Th1/Th2-regulated expression of arginase isoforms in murine macrophages and dendritic cells.

Activated murine macrophages metabolize arginine by two alternative pathways involving the enzymes inducible NO synthase (iNOS) or arginase. The balance between the two enzymes is competitively regulated by Th1 and Th2 T helper cells via their secreted cytokines: Th1 cells induce iNOS, whereas Th2 cells induce arginase. Whereas the role of macrophages expressing iNOS as inflammatory cells is well established, the functional competence of macrophages expressing arginase remains a matter of speculation. Two isoforms of mammalian arginases exist, hepatic arginase I and extrahepatic arginase II. We investigated the regulation of arginase isoforms in murine bone marrow-derived macrophages (BMMPhi) in the context of Th1 and Th2 stimulation. Surprisingly, in the presence of either Th2 cytokines or Th2 cells, we observe a specific induction of the hepatic isoform arginase I in BMMPhi. Induction of arginase I was shown on the mRNA and protein levels and obeyed the recently demonstrated synergism among the Th2 cytokines IL-4 and IL-10. Arginase II was detectable in unstimulated BMMPhi and was not significantly modulated by Th1 or Th2 stimulation. Similar to murine BMMPhi, murine bone marrow-derived dendritic cells, as well as a dendritic cell line, up-regulated arginase I expression and arginase activity upon Th2 stimulation, whereas arginase II was never detected. In addition to revealing the unexpected expression of arginase I in the macrophage/monocyte lineage, these results uncover a further intriguing parallelism between iNOS and arginase: both have a constitutive and an inducible isoform, the latter regulated by the Th1/Th2 balance.     

Enhancer-mediated control of macrophage-specific arginase I expression

Arginase I expression in the liver must remain constant throughout life to eliminate excess nitrogen via the urea cycle. In contrast, arginase I expression in macrophages is silent until signals from Th2 cytokines such as IL-4 and IL-13 are received and the mRNA is then induced four to five orders of magnitude. Arginase I is hypothesized to play a regulatory and potentially pathogenic role in diseases such as asthma, parasitic, bacterial, and worm infections by modulating NO levels and promoting fibrosis. We show that Th2-inducible arginase I expression in mouse macrophages is controlled by an enhancer that lies -3 kb from the basal promoter. PU.1, IL-4-induced STAT6, and C/EBPbeta assemble at the enhancer and await the effect of another STAT6-regulated protein(s) that must be synthesized de novo. Identification of a powerful extrahepatic regulatory enhancer for arginase I provides potential to manipulate arginase I activity in immune cells while sparing liver urea cycle function.     

Gliadin activates arginase pathway in RAW264.7 cells and in human monocytes

Celiac disease (CD) is an autoimmune enteropathy triggered in susceptible individuals by the ingestion of gliadin-containing grains. Recent studies have demonstrated that macrophages play a key role in the pathogenesis of CD through the release of inflammatory mediators such as cytokines and nitric oxide (NO). Since arginine is the obliged substrate of iNOS (inducible nitric oxide synthase), the enzyme that produces large amount of NO, the aim of this work is to investigate arginine metabolic pathways in RAW264.7 murine macrophages after treatment with PT-gliadin (PTG) in the absence and in the presence of IFNγ. Our results demonstrate that, besides strengthening the IFNγ-dependent activation of iNOS, gliadin is also an inducer of arginase, the enzyme that transforms arginine into ornithine and urea. Gliadin treatment increases, indeed, the expression and the activity of arginase, leading to the production of polyamines through the subsequent induction of ornithine decarboxylase. This effect is strengthened by IFNγ. The activation of these pathways takes advantage of the increased availability of arginine due to a decreased system y⁺l-mediated efflux, likely ascribable to a reduced expression of Slc7a6 transporter. A significant induction of arginase expression is also observed in human monocytes from healthy subject upon treatment with gliadin, thus demonstrating that gluten components trigger changes in arginine metabolism in monocyte/macrophage cells.     

Inhibition of arginase I activity by RNA interference attenuates IL-13-induced airways hyperresponsiveness

Increased arginase I activity is associated with allergic disorders such as asthma. How arginase I contributes to and is regulated by allergic inflammatory processes remains unknown. CD4+ Th2 lymphocytes (Th2 cells) and IL-13 are two crucial immune regulators that use STAT6-dependent pathways to induce allergic airways inflammation and enhanced airways responsiveness to spasmogens (airways hyperresponsiveness (AHR)). This pathway is also used to activate arginase I in isolated cells and in hepatic infection with helminths. In the present study, we show that arginase I expression is also regulated in the lung in a STAT6-dependent manner by Th2-induced allergic inflammation or by IL-13 alone. IL-13-induced expression of arginase I correlated directly with increased synthesis of urea and with reduced synthesis of NO. Expression of arginase I, but not eosinophilia or mucus hypersecretion, temporally correlated with the development, persistence, and resolution of IL-13-induced AHR. Pharmacological supplementation with l-arginine or with NO donors amplified or attenuated IL-13-induced AHR, respectively. Moreover, inducing loss of function of arginase I specifically in the lung by using RNA interference abrogated the development of IL-13-induced AHR. These data suggest an important role for metabolism of l-arginine by arginase I in the modulation of IL-13-induced AHR and identify a potential pathway distal to cytokine receptor interactions for the control of IL-13-mediated bronchoconstriction in asthma.     

Mitochondrial adenine nucleotide translocator 3 is regulated by IL-4 and IFN-gamma via STAT-dependent pathways

IL-4 and IFN-γ are prototypical Th2 and Th1 cytokines, respectively. They reciprocally regulate a number of genes involved in Th1 vs Th2 immune balance. Using DD-PCR analysis, adenine nucleotide translocase (ANT) 3, an enzyme which exchanges ATP and ADP through mitochondrial membrane, has been identified as a novel target counter-regulated by IL-4 and IFN-γ. We have observed that IL-4 and IFN-γ each up-regulates ANT3 in T cells both at mRNA and protein levels, while cotreatment of IL-4 and IFN-γ counter-regulates ANT3 expression. In contrast, other isoforms of ANT were not affected by IL-4 or IFN-γ. Emplyoing transfection and overexpression of STAT6 and STAT1 in STAT-deficient cells, we demonstrate that induction of ANT3 by IL-4 and IFN-γ proceeds via pathways involving STAT6 and STAT1, respectively. Furthermore, regulation of ANT3 expression by IL-4 and IFN-γ correlated with the modulation T cell survival by these cytokines from dex-induced apoptosis. Considering the critical role of mitochondrial ANTs in energy metabolism and apoptosis, ANT3 regulation by IL-4 and IFN-γ may have a functional implication in cytokine-mediated T cell survival.     

Distribution and properties of arginase in the salivary glands of four species of laboratory mammals

Important progress in arginine metabolism includes the discovery of widespread expression of two isoforms of arginase, arginase I and II, not only in hepatic cells but also in non-hepatic cells, and the formation of nitric oxide, a widely distributed signal-transducing molecule, from arginine by nitric oxide synthase. Possible physiological roles of arginase may therefore include regulation of nitric oxide synthesis through arginine availability for nitric oxide synthase. In this paper, arginase was investigated in the submandibular, sublingual, and parotid glands of rat, mouse, guinea pig, and rabbit. From their arginase contents, the salivary glands of these species were divided into two groups. Variable levels of arginase activity were detected in the salivary glands of mouse and rat. However, salivary glands of rabbit and guinea pig had almost no arginase activity. The presence of nitric oxide synthase has been reported in all the salivary glands used in this study. Therefore, one of the important findings was the presence of species specificity in the co-localization of arginase and nitric oxide synthase in the salivary glands of the four species. The highest specific activity of arginase was found in mouse parotid gland. In rat, considerable arginase activity was detected in all three glands, at 3.6–7.3% of that in rat liver. In rat submandibular gland, arginase was detected in both cytosolic and particulate fractions. In addition, arginase was detected in isolated acinar cells, but not in duct cells. Experiments on the intracellular distribution and the effects of the arginase inhibitors ornithine and N-hydroxy-L-arginine (NOHA), suggested the presence of both arginase I and arginase II in rat submandibular gland.Abbreviations cGMP cyclic guanosine 3,5-monophosphate - NO nitric oxide - NOHA N-hydroxy-L-arginine - NOS nitric oxide synthase Communicated by I.D. Hume     

Cutting edge: Stat6-dependent substrate depletion regulates nitric oxide production

The cytokines IL-4 and IL-13 inhibit the production of NO from activated macrophages through an unresolved molecular mechanism. We show here that IL-4 and IL-13 regulate NO production through depletion of arginine, the substrate of inducible NO synthase (iNOS). Inhibition of NO production from murine macrophages stimulated with LPS and IFN-gamma by IL-4 or IL-13 was dependent on Stat6, cell density in the cultures, and pretreatment for at least 6 h. IL-4/IL-13 did not interfere with the expression or activity of iNOS but up-regulated arginase I (the liver isoform of arginase) in a Stat6-dependent manner. Addition of exogenous arginine completely restored NO production in IL-4-treated macrophages. Furthermore, impaired killing of the intracellular pathogen Toxoplasma gondii in IL-4-treated macrophages was overcome by supplementing L-arginine. The simple system of regulated substrate competition between arginase and iNOS has implications for understanding the physiological regulation of NO production.     

