L-arginine metabolism in myeloid cells controls T-lymphocyte functions

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L-arginine metabolism in myeloid cells controls T-lymphocyte functions Vincenzo Bronte1, Paolo Serafini1, Alessandra Mazzoni2, David M. Segal2 and Paola Zanovello1 1 2

Department of Oncology and Surgical Sciences, Via Gattamelata 64, 35128 Padua, Italy Experimental Immunology Branch, NCI-NIH, NIH, 9000 Rockville Pike, Bldg 10, Room 4B36 Bethesda, MD 20892-1360, USA

Although current attention has focused on regulatory T lymphocytes as suppressors of autoimmune responses, powerful immunosuppression is also mediated by a subset of myeloid cells that enter the lymphoid organs and peripheral tissues during times of immune stress. If these myeloid suppressor cells (MSCs) receive signals from activated T lymphocytes in the lymphoid organs, they block T-cell proliferation. MSCs use two enzymes involved in arginine metabolism to control T-cell responses: inducible nitric oxide synthase (NOS2), which generates nitric oxide (NO) and arginase 1 (Arg1), which depletes the milieu of arginine. Th1 cytokines induce NOS2, whereas Th2 cytokines upregulate Arg1. Induction of either enzyme alone results in a reversible block in T-cell proliferation. When both enzymes are induced together, peroxynitrites, generated by NOS2 under conditions of limiting arginine, cause activated T lymphocytes to undergo apoptosis. Thus, NOS2 and Arg1 might act separately or synergistically in vivo to control specific types of T-cell responses, and selective antagonists of these enzymes might prove beneficial in fighting diseases in which T-cell responses are inappropriately suppressed. This Opinion is the second in a series on the regulation of the immune system by metabolic pathways. It has been well established that intense burdens on the immune system induces a profound immunosuppression of adaptive responses. The principal mediator of this suppression is a specialized myeloid suppressor cell (MSC) that expresses the Gr-1 and CD11b markers. Increased numbers of Gr-1þCD11bþ MSCs are recruited to secondary lymphoid organs of mice under conditions associated with impaired immune reactivity, such as severe parasitosis, bacterial infections, exposure to superantigens and immunization with strong recombinant vaccines [1]. MSCs are generated from bone-marrow hemopoietic precursors in response to several cytokines, including granulocyte – macrophage-colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and vascular endothelial growth factor (VEGF), which are released systemically under these conditions [1]. MSCs represent a phenotypically heterogeneous cell population that includes mature and immature myeloid cells as well as cells expressing immature Corresponding author: Vincenzo Bronte ([email protected]).

dendritic cell (DC) markers [2 – 5]. Whereas DCs control peripheral tolerance to tissue-specific antigens under steady-state conditions [6], MSCs act as sensors of T-cell activation during heightened immune responses. Indeed, MSCs block T-cell expansion only after receiving a triggering signal from activated T lymphocytes. MSCs negatively regulate the development of cytotoxic T lymphocytes (CTLs) and are frequently found systemically in mice bearing advanced tumors, where they have been hypothesized to favor tumor escape from immune recognition [1– 4]. MSCs are also detected in tumor infiltrates and inhibit effector phase lytic functions of CD8þ tumorinfiltrating lymphocytes [7]. L-arginine metabolism in MSCs: arginase and nitric oxide synthase Recent studies of the inhibitory pathways involved in MSC-mediated immune suppression have shown that MSCs exploit the metabolism of L-arginine (L-Arg) to render lymphocytes unresponsive to antigen stimulation. L-Arg is metabolized by myeloid cells (macrophages, granulocytes and DCs) by two enzymes: (1) nitric oxide synthase (NOS), which oxidizes L-Arg in two steps that generate NO and citrulline; and (2) arginase, which converts L-Arg into urea and L-ornithine [8,9] (Fig. 1). Separate genes encode three different isoforms of NOS in mammalian cells: NOS1 and NOS3 are constitutively expressed in neuronal tissue and endothelium, respectively, and NOS2, the inducible form of NOS, is expressed in multiple tissues and cell types, including vascular endothelium, DCs and macrophages. Induction of NOS2 is controlled mainly by Th1 cytokines, such as interferon-g (IFN-g) and tumor necrosis factor-a (TNF-a). The enzymatic activity of NOS2 is sustained over a long period of time, enabling the production of 1000-fold higher amounts of NO than those produced by NOS1 and NOS3 [9]. Two distinct isoforms of arginase, encoded by two genes, are found in mammalian cells: type I arginase (Arg1), a cytosolic enzyme expressed at high levels in the liver as a component of the urea cycle, and type II arginase (Arg2), a mitochondrial enzyme expressed at lower levels in kidney, brain, small intestine and mammary gland, with limited expression in liver. Both isoforms are expressed in murine macrophages: Arg1 is induced by Th2 cytokines including IL-4, IL-13, transforming growth factor-b (TGF-b) and IL-10, whereas Arg2 is induced by lipopolysaccharide

