes Because IFN-c promotes antibody isotype switching to IgG2b, reflective of Th1 response, we checked whether the treatment would induce a switch to IgG1 in sera, reflective of Th2 response (Fig. 9). NP-PEG-TAM treatment induced a significant up-regulation of specific IgG1 isotype response (p=0.001) compared to NP-PEGinjected controls with no effect on IgG2b isotype.

3 Discussion Tamoxifen has been used for the treatment of selected patients with breast cancer for its anti-estrogenic effect [10, 11]. In autoimmune diseases, the role of estrogen is complex, inducing either increased susceptibility or protection depending on the level of hormone [19, 20]. In this report, we present data demonstrating, for the first time, that tamoxifen may be of great benefit in the treatment of EAU, a rat model of ocular inflammation, but only if the drug is loaded onto nanoparticles (NP-PEG-

Fig. 8. Proliferative response to S-Ag in inguinal lymph node cells from treated and control rats collected 16 days after immunization and pooled within each group. Triplicate cultures were stimulated in vitro with S-Ag (50, 5, 0.5 lg/ ml) and Con A (5 lg/ml). (A) Proliferation was measured after 60 h as [3H]thymidine uptake. The response from each group is expressed as cpm. (B) The effect of NP-PEG-TAM treatment on IFN-c production was evaluated in the supernatants of lymph node cells stimulated in vitro with S-Ag (50 lg/ml) or Con A (5 lg/ml) and measured by an ELISA kit. f 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Fig. 9. Anti-S-Ag antibody IgG1 and IgG2b isotypes were quantified by isotype-specific ELISA in the serum of treated (n=8), control (n=7), and normal (n=5) rats at day 16 after immunization. www.eji.de