Control of Early Theiler's Murine Encephalomyelitis Virus Replication in Macrophages by Interleukin-6 Occurs in Conjunction with STAT1 Activation and Nitric Oxide Production

During Theiler's murine encephalomyelitis virus (TMEV) infection of macrophages, it is thought that high interleukin-6 (IL-6) levels contribute to the demyelinating disease found in chronically infected SJL/J mice but absent in B10.S mice capable of clearing the infection. Therefore, IL-6 expression was measured in TMEV-susceptible SJL/J and TMEV-resistant B10.S macrophages during their infection with TMEV DA strain or responses to lipopolysaccharide (LPS) or poly(I · C). Unexpectedly, IL-6 production was greater in B10.S macrophages than SJL/J macrophages during the first 24 h after stimulation with TMEV, LPS, or poly(I · C). Further experiments showed that in B10.S, SJL/J, and RAW264.7 macrophage cells, IL-6 expression was dependent on extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) and enhanced by exogenous IL-12. In SJL/J and RAW264.7 macrophages, exogenous IL-6 resulted in decreased TMEV replication, earlier activation of STAT1 and STAT3, production of nitric oxide, and earlier upregulation of several antiviral genes downstream of STAT1. However, neither inhibition of IL-6-induced nitric oxide nor knockdown of STAT1 diminished the early antiviral effect of exogenous IL-6. In addition, neutralization of endogenous IL-6 from SJL/J macrophages with Fab antibodies did not exacerbate early TMEV infection. Therefore, endogenous IL-6 expression after TMEV infection is dependent on ERK MAPK, enhanced by IL-12, but too slow to decrease viral replication during early infection. In contrast, exogenous IL-6 enhances macrophage control of TMEV infection through preemptive antiviral nitric oxide production and antiviral STAT1 activation. These results indicate that immediate-early production of IL-6 could protect macrophages from TMEV infection.     

Cell proliferation and STAT6 pathways are negatively regulated in T cells by STAT1 and suppressors of cytokine signaling

Suppressor of cytokine signaling (SOCS) proteins have emerged as important regulators of cytokine signals in lymphocytes. In this study, we have investigated regulation of SOCS expression and their role in Th cell growth and differentiation. We show that SOCS genes are constitutively expressed in naive Th cells, albeit at low levels, and are differentially induced by Ag and Th-polarizing cytokines. Whereas cytokines up-regulate expression of SOCS1, SOCS2, SOCS3, and cytokine-induced Src homology 2 protein, Ags induce down-regulation of SOCS3 within 48 h of Th cell activation and concomitantly up-regulate SOCS1, SOCS2, and cytokine-induced Src homology 2 protein expression. We further show that STAT1 signals play major roles in inducing SOCS expression in Th cells and that induction of SOCS expression by IL-4, IL-12, or IFN-gamma is compromised in STAT1-deficient primary Th cells. Surprisingly, IL-4 is a potent inducer of STAT1 activation in Th2 but not Th1 cells, and SOCS1 or SOCS3 expression is dramatically reduced in STAT1(-/-) Th2 cells. To our knowledge, this is the first report of IL-4-induced STAT1 activation in Th cells, and suggests that its induction of SOCS, may in part, regulate IL-4 functions in Th2 cells. In fact, overexpression of SOCS1 in Th2 cells represses STAT6 activation and profoundly inhibits IL-4-induced proliferation, while depletion of SOCS1 by an anti-sense SOCS1 cDNA construct enhances cell proliferation and induces constitutive activation of STAT6 in Th2 cells. These results are consistent with a model where IL-4 has dual effects on differentiating T cells: it simulates proliferation/differentiation through STAT6 and autoregulates its effects on Th2 growth and effector functions via STAT1-dependent up-regulation of SOCS proteins.