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Fig. 1. L-arginine metabolism in myeloid cells. Unbroken lines indicate activation whereas broken lines indicate inhibition. Pathways activated by Th1-type cytokines are indicated in blue; in red are the Th2-dependent pathways. Abbreviations: Arg1, arginase 1; IFN-g, interferon-g; IL-4, interleukin-4; NO, nitric oxide; NOHA, NG-hydroxyarginine; NOS2, inducible nitric oxide synthase; ODC, ornithine decarboxylase; TNF-a, tumor necrosis factor-a; TGF-b, transforming growth factor-b.

(LPS), cAMP and intact bacteria [8,10]. Discrepancies have been reported on the ability of LPS to induce Arg1, which probably reflect the use of either macrophage-like cell lines or primary cultured macrophages [10 –12]. NOS2 blocks T-cell function by interfering with the IL-2 pathway Induction of NOS2 alone in MSCs, with subsequent release of NO, is responsible for inhibition of T-cell responses in some experimental settings, demonstrated by complete reversal of immunosuppression with specific NOS2 inhibitors [13– 16]. Moreover, NO, when added directly to cultures, is an extremely potent inhibitor of T-cell proliferation. IFN-g released by activated T lymphocytes and a yet to be identified contact between T lymphocytes and MSCs is necessary for NO production and inhibition of immune functions [16 – 18]. In some models, the complete dependence on NOS2 and NO for suppression has been unequivocally established by experiments showing that inhibitory cells from NOS2 deficient mice are devoid of immunosuppressive properties [13,14,16,19]. We, and others, have shown that NO does not impair the early events triggered by T-cell receptor (TCR) crosslinking, but acts instead at the level of IL-2 receptor signaling, blocking the phosphorylation and activation of several signaling molecules, including Janus kinases (JAKs) 1 and 3, STAT5 (signal transducer and activator of transcription 5), Erk and Akt [13,16] (Fig. 2a). Previous studies demonstrate that JAK3 is oxidized and inactivated following direct exposure to NO [20], and the enzymatic activities of several other intracellular signaling proteins are also negatively regulated by NO either directly, by S-nitrosylation of crucial cysteine residues, or indirectly, through activation of guanylyl cyclase [8]. http://treimm.trends.com

NO-dependent inhibition of T-cell proliferation is reversible during the first 24 – 48 h of stimulation [16], suggesting that this inhibitory pathway prevents activated T lymphocytes from entering the cell cycle without killing them (Fig. 2a). However, sustained release or elevated concentrations of NO at the tumor site might results in apoptosis of infiltrating T lymphocytes [21]. Arg1 depletes L-Arg from the local environment Depletion of nutrients, such as L-Arg, is a strategy used throughout nature to control growth of organisms competing for the same biological niche. Lower organisms exploit L-Arg starvation as a strategy for survival, by using their own arginase or taking advantage of the derived enzyme of the host (reviewed in Ref. [22]). Higher organisms have evolved to use localized L-Arg starvation to control the growth of particular cell types, for example, T lymphocytes in higher vertebrates. L-Arg depletion is mediated by arginases, a family of enzymes that is highly conserved across species. In this regard, myeloid arginase behaves as the macrophage enzyme indoleamine 2,3-dioxygenase (IDO) that metabolizes the amino acid tryptophan. IDO is responsible for tolerance induction by macrophages and, as more recently described, by a subpopulation of mouse DCs [23 – 25]. These mechanisms have been reviewed in another article of the series ‘Regulation of the immune system by metabolic pathways’ in Trends in Immunology [26]. Amino acid-depleting enzymes are thus potential regulators of immunity. In myeloid cells, coordinated induction of Arg1 and a cationic amino acid transporter by IL-4 and IL-13 conveys L-Arg from the extracellular milieu into the cells where it is degraded [27] (Fig. 2b). The first evidence that arginase could have a role in suppression of T-lymphocyte responses

