Eur. J. Immunol. 2011. 41: 2997–3005 Immunomodulation 3001 Figure 3. Inhibition of arginase shortens corneal allograft survival but not skin graft survival. (A) BALB/c (H2d) mice received unilateral C57BL/6 (H2b) donor corneal allografts and intraperitoneal injections of either nor- NOHA at 100 mg/kg/day (n 5 14) or PBS (n 5 13) from day 1 to day 21. Pooled results from two independent experiments are shown. Allograft survival was assessed by determining the opacity of the cornea using an objective scale, as described in [44]. (B) RT-PCR analysis of arginase expression in donor tail skin and recipient shank skin. cDNA was prepared from tissues, and PCR was carried out using HPRT, arginase I and II primers and the product was run on a 1.5% agarose gel. Data are representative of three independent experiments. (C) B6 female mice received B6 male donor skin allografts and intraperitoneal injections of either nor-NOHA at 100 mg/kg/day (n 5 8) or PBS (n 5 8) from day 1 to day 21. absence of nor-NOHA. There are conﬂicting reports regarding the role of arginase in regulating NO synthesis. Some studies suggest that arginase activity regulates the availability of L-Arg as a substrate for NOS in endothelial cells [33, 34], while others show arginase activity has no effect on NO production in macrophages [35]. These differences may reﬂect whether arginine is limiting in particular systems. In higher organisms, the control of amino acid metabolism is a strategy for limiting the unwanted proliferation of cells, including T cells [36]. L-Arg is not the only amino acid with effects on the immune system. Indoleamine 2,3-dioxygenase (IDO) is another enzyme metabolizing the essential amino acid L-tryptophan that can modulate the function of the immune system [37]. IDO has been involved in the maintenance of immune privilege in the placenta [38]. We previously found expression in murine cornea of functional IDO; overexpression of IDO in the donor corneas prolonged allograft survival. However, it is noteworthy that inhibition of IDO had no effect on corneal allograft survival [39]. This report of arginase activity is another example of the ability of the eye to inhibit the proliferation of inﬁltrating T cells by enzyme-mediated amino acid starvation, contributing to the immune privilege of corneal allografts. Materials and methods Cell culture SV-40-immortalized BALB/c corneal endothelial cells (MCECs) [40], a gift of Dr J. Y. Niederkorn, Dallas, TX, were maintained in Eagle’s MEM supplemented with 1% (vol) penicillin and streptomycin and non-essential amino acids (Gibco-BRL, Paisley, UK) and 10% heat-inactivated FCS (Gibco-BRL) [22]. RNA extraction and reverse transcription Corneal or other tissues were ground using the beads and reagent supplied with the Fast RNA Pro Green Kit (QBiogene, Carlsbad, CA, USA). Corneal tissue was separated by dissection surgically into epithelium, and endothelium. RNA extraction and quantiﬁ- cation was performed as previously described [21]. Quantitative PCR As arginase and NOS share L-arginine as their primary substrate [11], it is possible that inhibition of arginase might increase the production of NO. Inﬂammatory cytokines induce apoptosis of corneal endothelial cells in an NO-dependent manner [22], and it is conceivable that arginase inhibition might potentiate this effect. However, there was no difference in either NO levels generated by MCEC cells, or in their apoptosis, following stimulation by a cocktail of cytokines in the presence or The PCR protocol was performed using an Applied Biosystems machine and the Power SYBR Green mastermix (Applied Biosystems, UK) with an initial denaturation step at 951C for 5 min followed by 40 cycles of ampliﬁcation (denaturation at 951C for 5 s, annealing at 601C for 10 s, elongation at 721C for 13 s), and quantiﬁcation at 811C [39]. Quantiﬁcation of arginase I mRNA was carried out using the primers 50-ACCTGGC CTTTGTTGATGTC-30 (forward) and 50-ACTGCCAGACTGTGGTCTCC-30 (reverse) spanning 160 bp of & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu