HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 19/8/1000 04-3416fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kropf, P. Articles by Müller, I. Search for Related Content PubMed PubMed Citation Articles by Kropf, P. Articles by Müller, I.

Arginase and polyamine synthesis are key factors in the regulation of experimental leishmaniasis in vivo

Pascale Kropf^*, José M. Fuentes, Eva Fähnrich, Luis Arpa, Shanthi Herath^*, Verena Weber, Germán Soler^||, Antonio Celada^||, Manuel Modolell^,1 and Ingrid Müller^*,1,2

^* Department of Immunology, Faculty of Medicine, Imperial College London, UK;
Departamento de Bioquimica y Biologia Molecular, E. U. Enfermería y T. O., Universidad de Extremadura, Caceres, Spain;
Max-Planck-Insitute für Immunbiologie, Freiburg, Germany;
Group of Macrophage Biology, Barcelona Science Park, University of Barcelona, Spain; and
^|| Departamento de Bioquimica y Biologia Molecular, Facultad de Veterinaria, Universidad de Extremadura, Caceres, Spain

²Correspondence: Imperial College London, Department of Immunology, Norfolk Pl., London W2 1PG, UK. E-mail: i.muller{at}imperial.ac.uk

SPECIFIC AIMS

Arginase 1, an enzyme induced by Th2 cytokines, is a hallmark of alternatively activated macrophages and is responsible for the hydrolysis of L-arginine into ornithine, the building block for production of polyamines. Up-regulation of arginase 1 has been observed in a variety of diseases, but the mechanisms by which arginase contributes to pathology are not well understood. The aim of this study was to characterize the role for arginase 1 in the pathogenesis of nonhealing leishmaniasis, a prototype Th2 disease.

PRINCIPAL FINDINGS

1. The parasite load at the site of infection correlates with the activity of arginase
We first determined the levels of arginase activity at the site of pathology during the course of L. major infection in healer (CBA) and nonhealer (BALB/c) strains of mice. As shown in Fig. 1 A, B, CBA mice controlled cutaneous lesions and the parasite replication. The arginase activity at the site of lesions was detectable during the time of active disease, decreased with healing, and when the lesions were resolved and most of the parasites cleared, the arginase activity had almost reached background levels (Fig. 1C ). In sharp contrast, the size of the cutaneous lesions and the number of viable parasite in the lesions of BALB/c mice increased progressively with time until the lesions eventually ulcerated (Fig. 1A, B ). Strikingly, the uncontrolled parasite replication was paralleled by a strong increase in arginase activity (Fig. 1C ). These results show a clear, direct correlation between the parasite load and the arginase activity in the lesions of nonhealer mice.

View larger version (11K): [in this window] [in a new window]   Figure 1. Lesion development in leishmaniasis correlates with arginase activity. Groups of BALB/c and CBA mice (n=12) were infected with 2 x 106 L. major promastigotes in one hind footpad. A) The lesion development was monitored by measuring the increase in footpad thickness at regular intervals. B) The parasite load and (C) the arginase activity were determined in the lesions of BALB/c mice.

Since it is not possible to determine in vivo whether the arginase activity is due to the isoform 1 or 2 of the macrophages or to that of the parasite, we performed in vitro experiments with macrophages from BALB/c mice. IL-4 and, in particular, the combination of IL-4 and IL-10 induced pronounced arginase activities which were increased even further by L. major infection. However, only mouse arginase 1 mRNA was significantly increased, but not mouse arginase 2 mRNA or L. major arginase. These experiments show that most of the arginase activity is due to Th2 cytokine-induced arginase 1. To confirm the presence of arginase 1 in the lesions, we determined its expression by real time PCR and by Western blot and we showed that there was a clear increase in mouse arginase 1 in the footpads of infected BALB/c mice as compared with naive BALB/c mice. No increase in arginase 1 mRNA was detected in the footpads of infected CBA mice at this time point.

These results show that the high parasite burden found in the lesions of nonhealer BALB/c mice correlates with high arginase activity that is likely to be due to the inducible mouse arginase 1.

2. Inhibition of arginase activity results in reduced pathology and more efficient control of the parasite replication
To demonstrate that the high arginase activity is responsible for uncontrolled parasite replication in BALB/c mice, the activity of this enzyme was inhibited in vivo during the course of L. major infection by administering a selective competitive inhibitor of arginase, N-hydroxy-nor-L-arginine (nor-NOHA). In striking contrast to the progressive nonhealing lesion development of control infected BALB/c mice, BALB/c mice treated with nor-NOHA throughout the course of infection developed significantly smaller lesions (Fig. 2 A) and 32 days postinfection, one mouse of six developed an ulcer; all other mice showed no signs of pathology. The lesions of mice treated with nor-NOHA harbored significantly fewer parasites as compared with those of the control infected mice (Fig. 2B ), and the activity of arginase at the site of infection was significantly lower (Fig. 2C ).