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Fig. 2. L-Arg metabolism in MSCs and regulation of T-lymphocyte activation. According to the prevailing cytokine response, NOS2, Arg1 or both enzymes are induced in myeloid cells and restrain T-lymphocyte activation. (a) IFN-g released by Th1 lymphocytes induces NOS2 and its product, NO. By interfering with the IL-2R signaling pathway, NO prevents proliferation but does not block the initial TCR-dependent events of activation, such as CD69 expression and IL-2 release. This block is reversible within the first day or two of stimulation. (b) Th2 cells secrete IL-4 and IL-13, which induce in MSCs the expression of Arg1 and the transporter CAT-2B. MSCs then deplete the local environment of L-Arg, which results in an impaired expression of the CD3 z chain, particularly in CD8þ T lymphocytes. The signal transduction from the TCR is thus altered leading to a proliferative arrest. (c) When both enzymes are activated, in addition to the previously described pathways, production of ONOO2 is also triggered. By nitrating tyrosines on several proteins, peroxynitrites induce T-lymphocyte apoptosis. Abbreviations: Arg1, type 1 arginine; CAT-2B, cationic amino acid transporter 2B; Cyt C, cytochrome C; IFN-g, interferon-g; IL-4, interleukin-4; IL-2R, IL-2 receptor; L-Arg, L-arginine; MSCs, myeloid suppressor cells; NO, nitric oxide; NOS2, inducible nitric oxide synthase; ONOO2, peroxynitrite; STAT5, signal transducer and activator of transcription 5; TCR, T-cell receptor.

by myeloid cells was the discovery that macrophagemediated suppression was accompanied by a profound depletion of L-Arg in the supernatants of alloantigenstimulated T lymphocytes and that suppression was reversed by adding L-Arg to the medium [28]. Like the suppressor macrophages of this earlier study, we found that a cloned MSC line, which constitutively expressed Arg1 (but not Arg2), blocked alloreactive T lymphocytes through a pathway that was inhibited by either Nv-hydroxy-L-arginine (NOHA, a selective inhibitor of arginases) or by addition of excess L-Arg to the culture (P. Serafini, unpublished). Proliferating mouse CD8þ T lymphocytes are more sensitive to L-Arg depletion than CD4þ T lymphocytes. In the CD8þ population, ample L-Arg is necessary for CD3 and CD8 expression, optimal use of IL-2 and the development of a memory population [29]. Human Jurkat T lymphocytes cultured in the absence of L-Arg lose expression of the CD3 z chain, the principal signal transduction element of the TCR, and undergo a decrease in cell proliferation [30]. This suggests that depletion of L-Arg in the microenvironment would block TCR signaling and modulate T-cell function at an early stage of lymphocyte activation (Fig. 2b). Loss of CD3 z chain in peripheral blood lymphocytes (PBLs) has been reported in patients under conditions that might lead to http://treimm.trends.com

