2998 Hongmei Fu et al. Eur. J. Immunol. 2011. 41: 2997–3005 which are important in vascular homeostasis [11] and macro- phage cytotoxicity [12]. The two identiﬁed arginase isoforms, arginase I and arginase II, differ in tissue distribution, subcellular localization and regu- lation [13]. Arginase-mediated L-Arg depletion has been shown to down-regulate T-cell functions in various systems. Thus, tumour- associated myeloid cells express arginase I, and inhibition of the enzyme can slow tumour growth, suggesting that arginase I in the tumour microenvironment may be important for tumour escape [14, 15]. T-cell functions are also suppressed by human granu- locyte arginase [16]. In human pregnancy, arginase activity is enhanced in both the peripheral blood and term placenta, resulting in T-cell hyporesponsiveness [17]. In this study, we investigated the hypothesis that arginase is involved in the maintenance of immune privilege in the eye. Our data show that both murine corneal endothelium and epithelium express functional arginase capable of down-regulating T-cell proliferation in an in vitro culture system. In a murine model of corneal allograft transplantation, inhibition of arginase in vivo accelerated corneal allograft rejection, but had no effect on the survival of transplanted donor skin that expresses little arginase. Therefore, arginase plays an important role in the maintenance of immune privilege in the eye by contributing to allograft acceptance. Results Functional arginase is expressed in murine corneas We examined the expression of arginase in whole murine corneas. BALB/c corneas were homogenized and tissue extracts were assessed for arginase expression at the mRNA and protein levels by real-time PCR and Western blotting, respectively. The expression of arginase was compared with that in liver, heart, lung, kidney, spleen and intestine. Arginase I mRNA was detectable in resting murine corneas (Fig. 1A). The level of arginase I in resting corneas was nearly a tenth of that in liver, which has the highest expression of arginase I. No expression of arginase I mRNA was found in heart, lung, spleen and intestine. The mRNA expression of arginase I was consistent with arginase I protein expression shown by Western blot using anti-arginase I antibody (Fig. 1B). In contrast, we were unable to detect arginase II mRNA or protein expression in the cornea. Strongest arginase II expression was seen in the intestine, followed by kidney and liver (Fig. 1A and B). To test whether the arginase present in the corneas was functional, we measured arginase enzymatic activity in murine corneas and compared it with other tissues. Arginase activity in unmodiﬁed resting murine corneas was easily detectable. It was lower than that in liver, intestine and kidney, but signiﬁcantly higher than that in heart, lung and spleen (Fig. 1C). To address, in part, the question of whether arginase function in vivo in the cornea might be altered during inﬂammation, corneas were cultured in the presence of murine cytokines IL-4, & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim TNF-a and IFN-g. These are reported to induce arginase expression in murine macrophages [18]. A marginal increase in the mRNA expression of arginase I was seen in corneas stimulated with either IL-4 or TNF-a, but no change was seen with IFN-g (Fig. 1D). No arginase II expression was seen under any conditions. Immunohistochemistry was used to localize expression of arginase I and II in the cornea. Staining for arginase I but not arginase II was found in the endothelium and epithelium of the cornea (Fig. 1E). This was conﬁrmed using RT-PCR analysis, which showed that arginase I mRNA was present in both the epithelium and endothelium of the murine cornea (Fig. 1F). Arginase in corneas has inhibitory effect on T-cell proliferation To examine the functional effect of corneal arginase, corneas were homogenized and the tissue extracts were added to puriﬁed CD41 T cells stimulated with anti-CD3 and anti-CD28 beads in the presence or absence of competitive arginase inhibitors, N-hydroxy-nor-L-arginine (nor-NOHA), (S)-(2-Boronoethyl)-L- cysteine (BEC) or 0.1 mM L-arginine. 3H thymidine was added to T cells on day 4 and cells were harvested 16 h later. As shown in Fig. 2, addition of corneal extracts inhibited the proliferation of the T cells; this inhibition could be partially restored by addition of either nor-NOHA, BEC or L-arginine (po0.05). These data indicate that arginase in corneal extracts is, in part, responsible for the observed inhibition of T-cell proliferation. A similar result was also seen with liver extracts. Addition of nor-NOHA or BEC had no effect on T-cell proliferation in the absence of tissue extracts. In contrast, addition of nor-NOHA had no effect on the inhibition of T-cell proliferation caused by lysates of heart tissue. Inhibition of arginase shortens corneal allograft survival Having found that the cornea expresses arginase I, which inhibits in vitro T-cell proliferation, we then tested the possibility that functional arginase expressed by the cornea might prevent or delay corneal allograft rejection. As arginase knock-out mice do not live beyond 12 days [19], we used a competitive arginase inhibitor, nor-NOHA, in vivo. C57BL/6 (B6) strain mouse corneas were grafted as orthotopic transplants to BALB/c recipients randomized to receive daily intraperitoneal injections of either nor-NOHA at 100 mg/kg/day or control PBS from day 1 to day 21 post-transplantation. The rejection of B6 corneas in the nor-NOHA treatment group was accelerated compared with that of control group (p 5 0.014). Graft rejection onset ranged from day 11 to day 35, with 22% long-term survival. In contrast, control graft rejection ranged between days 25 and 33 with 61% long-term survival (Fig. 3A). Syngeneic grafts survived indeﬁnitely. www.eji-journal.eu