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TAM). Indeed, no influence on EAU was observed after intravitreal treatment with free tamoxifen. This could be related to the encapsulation of molecules within biodegradable polymeric nanoparticles, which enables their progressive release and also prevents their in vivo degradation and rapid metabolization [18]. In order to determine the ocular tissues and cells concerned with tamoxifen delivery by nanoparticles, we injected NP-PEG-F into the vitreous of normal and EAU rats. In normal rats, relatively large numbers of nanoparticles penetrated the intraocular tissues from the vitreous body with a modest inflammatory reaction. NPPEG-F diffused through the retina to reach the RPE as previously reported [21]. An uptake of nanoparticles was observed at 24 h in retinal astrocytes and ED2+ uveal cells. NP-PEG-F accumulated in ocular tissue from 3 to 9 days in our study. In contrast, NP-PEG-F injected at the day of clinical onset of EAU were phagocytosed locally by the blood-borne ED1+ macrophages that had already begun to infiltrate all ocular tissues, and by activated resident ED2+ macrophages and OX42+ macrophages/microglial cells. Then free nanoparticles migrated into the ocular tissues, through the edematous retina to reach the subretinal space by 24 h accumulating at different sites, including the RPE, the ciliary epithelium, and the trabecular meshwork. The injection of NP-PEG-F into the inflamed eyes induced an earlier and more important accumulation of nanoparticles in ocular resident cells compared to injection into a normal eye. This could be related to the activation of these cells in inflammatory conditions. The detection of nanoparticles in ocular resident cells such as RPE and trabecular meshwork cells suggests that these cells may contribute to extended intraocular drug delivery. Since intravitreal injection of nanoparticles allowed the nanoparticles and macrophages loaded with nanoparticles to spread to all the ocular tissues within 24 h, treatment with NP-PEG-TAM was started on day 13 after immunization, i.e. 1–2 days before the expected disease onset as observed in control rats. Clinical and histopathological data showed that administration of NPPEG-TAM into the vitreous cavity significantly inhibited the course of the disease with a preservation of the retinal photoreceptors as compared to NP-PEG injection. Macrophages have been shown to play an important role in tissue destruction [2, 3]. It may be that macrophages containing NP-PEG-TAM release tamoxifen or its active products of degradation into the ocular tissues [22, 23]. Further, NP-PEG-TAM-containing macrophages could be deactivated and less deleterious for retinal tissues, as suggested by the reduction of f 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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inflammatory cytokines found in the eyes of the treated rats as reported below. The observation in the present study of the lack of effectiveness of NP-PEG-TAM injection performed at day 10 after immunization, i.e. too far in advance of the onset of the ocular inflammation, further indicates an important role of macrophages in the inhibitory effect of this treatment. Females develop increased immune responses compared to males that are responsible for enhanced development of autoimmunity. [24]. In the present study, E2 was observed to be responsible for an increase of EAU, which is in agreement with previous data [25] reporting that administration of E2 enhances antigenspecific CD4 T cell responses and promotes Th1 cell response. However, reduced clinical severity of experimental autoimmune encephalomyelitis and decreased secretion of Th1 cytokines were observed after feeding with E2, pointing out the complexity of the mechanisms at the origin of biological effects of estrogens in autoimmunity [26]. Tamoxifen is a non-steroidal estrogen receptor modulator which binds competitively with estrogen to the estrogen receptors [11, 13]. However, it can act as either estrogen agonist or antagonist depending on the target cells, the estrogen receptor subtype, and the promoter context [23, 24]. In our study, more severe disease was observed with E2 that was partially blocked with NP-PEG-TAM, suggesting that tamoxifen acts as a partial antagonist to E2. Tamoxifen treatment of experimental autoimmune diseases such as systemic lupus erythematosus (SLE) has been associated with the modulation of pro- and antiinflammatory cytokines [10, 12]. The treatment of adjuvant-induced arthritis in rats with an analogue of tamoxifen, idoxifene, has shown an anti-inflammatory effect associated with reduced serum IL-6 levels [27]. In addition, the intraperitoneal injection of E2 inhibited endotoxin-induced uveitis and reduced E-selectin and IL-6 mRNA levels in the iris-ciliary body [28]. In our study, NP-PEG-TAM treatment significantly downregulated TNF-a, IL-1b, and RANTES mRNA expression in the eye. These cytokines participate to the development of EAU, chemotaxis, and extravasation of inflammatory cells [29]. Inflammatory cytokines have been reported to induce the maturation of dendritic cells, resulting in an up-regulation of the MHC class II [14]. In the present study, the level of OX6 (MHC class II) expression on OX42+ and OX62+ cells was, indeed, substantially reduced in NP-PEG-TAM-treated rats compared to controls. Consequently, a reduction of local antigen presentation could occur, inhibiting infiltration of the eye by additional inflammatory cells. Furthermore, a trend to an increase of the immunosupwww.eji.de

