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Keywords:

  • Leishmania;
  • macrophages;
  • arginase;
  • ornithine;
  • proliferation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Leishmania spp. are intracellular protozoan parasites that invade and replicate within macrophages. In a previous report, we have demonstrated that the growth of intracellular amastigotes could be controlled by inhibition of arginase. This enzyme, induced in host cells by Th2 cytokines, synthesizes L-ornithine which can be used by parasites to generate polyamines and proliferate. In this study, we have designed experiments to better analyse the dependence of parasite proliferation on arginase induction in infected macrophages. Treatment of Leishmania major-infected BALB/c macrophages with interleukin (IL)-4, IL-10 or transforming growth factor-β, which are all inducers of arginase I in murine macrophages, led to a proportional increase in the number of intracellular amastigotes. Moreover, parasite proliferation and arginase activity levels in macrophages from the susceptible BALB/c mice were significantly higher than those from infected C57BL/6 cells when treated with identical doses of these cytokines, indicating that a strong correlation exist between the permissibility of host cells to L. major infection and the induction of arginase I in macrophages. Specific inhibition of arginase by Nω-hydroxy-nor-L-arginine (nor-LOHA) reverted growth, while L-ornithine and putrescine promoted parasite proliferation, indicating that the parasite cell division depends critically on the level of L-ornithine available in the host. Therefore, arginase induction in the context of a Th2 predominant response might be a contributor to susceptibility in leishmaniasis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Leishmania parasites, the causative agent of leishmaniasis, require polyamines for growing and differentiation as well as for the synthesis of trypanothione, the equivalent of gluthatione in mammals (1,2). The hydrolysis of L-arginine to L-ornithine, catalysed by arginases, provides the substrate for ornithine decarboxilase, the key enzyme in polyamine biosynthesis.

Macrophages are the major host cells for Leishmania infection and express two arginase isoforms, arginase I and arginase II. Arginase I is induced by the Th2-like cytokines, interleukin (IL)-4 and IL-10 (3,4) as well as by transforming growth factor (TGF)-β (5). Both arginase isoforms can be induced by other pharmacoactive substances, such as cyclic AMP analogs or prostaglandin E2 (6). It has also been reported that arginases are up-regulated in several human pathologies, such as glomerulonephritis (7) or breast cancer, in which arginase has been suggested to have a role in the growth and proliferation of cancer cells (8). More recently, it was shown that arginase is up-regulated in vivo in experimental trypanosomiasis (9) and its induction might be associated with the development of the disease. However, to date, the precise function of arginase induction in association with molecules of the immune system was poorly defined.

Leishmania parasites, via disruption of macrophage activation, are able to reduce MHC class II levels in infected macrophages (10) and inhibit cellular apoptosis to prolong their prevalence inside the host (11). Furthermore, parasites regulate cytokine synthesis towards those cytokines that suppress the killing capacities of macrophages. In this context, the most important mechanism analysed to date appears to be the inhibition of IL-12 synthesis in Leishmania-infected cells, which delays or prevents the secretion of interferon-γ (12). Moreover, it has been shown that Leishmania infection induces the synthesis of IL-10 and TGF-β, preventing macrophage activation, since Leishmania parasites are potent inhibitors of IL-12 production by antigen presenting cells (13).

We have recently shown that Nω-hydroxy-L-arginine, a stable intermediate in the synthesis of nitric oxide (14) and also a physiological inhibitor of arginases (15), was able to control cellular infection in Leishmaniamajor and Leishmaniainfantum-infected macrophages (16). In the present study, we have designed experiments to investigate the role of arginase I induction in infected cells. Ours results clearly demonstrate that the proliferation of intracellular Leishmania parasites triggered by IL-4, IL-10 and TGF-β depends on arginase I induction in macrophages. The increase in the pool of L-ornithine generated by the enzyme allows the generation of polyamines needed for the replication of parasites.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Medium and reagents

Macrophage cultures were performed in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mm L-glutamine, 60 µm 2-mercaptoethanol, 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco BRL, Paisley, UK). Recombinant murine IL-4, IL-10 and TGF-β were obtained from peproTech, EC Ltd (London, UK); L-ornithine and putrescine from Sigma (Madrid, Spain) and Nω-hydroxy-nor-L-arginine (nor-LOHA), from Alexis Corp (Lausen, Switzerland).

Animals, parasites and generation of bone marrow-derived macrophages (BMMφ)

Mice of strains BALB/c and C57BL/6, aged 6–8 weeks, were purchased from Janvier España, SL (Madrid, Spain) and tested routinely in the Parasitology Unit for murine pathogens.

L. major (WHO reference strain M/IR/–/173) promastigotes were isolated and propagated as published elsewhere (17). Macrophages were derived from bone marrow cells as previously described (3).