View larger version (13K): [in this window] [in a new window]   Figure 2. Inhibition of arginase activity results in the control of lesion development and the parasite replication. One group of L. major infected BALB/c mice (n=6) was treated 5 times/wk with i.p. injection of 100 µg nor-NOHA and one group (n=6) was control infected. The lesion development was monitored by measuring the increase in footpad thickness (A). 32 days postinfection, the number of viable parasites (B) and the arginase activity (C) were determined in the lesions of individual mice.

No significant change in the production of L. major-specific IFN-, IL-4, or IL-10 was detected between the two groups of mice.

These results show that inhibition of arginase activity resulted in more efficient control of parasites, despite the presence of a polarized Th2 response, and they demonstrate that arginase is a key factor in the replication of L. major parasites.

3. Arginase activity and production of polyamines are key factors in the regulation of parasite growth
We then tested whether the competitive inhibitor of L-arginine, nor-NOHA, acts by preventing the hydrolysis of L-arginine into ornithine, thereby depriving the parasites of the main intracellular source for the synthesis of the polyamines required for their growth. A well-defined in vitro system of bone marrow-derived BALB/c macrophages was used to follow the catabolism of L-arginine.

We showed that alternative activation of macrophages leads to the consumption of L-arginine by arginase and that infection with L. major resulted in an additional increase of the L-arginine catabolism, which was inhibited by the addition of nor-NOHA.

This consumption of L-arginine correlated with the production of ornithine and spermine in macrophages. This process was controlled by arginase as addition of the arginase inhibitor nor-NOHA reduced the amount of ornithine and spermine to control levels. These results demonstrate that nor-NOHA inhibits the conversion of L-arginine into ornithine and spermine.

To evaluate directly the effects of the reduction in polyamine synthesis on parasite growth, the number of viable parasites was assessed. Inhibition of the arginase activity by nor-NOHA clearly reduced parasite replication as compared with the control group. These results show that arginase activity is essential for parasite growth; indeed, addition of ornithine in the presence of nor-NOHA circumvents the lack of arginase activity as evidenced by the restoration of L. major proliferation back to normal levels.

To determine whether the synthetic arginase inhibitor nor-NOHA acts exclusively on the arginase of the macrophages or whether it also impairs the arginase of the parasites, we determined its effects on the L-arginine metabolism of L. major promastigotes. We demonstrated that the ability of freshly isolated promastigotes to metabolize L-arginine and to synthesize ornithine remained largely unaffected in the presence of this inhibitor. In contrast, the naturally occurring endogenous inhibitor of arginase, N-hydroxy-arginine (OH-arg), inhibited the degradation of L-arginine and reduced the synthesis of ornithine. Both inhibitors were not toxic and did not affect the viability of the promastigotes during the duration of these experiments.

These results demonstrate that the observed therapeutic effects of nor-NOHA on the parasite replication are due to the inhibition of the arginase of host macrophages.

CONCLUSIONS AND SIGNIFICANCE

We show here a direct correlation between the unrestricted replication of L. major parasites in susceptible hosts, the pathology, and the increasing levels of arginase activity at the site of lesion. We demonstrate that the activity of arginase, one of the hallmarks of alternatively activated macrophages, is responsible for the uncontrolled growth of Leishmania parasites in vivo. Indeed, treatment of nonhealer mice with the competitive arginase inhibitor nor-NOHA, which targets only the arginase of the macrophage and not that of Leishmania parasites, ameliorated the lesion pathology and reduced the growth of the parasites in vivo. To address the underlying mechanisms, the arginase-induced L-arginine catabolism was investigated and the results show that arginase regulates parasite growth directly by affecting the polyamine synthesis in macrophages.

Polyamines are not only beneficial for the growth of Leishmania, they have wider effects such as affecting signaling transduction pathways and modulating immune functions: for example, spermine and spermidine have been shown to inhibit the secretion of proinflammatory cytokines and spermine counter-regulates the innate immune response induced by the TLR4 ligand lipopolysaccharide. Therefore, increased polyamine levels in L. major infections could contribute to the inefficient host defense in BALB/c mice in vivo.

The exact role of host arginase 1 in extrahepatic cells and tissues is not clear; however, arginase plays a role in a variety of diseases, such as asthma, cancer, psoriasis, schistosomiasis, and trypanosomiasis. Arginase 1 can directly and indirectly control specific T cell functions. Thus, it is tempting to speculate that in the nonhealing progressive form of leishmaniasis the increased arginase expression by macrophages at the site of infection and pathology could result in a down-modulation of the effector functions of the infiltrating T cells. This ability to modulate T cell effector functions could not only be relevant for leishmaniasis but could also have serious implications for all diseases in which the host defense depends on cell-mediated immune responses.

View larger version (18K): [in this window] [in a new window]   Figure 3. L-arginine metabolism. L-arginine is the substrate for both arginase and NOS2. Th1 cytokines induce NOS2, which metabolizes L-arginine in a two-step procedure into NO, the metabolite responsible for parasite killing. Arginase is induced by Th2 cytokines and hydrolyses L-arginine into ornithine, the first building block in the synthesis of polyamines.