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pressive agent TGF-b2 was also observed. This is consistent with the amelioration of EAU observed during pregnancy and attributed to elevated levels of TGF-b [30]. In our study, NP-PEG-F also accumulated in different ocular resident cells: in RPE and in epithelium of the iris, ciliary body, and in trabecular meshwork cells. The uptake of fluorescent-labeled antigen by cells present in the iris, the iridocorneal angle, the suprachoroidal space, and around limbal-episcleral vessels has been reported [31, 32]. These antigen-bearing cells would deliver signals at the origin of the phenomenon known as anterior chamber-associated immune deviation, a manifestation of the immune privilege of the eye that protects the ocular tissues from the deleterious effects of ocular inflammation [33]. These results prompted us to examine the immune response in treated rats. We found that intravitreal injection of NP-PEG-TAM reduced specific lymphocyte proliferation and IFN-c production by lymph nodes cells stimulated in vitro by S-Ag and in vivo by DTH test to SAg. Tamoxifen treatment had no effect on the lymphocyte proliferative response to Con A, suggesting that the treatment affected the antigen presentation. Interestingly estrogen receptors are expressed in ocular tissues [28, 34], in macrophages, and lymphocytes [35, 36]. Therefore, the interaction of tamoxifen with estrogen receptors in macrophages could have altered the antigen presentation that induces DTH T cells. The treatment also up-regulated anti-S-Ag antibody IgG1 isotype, suggesting an antibody class switch to Th2 response. Previous data demonstrated that antigens leave the eye in a soluble form contributing to deviant immune responses [31, 32]. In our study, NP-PEG-F injected into the vitreous body were not detected in inguinal lymph nodes (not shown). Tamoxifen released continuously from ocular resident cells could reach the immune system through the blood, thus contributing to the deviated form of the immune response to S-Ag. In conclusion, the use of intravitreal administration of tamoxifen loaded onto nanoparticles enabled us to decrease the amount of the drug delivered: 250 ng/eye compared to 200–800 lg/mice in experimental SLE [12]. This should reduce possible ocular side effects as observed after systemic administration of high doses of tamoxifen [16]. Although new drugs with unique therapeutic properties may be discovered and synthesized, their administration for the purpose of treating ocular inflammation remains a major challenge. The present study on EAU clearly demonstrates that the use of nanotechnologies for the delivery of immunoregulatory molecules may open new perspectives for the treatment of uveitis. f 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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4 Materials and methods 4.1 Nanoparticles preparation and localization within the eye The copolymer PEG-PHDCA was synthesized by condensation of methoxy poly(ethylene glycol) cyanoacetate with nhexadecyl cyanoacetate in ethanol, in the presence of formalin and pyrrolidine as described elsewhere [17, 18]. NPPEG were prepared by the nanoprecipitation technique. Preparations of NP-PEG-TAM were made in the same way as the NP-PEG suspensions, except that 200 lg of tamoxifen was added to the organic phase containing the copolymer. Suspensions of NP-PEG-TAM containing 5 mg/ml of copolymer and 25 lg/ml of tamoxifen in sterile water were prepared and stored at 4 C. The size of the nanoparticles was measured at 20 C by Quasi-Elastic Light Scattering using a Nanosizer Coulterj N4MD (Coulter Electronics, Inc., FL). The mean diameters of the NP-PEG and the NP-PEGTAM were 9529 nm and 11225 nm, respectively (analysis in triplicate). To determine the localization of the nanoparticles within the eye, fluorescent nanoparticles were prepared using a core of fluorescent polystyrene nanoparticles (0.2 lm, FluorobriteTM; Polysciences, Inc., Warrington, PA) coated with PEG-PHDCA copolymer [17].

4.2 Induction of EAU Male and female Lewis rats (Charles River, Saint-Aubin-le`sElbeuf, France), 8–11 weeks old, were immunized into both hind footpads with S-Ag purified from bovine retinas [37], emulsified (1:1) in CFA (Difco, Detroit, MI), supplemented with 250 lg of Mycobacterium tuberculosis H37Ra (Difco). The care and use of the animals was in compliance with institutional guidelines.

4.3 Biodistribution of nanoparticles during EAU Nanoparticles-Fluoresbrite (NP-PEG-F) were injected into the vitreous cavity of both eyes (10 ll/eye, n=7) on the day of clinical onset of EAU. Eyes were taken at 8 h, 24 h, 3 days, and 9 days. The same number of nonimmunized control rats were injected, and eyes were processed in the same manner. To detect a possible systemic spread of NP-PEG-F, nanoparticles were injected into the vitreous 1 day before expected clinical onset of EAU, and submandibular, cervical, and inguinal lymph nodes, spleen, and liver were collected 3 days later. The samples were processed and immunohistochemistry was performed [2].