Infection of BMMφ with Leishmania major

Stationary-phase promastigotes were added to BMMφ cultures (106 cells per ml) in 24-well plates (Costar, Cambridge, MA, USA) with round cover slides, at parasite-to-cell ratio of 3 : 1 and transferred to a CO2 at 37°C. After 3 h, extracellular parasites were removed by washing and cultures were treated with increasing concentrations of IL-4, IL-10, TGF-β, L-ornithine and putrescine, as described in the results section, for an infection period of 48 h. Finally, the round cover slides were removed from the plates, mounted and stained with Giemsa (DiffQuick, QCA, Amposta, Spain). The number of amastigotes within macrophages was determined by microscopical examination, after counting 100 cells in the stained preparations.

Measurement of arginase activity in infected BMMφ

Arginase activity was measured in macrophage lysates, as previously described (3).

Briefly, cells were lysed with 100 µl of a buffer containing 0·1% Triton X-100 and 25 mm Tris-HCl, MnCl2 5 mm, pH 7,4. Arginine hydrolysis was performed by incubating the lysate with 100 µl of 0·5 m L-arginine (pH 9·7) at 37°C for 30 min. The reaction was stopped with 800 µl of H2SO4 (96%)/H3PO4 (85%)/H2O (1/3/6, v/v/v). The urea concentration was measured at 550 nm after addition of 40 µl of α-isonitrosopropiophenone (dissolved in 100% ethanol) followed by heating at 95°C for 30 min. One unit of enzyme activity is defined as the amount of enzyme that catalysed the formation of 1 µmol of urea per min.

Western blotting

Relative amounts of arginase I protein expressed in infected macrophages, were analysed by Western blotting. After incubation for 24 h with IL-4, IL-10 or TGF-β, culture supernatants were removed and cells washed twice with cold phosphate-buffered saline (PBS). The lysis were directly performed by boiling the samples in electrophoresis sample buffer (62·5 mm Tris-HCl pH 6·8, 2% SDS, 25% glycerol, 0·01% bromophenol blue and 2%β-mercaptoethanol). The protein content in the lysate was quantified by the Bradford method. Equal samples (15 µg protein of each lysate) were subjected to SDS-PAGE. Electrophoresis was performed on Criterion precast gels (Bio-Rad, Hercules, CA, USA) 10–20%. After semidry electrotransfer (Bio-Rad), the polyvinilidene difluoride membrane (Bio-Rad) was blocked with PBS-0·1% (wt/v) Tween-20, supplemented with 2% bovine serum albumin and incubated with an antimouse arginase I monoclonal antibody (dilution 1 : 1000, Transduction Laboratory, Lexington, KY, USA) followed by incubation with horseradish peroxidase-conjugated antimouse immunoglobulin G antibody (Sigma). The bands were detected by an enhanced chemiluminiscence Western blotting detection kits (Pierce, IL, USA) according to the manufacturer’s directions.

Statistical analysis

Data reported are means ± SD (n = 4) from at least three independent experiments. P < 0·05 was considered statistically significant using Student’s paired t-test.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Induction of arginase by the Th2-like cytokines, IL-4 and IL-10, promotes the growth of intracellular amastigotes

It has been previously reported that Leishmania parasites are able to induce in the host the anti-inflammatory cytokines IL-4, IL-10 and TGF-β (13). Because these molecules are also inducers of arginases (3,5), our first experimental approach was to compare the levels of arginase induction with the growth of intracellular amastigotes. The results, presented in Figure 1, clearly show that when infected macrophages from BALB/c mice were treated with increasing amounts of IL-4 (Figure 1a), which resulted in a proportional increment in arginase activity, the number of amastigotes within the macrophages gradually increased. These data are in agreement with previous findings indicating that the early priming of a T cell response towards a polarized Th2 cell subset determines the growth of Leishmania inside macrophages (18). In fact, in BALB/c mice, susceptible to Leishmania infection, the early production of IL-4 is necessary and sufficient to instruct Th2 cell development (19). Moreover, mice with a targeted disruption of the IL-4 gene contained the infection and did not develop progressive disease (20), while the ample production of IL-4 in C57BL/6 mice, due to the presence of an IL-4 transgene, created a susceptible phenotype (21). The contribution of IL-4 induction to the disease has been recently confirmed by the demonstration that mice with a disruption in the gene for STAT-6 transcription factor, which mediates in IL-4 signalling, became resistant to Leishmania infection (22) and, coincidently, macrophages from these mice did not induce arginase I in response to IL-4, as expected (23). All these findings, together with the present results, point to a new role of IL-4 in infected cells, which is to support the growth of Leishmania via induction of arginase I.

image

Figure 1. Th2-derived cytokines dose-dependently promote parasite proliferation and increase arginase activity. BMMΦ from BALB/c mice were infected with L. major promastigotes and treated with increasing amounts of (a) IL-4 or (b) IL-10. After 48 h, infected cells were used to determine intracellular parasites (▪) and, in replicate cultures, to measure arginase activity (•).

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Similarly, when infected cells were treated with increasing doses of IL-10 (Figure 1b), we observed that, together with an increase in macrophage arginase activity, the number of parasites also increased. In agreement with our results, it has been recently shown that macrophages from BALB/c mice infected in vitro with L. major amastigotes, produce IL-10, which reduces the production of IL-12 and tumour necrosis factor-α, prevents parasite killing by activated cells and enhances the survival of parasites (24). In addition to this, our results show that IL-10 also contributes to disease progression by arginase I induction, which results in intracellular parasite proliferation.