Arg1 activation, including tumor growth, overwhelming infections, liver transplantation and trauma [30,31]. Although decreased levels of L-Arg have been detected in wounds, in liver transplanted individuals and in patients with acute bacterial peritonitis ([32,33] and discussed in Ref. [30]), it remains to be determined whether L-Arg depletion in vivo is sufficient to affect T-lymphocyte functions. Arg1 and NOS2 synergism in MSC-dependent suppression In macrophages, Thl cytokines induce NOS2 but inhibit Arg1, whereas the reverse is true for Th2 cytokines [34,35]. This reciprocal regulation is also promoted by biochemical feedback mechanisms: NOHA, a byproduct of the conversion of L-Arg to NO by NOS2, inhibits arginase [36], whereas L-Arg depletion by Arg1 can limit NOS2dependent synthesis of NO. Also, polyamines, final products of the Arg1 –ornithine decarboxylase (ODC) pathway, downregulate NOS2 expression [12,37] (Fig. 1). Because Th1 cytokines polarize macrophages to a cytotoxic, inflammatory ‘M1’ phenotype, and Th2 cytokines induce an anti-inflammatory, ‘M2’ phenotype, it has been suggested that NOS2 and arginase pathways operate in distinct macrophage subsets [38,39]. However, the

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paradigm of a strict separation of Arg1 and NOS2 in myeloid subpopulations is challenged by studies with cloned cell lines. LPS treatment of either mouse or rat macrophages upregulated both NOS2 and Arg1 [11,12], suggesting that under some circumstances both enzymes can work in the same cellular environment. More importantly, cloned MSC lines require both Arg1 and NOS2 to block allogeneic T-cell responses in tumor-bearing mice [18]. The requirement for both enzymes is puzzling because if arginase acts by depleting L-Arg as an essential nutrient of T lymphocytes, then it should also block NOS2 by depleting one of its substrates. However, it has been established that under conditions of low L-Arg concentrations, the reductase domain of NOS2 generates superoxide ion (O2 2 ) [40,41]. This has been demonstrated in macrophages in which targeted expression of Arg1 increased the production of O2 2 , which was blocked by a selective inhibitor of the NOS2 reductase domain. This inhibitor also restored alloreactive T-cell responses in mice bearing a highly immunosuppressive colon carcinoma [18]. In a different colon carcinoma model, T-cell activation by CD3 and CD28 stimulation was impaired by Gr-1þ cells isolated from the spleen or bone marrow of tumor-bearing mice. The responsiveness was restored by either depletion of Gr-1þ cells or by addition of a NOS2 inhibitor together with a superoxide dismutase mimetic able to scavenge O2 2 [42]. These results are consistent with a model in which Arg1 lowers the L-Arg concentration in the microenvironment, thus inducing NOS2 to produce O2 2 in addition to NO, the exclusive product of NOS2 at higher L-Arg concentrations. NO reacts with O2 2 , giving rise to peroxynitrite (ONOO2), a highly reactive oxidizing agent that nitrates tyrosines on proteins. Peroxynitrites can induce apoptosis in T lymphocytes by inhibiting activationinduced protein tyrosine phosphorylation [43] or by nitrating a component of the mitochondrial permeability transition pore, which causes release of death-promoting factors, such as cytochrome C [44] (Fig. 2c). Conclusion and therapeutic perspectives It is probable that the main function of MSCs in vivo is in the restraint of runaway immune responses, however, in some cases, MSC might favour disease progression, for example, MSCs frequently hinder anti-tumor T-cell responses. Drugs controlling Arg1 and NOS2, the enzymes mediating suppression in MSCs, might, therefore, represent a novel class of immune modulators that would act by limiting the effects of MSC activity in vivo. Such drugs could be used in combination with cancer vaccines for the treatment of advanced neoplastic diseases or to restore immune function in diseases in which the immune system has been inappropriately suppressed. Because L-Arg is a substrate for both arginase and NOS, the synthesis of inhibitors affecting both enzymes should be a realistic task, providing that ways of regulating immunity without blocking essential metabolic pathways (e.g. the urea cycle) could be found. Novel L-Arg analogues are currently being developed as isozyme-specific inhibitors for either NOS or arginases [36,45]. Such inhibitors would be expected to have few side effects, and in view of the different susceptibilities of T-cell subsets to arginase and NO, it might http://treimm.trends.com