4.4 EAU treatment The administration in both eyes of NP-PEG-TAM and tamoxifen (10 ll/eye containing 0.25 lg of tamoxifen), or www.eji.de

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NP-PEG and pyrogen-free sterile water as controls, respectively, was performed by intravitreal injection in five separate experiments (n=4–5 rats/group) using male Lewis rats and one using female Lewis rats. Eyes were taken for analysis at days 16–19 after immunization (i.e. 2–5 days after disease onset in controls). Several experimental conditions were tested.

mild to (2) moderate impairment of pupillary dilatation (posterior synechiae); (3) severe posterior synechiae with fibrin and cellular infiltration of the anterior chamber of the eye (hypopion); and (4) no dilation of the iris, hypopion, and a fibrin plug within the pupil, impeding view of the vitreous body.

In an initial set of experiments, we tested two treatment protocols: (A) on day 10 after immunization, one intravitreal injection of NP-PEG-TAM or control NP-PEG; and (B) on day 13 after immunization (i.e. 1–2 days before expected disease onset, occurring at median day 14 in NP-PEG-injected controls), one intravitreal injection of NP-PEG-TAM or control NP-PEG.

4.5.2 Histopathology and EAU grading

The injection of NP-PEG-TAM performed on day 10 after immunization did not result in EAU protection in treated rats (n=3) versus controls (n=3) (mean histological severity grade = 2.830.3 versus 3.10.2, respectively; p=0.6; not shown). In the subsequent treatment experiments, injection of NPPEG-TAM was performed on day 13. To determine the efficacy of tamoxifen encapsulation into nanoparticles, injection of 10 ll/eye containing 0.20 lg of tamoxifen and pyrogen-free sterile water as control was performed at day 13 after immunization in two separated experiments. Comparison between NP-PEG-TAM and tamoxifen was done in the same experiment. After enucleation, the eyes were fixed in paraformaldehyde or Bouin’s solution for immunohistochemistry and histopathology, respectively, or were taken for RT-PCR analysis of cytokine mRNA expression. To determine whether tamoxifen is acting as an agonist or antagonist to the estrogen receptors, rats were injected into the vitreous with one of the following: 10 ll containing NPPEG-TAM loaded with 0.25 lg of tamoxifen associated with 2 lg of E2 benzoate, or 10 ll of NP-PEG with 2 lg of E2 benzoate. E2 benzoate was chosen because of its better water solubility than E2, the dose used corresponding to the maximum allowed according to the water solubility of this molecule. These groups of rats were compared to rats injected with NP-PEG and NP-PEG-TAM. To determine whether the gender of the animals would have an influence on the therapeutic effect, intravitreal treatment with NP-PEGTAM on day 13 after immunization was also investigated in female rats.

4.5 Clinico-pathological assessment of EAU 4.5.1 Clinical examination Eyes were examined daily from day 10 after immunization with a slit-lamp biomicroscope [37, 38]. Briefly, the ocular inflammation was scored from 0 to 4 as follows: (0) normal iris dilation after instillation of a mydriatic drug (Tropicamide 1%) and no inflammatory cells in the intraocular media; (1) f 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

At the time of sacrifice, i.e. 17–19 days after immunization, enucleated rat eyes were processed, paraffin sections cut through the pupillary-optic nerve plane, and stained with hematoxylin-eosin for histological evaluation [37]. Sections were examined by a masked investigator who scored the severity of EAU on a semiquantitative scale from 0 to 7 [38] as follows: (0) no tissue destruction and (1) to (7) limited to total destruction of the various layers of the retina: (1–2) destruction of outer segments of rods and cones, (3–4) destruction of the outer nuclear layer, (5–6) destruction of the inner nuclear layer, and (7) destruction of the ganglion cell layer.

