3000 Hongmei Fu et al. Eur. J. Immunol. 2011. 41: 2997–3005 Figure 2. Effect of corneal arginase on T-cell proliferation. Tissues from cornea, heart and liver were homogenized and the supernatants (10 mL) were added to puriﬁed CD41 T cells stimulated with anti-CD3 and -CD28 beads in the presence or absence of the arginase inhibitors, nor-NOHA, BEC, or 0.1 mM L-arginine in 96-well plates. T-cell prolifera- tion was determined by 3H thymidine incorporation after 3 days ( po0.05, Student’s t-test). Data are representative of three independent experiments, and in each group triplicates were performed. Error bars indicate SD. The inhibition of arginase does not alter NO- dependent apoptosis of corneal endothelial cells Non-dividing corneal endothelial cells are critical for the maintenance of corneal transparency and thus are essential to graft survival [21]. We have shown that the pro-inﬂammatory cytokines, IL-1, TNF-a and IFN-g, induce apoptosis of corneal endothelium through an NO-dependent pathway [22]. Since arginase and NOS share the same substrate [11], it is possible that the arginase inhibition would result in an increase in NO production through the NOS pathway, thus accelerating graft destruction. To investigate this, murine corneal endothelial cells (MCECs) were exposed to different concentrations of cytokines in the presence of either the arginase inhibitor nor-NOHA, or the NOS inhibitor L-NAME (5 mM). NO level in the supernatant of cultured cells was subsequently measured. As shown in Fig. 5A, the cytokines increased NO production by MCECs, as previously shown. L-NAME signiﬁcantly reduced the production of NO; however, nor-NOHA did not alter the production of NO. Therefore, L-Arg was probably not limiting for NO production by NOS in this setting. As expected, considerable cell apoptosis was observed in MCECs stimulated with IL-1, TNF-a and IFN-g. The NOS inhibitor L-NAME, but not the arginase inhibitor nor- NOHA, inhibited cytokine-induced apoptosis (Fig. 5B). Discussion Corneal transplantation is highly successful without tissue matching and systemic immunosuppressive therapy due to the immune privilege of the eye, with around 90% of grafts surviving 1 year, though this drops to 75% at 5 years [23]. Lack of lymphatic drainage [3], the atypical nature of the APCs within & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim the cornea [7], limited MHC expression [5, 6], FasL expression [4], the tendency to induce anterior chamber-associated immune deviation [8] and immunosuppressive cytokines [24, 25] have all been associated with immune privilege. Here, we reveal for the ﬁrst time that the murine cornea expresses functional arginase I that plays an important role in the maintenance of immune privilege of corneal allografts. Arginase is expressed in various tissues and organs. Consistent with previous reports [26], we found that the murine liver expresses high levels of arginase I and low levels of arginase II. The kidney, intestine and spleen express only arginase II. Argi- nase, by virtue of its ability to deplete arginine, can be an effector mechanism of the immune system. Arginase expression by tumour and human term placenta have been found to suppress T-cell responses [14, 17]. This is due to the L-Arg depletion by arginase. It has been demonstrated that L-Arg availability can modulate T-cell function. Arginase-mediated L-Arg depletion induces down-regulation of CD3z, the main signalling chain of TCR [27]. Therefore, arginase has been suggested to play a role in local immune suppression. This is the ﬁrst report of functional arginase I expression in the cornea and several lines of evidence suggest that arginase may be an additional factor in the comparative immune privilege of this tissue. We found that both corneal endothelium and epithelium expressed arginase I. We also found that tissue extracts from murine corneas inhibited T-cell proliferation, which could be partly reversed by either nor-NOHA or BEC or by supplementa- tion of the medium with L-arginine, thus demonstrating the presence of functional arginase in the cornea. When the compe- titive arginase inhibitor, nor-NOHA, was systemically adminis- tered, the rejection of corneal allografts was accelerated. Consistent with our ﬁnding, it has been reported that arginase I mRNA was present in murine cornea but arginase II mRNA was not detected. Expression of both arginase I and arginase II were elevated in the cornea at late stages of ocular herpes simplex virus type-1 infection [28]. Arginase can operate either locally or systemically to suppress the immune response [20]. Therefore, the shortening of graft survival may be due to systemic, rather than local, effects. Consistent with another report [29], there is no arginase in the murine skin, although expression has been seen in human skin [30]. However, in skin allografts, which do not express arginase I, there was no effect of nor-NOHA treatment. Furthermore, inhi- bition of arginase had no effect on the T-cell proliferation following systemic immunization. Therefore, it is likely that arginase is operating locally to down-regulate the rejection in the tissue, rather than affecting the priming or initial activation of the alloreactive T cells. This is the ﬁrst report of arginase function in transplantation immunology. While we have shown that arginase has a role in preventing corneal graft rejection, though not skin graft rejection, it is interesting to speculate that it may also have a role in other organ allografts. This is especially the case for the liver, which has high levels of arginase and allografts of which can undergo spontaneous acceptance in animal models [31, 32]. www.eji-journal.eu