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prove possible to relieve suppression of specific T-cell compartments with isozyme-specific antagonists. References 1 Bronte, V. et al. (2001) Tumor-induced immune dysfunctions caused by myeloid suppressor cells. J. Immunother. 24, 431 – 446 2 Bronte, V. et al. (1998) Apoptotic death of CD8þ T lymphocytes after immunization: induction of a suppressive population of Mac-1þ/Gr-1þ cells. J. Immunol. 161, 5313 – 5320 3 Bronte, V. et al. (1999) Unopposed production of granulocyte – macrophage colony-stimulating factor by tumors inhibits CD8þ T cell responses by dysregulating antigen-presenting cell maturation. J. Immunol. 162, 5728– 5737 4 Bronte, V. et al. (2000) Identification of a CD11bþ/Gr-1þ/CD31þ myeloid progenitor capable of activating or suppressing CD8þ T cells. Blood 96, 3838 – 3846 5 Kusmartsev, S. and Gabrilovich, D.I. (2002) Immature myeloid cells and cancer-associated immune suppression. Cancer Immunol. Immunother. 51, 293 – 298 6 Steinman, R.M. and Nussenzweig, M.C. (2002) Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc. Natl. Acad. Sci. U. S. A. 99, 351– 358 7 Radoja, S. et al. (2001) CD8(þ) tumor-infiltrating T cells are deficient in perforin-mediated cytolytic activity due to defective microtubule-organizing center mobilization and lytic granule exocytosis. J. Immunol. 167, 5042– 5051 8 Wu, G. and Morris, S.M. Jr (1998) Arginine metabolism: nitric oxide and beyond. Biochem. J. 336, 1 – 17 9 Bogdan, C. (2001) Nitric oxide and the immune response. Nat. Immunol. 2, 907 – 916 10 Morris, S.M. Jr et al. (1998) Differential regulation of arginases and inducible nitric oxide synthase in murine macrophage cells. Am. J. Physiol. 275, E740 – 747 11 Salimuddin, A. et al. (1999) Regulation of the genes for arginase isoforms and related enzymes in mouse macrophages by lipopolysaccharide. Am. J. Physiol. 277, E110– 117 12 Sonoki, T. et al. (1997) Coinduction of nitric-oxide synthase and arginase I in cultured rat peritoneal macrophages and rat tissues in vivo by lipopolysaccharide. J. Biol. Chem. 272, 3689– 3693 13 Bingisser, R.M. et al. (1998) Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway. J. Immunol. 160, 5729 – 5734 14 Koblish, H.K. et al. (1998) Immune suppression by recombinant interleukin (rIL)-12 involves interferon g induction of nitric oxide synthase 2 (iNOS) activity: inhibitors of NO generation reveal the extent of rIL-12 vaccine adjuvant effect. J. Exp. Med. 188, 1603– 1610 15 Angulo, I. et al. (2000) Early myeloid cells are high producers of nitric oxide upon CD40 plus IFN-g stimulation through a mechanism dependent on endogenous TNF-a and IL-1a. Eur. J. Immunol. 30, 1263– 1271 16 Mazzoni, A. et al. (2002) Myeloid suppressor lines inhibit T cell responses by an NO-dependent mechanism. J. Immunol. 168, 689 – 695 17 Apolloni, E. et al. (2000) Immortalized myeloid suppressor cells trigger apoptosis in antigen-activated T lymphocytes. J. Immunol. 165, 6723– 6730 18 Bronte, V. et al. (2003) IL-4-induced arginase 1 suppresses alloreactive T cells in tumor-bearing mice. J. Immunol. 170, 270 – 278 19 Goni, O. et al. (2002) Immunosuppression during acute Trypanosoma cruzi infection: involvement of Ly6G (Gr1(þ))CD11b(þ ) immature myeloid suppressor cells. Int. Immunol. 14, 1125– 1134 20 Duhe, R.J. et al. (1998) Nitric oxide and thiol redox regulation of Janus kinase activity. Proc. Natl. Acad. Sci. U. S. A. 95, 126 – 131 21 Saio, M. et al. (2001) Tumor-infiltrating macrophages induce apoptosis in activated CD8(þ) T cells by a mechanism requiring cell contact and mediated by both the cell-associated form of TNF and nitric oxide. J. Immunol. 167, 5583– 5593 22 Vincendeau, P. et al. (2003) Arginases in parasitic diseases. Trends Parasitol. 19, 9 – 12 23 Mellor, A.L. and Munn, D.H. (1999) Tryptophan catabolism and T-cell tolerance: immunosuppression by starvation? Immunol. Today 20, 469– 473 24 Fallarino, F. et al. (2002) Functional expression of indoleamine