4.5.3 Immunohistochemistry Eyes were processed, cryostat sections cut and stained for cell surface markers as described previously [2], using mAb (Serotec, Oxford, UK) diluted 1:100: ED1 (macrophages and dendritic cells), ED2 (resident tissue macrophages), OX42 (C3bi receptor on macrophages, microglia, dendritic cells, and polymorphonuclear leukocytes), OX62 (veiled dendritic cells expressing the subunit a E2 of integrin) followed by Texas Red-labeled anti-mouse IgG (H+L) (Jackson ImmunoResearch, West Grove, PA). MHC class II expression was detected by double immunostaining for cell markers (ED1, ED2, OX42, or OX62) followed by biotin-labeled anti-rat OX6 (Serotec), and identified by fluorescein-conjugated streptavidin (Amersham, Little Chalfont, UK). Control sections, incubated without primary antibodies, were negative. GFAP expression was determined with rabbit polyclonal anti-GFAP (Dakopatts, Trappes, France), then incubated with Texas Red-conjugated anti-rabbit IgG (Jackson ImmunoResearch), both diluted 1:100. The sections were examined with an optical microscope (Microphot-FXA; Nikon, Tokyo, Japan) and viewed with appropriate filters of a photomicroscope (FXA; Nikon). Confocal micrographs were obtained with a scanning laser confocal microscope system (LSM 510; Carl Zeiss, Oberkochen, Germany).

4.6 RNA isolation and reverse transcription PCR Total RNA was isolated from enucleated eyes using the acid guanidinium thiocyanate-phenol-chloroform method, as www.eji.de

Eur. J. Immunol. 2004. 34: 3702–3712 described previously [38]. TNF-a sense and anti-sense primers were obtained from Clontech Laboratories (Palo Alto, CA) and the PCR amplification was performed according to the manufacturer’s instructions; IL-1b, IL-6, IL-10, RANTES, TGF-b1, TGF-b2, IFN-c, MCP-1, MIP-1a, Eselectin, and b-actin sense and anti-sense primers were obtained from GENSET (Paris, France). These primers were designed to specifically amplify cDNA fragments representing mature mRNA transcripts of 295 bp for TNF-a, 264 bp for IL-1b, 211 bp for RANTES, 315 bp for TGF-b2, and 245 bp for b-actin. The PCR fragments were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. The b-actin gene was used to assess the amount and integrity of RNA samples. The quantities of cytokine mRNA present were expressed relative to the quantity of the housekeeping gene b-actin. These data were obtained by determining the ratio of intensity of signals obtained by scanning the bands of the cytokine and b-actin amplified fragments in agarose gel using the NIH1.57 software package.

Treatment of EAU by tamoxifen-loaded nanoparticles

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4.8 Statistical analysis Data are presented as mean  standard error of the mean (SEM). Statistical analysis of EAU scoring was performed using the non-parametric Mann-Whitney sum test, or the Kruskal-Wallis non-parametric ANOVA test, followed by the Bonferroni multiple comparison test. Analysis of lymphocyte proliferation, cytokine production, and anti-S-Ag serum antibodies was performed using Student’s t-test. A p value, adjusted by a multiple comparison test, was calculated for each experiment. Values of p less than or equal to 0.05 were considered statistically significant.

Acknowledgements: This work was supported by grants from INSERM (Institut National de la Sante´ et de la Recherche Me´dicale), CNRS (Centre National de la Recherche Scientifique), and Association Retina France. We thank Drs. Pilar Calvo and Ire`ne Brigger for their participation to this work. We wish to express our gratitude to Dr. Claudie Verwaerde and Dr. Justine Smith for revising the manuscript and to Dr. Michel Renoir for helpful discussion on the effect of tamoxifen and estradiol receptors.

4.7 Immune response estimation DTH was estimated by ear assay measuring the specific antiS-Ag response [8]. Lymphocyte proliferation was measured by [3H]thymidine incorporation by antigen-stimulated T cells from inguinal lymph nodes [37]. Lymph node cells were pooled within the groups from two treated rats that presented a clinical inhibition of EAU (grade 0) by intravitreal injection of NP-PEG-TAM and two control rats that presented a clinical EAU (grade 3.5–4) by intravitreal injection of NP-PEG. Cytokine production was measured by ELISA kits [8], according to the manufacturer’s instructions (BioSource International, Inc., Camarillo, CA). The limit of sensitivity of the ELISA test is