FOOTNOTES

¹ These authors share senior authorship.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3416fje; doi: 10.1096/fj.04-3416fje

This article has been cited by other articles:

				U. Gaur, S. C. Roberts, R. P. Dalvi, I. Corraliza, B. Ullman, and M. E. Wilson An Effect of Parasite-Encoded Arginase on the Outcome of Murine Cutaneous Leishmaniasis J. Immunol., December 15, 2007; 179(12): 8446 - 8453. [Abstract] [Full Text] [PDF]

				U. Muller, W. Stenzel, G. Kohler, C. Werner, T. Polte, G. Hansen, N. Schutze, R. K. Straubinger, M. Blessing, A. N. J. McKenzie, et al. IL-13 Induces Disease-Promoting Type 2 Cytokines, Alternatively Activated Macrophages and Allergic Inflammation during Pulmonary Infection of Mice with Cryptococcus neoformans J. Immunol., October 15, 2007; 179(8): 5367 - 5377. [Abstract] [Full Text] [PDF]

				J. Ehrchen, L. Helming, G. Varga, B. Pasche, K. Loser, M. Gunzer, C. Sunderkotter, C. Sorg, J. Roth, and A. Lengeling Vitamin D receptor signaling contributes to susceptibility to infection with Leishmania major FASEB J, October 1, 2007; 21(12): 3208 - 3218. [Abstract] [Full Text] [PDF]

				F. J. S. Rocha, U. Schleicher, J. Mattner, G. Alber, and C. Bogdan Cytokines, Signaling Pathways, and Effector Molecules Required for the Control of Leishmania (Viannia) braziliensis in Mice Infect. Immun., August 1, 2007; 75(8): 3823 - 3832. [Abstract] [Full Text] [PDF]

				N. Wanasen, C. L. MacLeod, L. G. Ellies, and L. Soong L-Arginine and Cationic Amino Acid Transporter 2B Regulate Growth and Survival of Leishmania amazonensis Amastigotes in Macrophages Infect. Immun., June 1, 2007; 75(6): 2802 - 2810. [Abstract] [Full Text] [PDF]

				P. Smith, N. E. Mangan, C. M. Walsh, R. E. Fallon, A. N. J. McKenzie, N. van Rooijen, and P. G. Fallon Infection with a Helminth Parasite Prevents Experimental Colitis via a Macrophage-Mediated Mechanism J. Immunol., April 1, 2007; 178(7): 4557 - 4566. [Abstract] [Full Text] [PDF]

				M. Munder, H. Schneider, C. Luckner, T. Giese, C.-D. Langhans, J. M. Fuentes, P. Kropf, I. Mueller, A. Kolb, M. Modolell, et al. Suppression of T-cell functions by human granulocyte arginase Blood, September 1, 2006; 108(5): 1627 - 1634. [Abstract] [Full Text] [PDF]

				M. T. Talaue, V. Venketaraman, M. H. Hazbon, M. Peteroy-Kelly, A. Seth, R. Colangeli, D. Alland, and N. D. Connell Arginine Homeostasis in J774.1 Macrophages in the Context of Mycobacterium bovis BCG Infection J. Bacteriol., July 1, 2006; 188(13): 4830 - 4840. [Abstract] [Full Text] [PDF]

				C. Allenbach, C. Zufferey, C. Perez, P. Launois, C. Mueller, and F. Tacchini-Cottier Macrophages Induce Neutrophil Apoptosis through Membrane TNF, a Process Amplified by Leishmania major. J. Immunol., June 1, 2006; 176(11): 6656 - 6664. [Abstract] [Full Text] [PDF]

				A. Yeramian, L. Martin, N. Serrat, L. Arpa, C. Soler, J. Bertran, C. McLeod, M. Palacin, M. Modolell, J. Lloberas, et al. Arginine Transport via Cationic Amino Acid Transporter 2 Plays a Critical Regulatory Role in Classical or Alternative Activation of Macrophages J. Immunol., May 15, 2006; 176(10): 5918 - 5924. [Abstract] [Full Text] [PDF]

				L. Martin, M. Comalada, L. Marti, E. I. Closs, C. L. MacLeod, R. Martin del Rio, A. Zorzano, M. Modolell, A. Celada, M. Palacin, et al. Granulocyte-macrophage colony-stimulating factor increases L-arginine transport through the induction of CAT2 in bone marrow-derived macrophages Am J Physiol Cell Physiol, May 1, 2006; 290(5): C1364 - C1372. [Abstract] [Full Text] [PDF]

				C. Holscher, B. Arendse, A. Schwegmann, E. Myburgh, and F. Brombacher Impairment of Alternative Macrophage Activation Delays Cutaneous Leishmaniasis in Nonhealing BALB/c Mice J. Immunol., January 15, 2006; 176(2): 1115 - 1121. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 19/8/1000 04-3416fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kropf, P. Articles by Müller, I. Search for Related Content PubMed PubMed Citation Articles by Kropf, P. Articles by Müller, I.