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2,3-dioxygenase by murine CD8a(þ) dendritic cells. Int. Immunol. 14, 65 – 68 Grohmann, U. et al. (2002) CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat. Immunol. 3, 1097 – 1101 Grohmann, U. et al. (2003) Tolerance, DCs and tryptophan: much ado about IDO. Trends Immunol. 24 Louis, C.A. et al. (1999) Regulation of arginase isoforms I and II by IL-4 in cultured murine peritoneal macrophages. Am. J. Physiol. 276, R237 – 242 Kung, J.T. et al. (1977) Suppression of in vitro cytotoxic response by macrophages due to induced arginase. J. Exp. Med. 146, 665– 672 Ochoa, J.B. et al. (2001) Effects of L-arginine on the proliferation of T lymphocyte subpopulations. JPEN J. Parenter. Enteral Nutr. 25, 23 – 29 Rodriguez, P.C. et al. (2002) Regulation of T cell receptor CD3z chain expression by L-arginine. J. Biol. Chem. 277, 21123– 21129 Kiessling, R. et al. (1999) Tumor-induced immune dysfunction. Cancer Immunol. Immunother. 48, 353– 362 Shearer, J.D. et al. (1997) Differential regulation of macrophage arginine metabolism: a proposed role in wound healing. Am. J. Physiol. 272, E181 – 190 Suh, H. et al. (1997) Decreased L-arginine during peritonitis in ESRD patients on peritoneal dialysis. Adv. Perit. Dial. 13, 205 – 209 Munder, M. et al. (1998) Alternative metabolic states in murine macrophages reflected by the nitric oxide synthase/arginase balance: competitive regulation by CD4þ T cells correlates with Th1/Th2 phenotype. J. Immunol. 160, 5347 – 5354 Mills, C.D. et al. (2000) M-1/M-2 macrophages and the Th1/Th2 paradigm. J. Immunol. 164, 6166– 6173

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36 Colleluori, D.M. and Ash, D.E. (2001) Classical and slow-binding inhibitors of human type II arginase. Biochemistry 40, 9356 – 9362 37 Mossner, J. et al. (2001) Concomitant down-regulation of L-arginine transport and nitric oxide (NO) synthesis in rat alveolar macrophages by the polyamine spermine. Pulm. Pharmacol. Ther. 14, 297 – 305 38 Mantovani, A. et al. (2002) Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 23, 549– 555 39 Gordon, S. (2003) Alternative activation of macrophages. Nat. Rev. Immunol. 3, 23 – 35 40 Xia, Y. and Zweier, J.L. (1997) Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc. Natl. Acad. Sci. U. S. A. 94, 6954 – 6958 41 Xia, Y. et al. (1998) Inducible nitric-oxide synthase generates superoxide from the reductase domain. J. Biol. Chem. 273, 22635 – 22639 42 Kusmartsev, S.A. et al. (2000) Gr-1þ myeloid cells derived from tumorbearing mice inhibit primary T cell activation induced through CD3/CD28 costimulation. J. Immunol. 165, 779– 785 43 Brito, C. et al. (1999) Peroxynitrite inhibits T lymphocyte activation and proliferation by promoting impairment of tyrosine phosphorylation and peroxynitrite-driven apoptotic death. J. Immunol. 162, 3356– 3366 44 Aulak, K.S. et al. (2001) Proteomic method identifies proteins nitrated in vivo during inflammatory challenge. Proc. Natl. Acad. Sci. U. S. A. 98, 12056 – 12061 45 Vallance, P. and Leiper, J. (2002) Blocking NO synthesis: how, where and why? Nat. Rev. Drug Discov. 1, 939 – 950

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