Effects of TGF-β on parasite growth and arginase activity in infected BALB/c macrophages

To gain further insight on the effect of arginase in infected cells, we used TGF-β. This molecule is also a potent inducer of arginase in rodent macrophages. Coincidently, this cytokine has several down-regulatory functions on the immune system, including the inhibition of macrophage activation. It is induced in infected cells, probably acting as one of the escape mechanisms evolved by Leishmania to avoid the killing capacities of macrophages. It therefore has been recognized as disease promoting in leishmaniasis (25).

As indicated in Figure 2, TGF-β treatment of infected cells increases parasite proliferation and, at the same time, induces a dose-dependent increment in arginase specific activity in macrophages. Thus, via TGF-β-induced arginase, we confirm the same pattern obtained by Th2 cytokines: ornithine generation by host cells, resulting in the growth of Leishmania.

image

Figure 2. TGF-β induces arginase activity and parasite proliferation. BALB/c cells were infected with L. major promastigotes in the presence of increasing amounts of TGF-β for 48 h. Macrophages were used to determine both intracellular parasites and arginase activity.

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Arginase I is the isoform induced in infected macrophages by Th2-like cytokines

Having demonstrated that murine macrophages express arginase I in response to Th2 cytokines, we wanted to determine the arginase isoform that was induced in L. major-infected macrophages after incubation for 48 h with either IL-4, IL-10 or TGF-β (Figure 3). As can be appreciated, arginase I is indeed the isoform induced by these cytokines, with IL-4 being the cytokine which produces the higher levels of the enzyme, in an amount that is close to that found in mouse liver, in which this isoform is constitutive and has the highest levels of specific activity.

image

Figure 3. Arginase I is the isoform induced in infected macrophages by either IL-4, IL-10 or TGF-β. Lane 1: mouse liver homogenate used as positive control; lane 2: control infected cells; lane 3: L. major-infected macrophages treated for 48 h with 2·5 ng/ml of IL-4; lane 4: L. major-infected macrophages treated for 48 h with 20 ng/ml of IL-10; lane 5: L. major-infected macrophages treated for 48 h with 10 ng/ml of TGF-β.

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Macrophages from BALB/c mice, susceptible to Leishmania infection, produce more arginase I activity than those from the resistant C57BL/6 mice in response to Th2-like cytokines

It has been reported that there is a difference in the levels of arginase I induction in macrophages, depending on the genetic background of the mice; those that develop a predominant Th1 response produce less arginase I activity in response to lipopolysaccharide than those evolving under a Th2 predominant response. These cells have been called M1 and M2 macrophages, respectively (26). Here, we confirm this model by showing the results obtained in infected cells from BALB/c and C57BL/6 mice exposed to Th2-like cytokines. As shown in Figure 4, the constitutive levels of arginase activity/infection are already higher in susceptible compared to resistant cells. This difference in arginase and parasite proliferation is much more prominent in infected cells from the two strains treated with identical doses of IL-4, IL-10 and TGF-β. BALB/c mice respond with higher levels of arginase I/infection than those from C57BL/6 mice. Thus, this finding suggests that there is a strong correlation between arginase I levels and the growth of Leishmania in the host. Therefore, this degree of macrophage allowance observed in vitro between the two strains might explain the differences observed in an infection in vivo, where C57BL/6 mice have less parasites in lesions compared to BALB/c mice (27).

image

Figure 4. The susceptibility and resistance to Leishmania infection of both BALB/c and C57BL/6 strains correlate with differences in arginase induction by IL-4, IL-10 and TGF-β. Macrophages from the two strains were infected in the presence of 2·5 ng/ml IL-4, 20 ng/ml IL-10 and 10 ng/ml TGF-β for 48 h. The results represent (a) the number of amastigotes per 100 cells and (b) arginase induction in these cells. *P < 0·05 by Student’s paired t-test.

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Nor-LOHA, a specific competitive inhibitor of arginase inhibits both, enzyme activity and parasite growth in the presence of Th2-like cytokines

Since the data shown in our study strongly suggest that a correlation exists between the levels of macrophage arginase I induction and the growth of intracellular Leishmania parasites, we designed an additional experiment to directly involve arginase I induction as being responsible for the growth of parasites triggered by these Th2-like cytokines. For that purpose, we used nor-LOHA, which is the most specific arginase competitive inhibitor available (28). It should be noted that macrophages could also produce Nω-hydroxy-L-arginine (LOHA), the physiological inhibitor of the enzyme, when they are activated by Th1-like cytokines that induce nitric oxide synthase II (NOS II). LOHA is a stable intermediate in the NOS reaction and can further be metabolized to NO and citrulline (14). In a previous study, we showed that LOHA could control parasite infection by inhibiting arginase activity although, in these studies, the involvement of the NO pathway could not be excluded (16). In contrast to LOHA, nor-LOHA is neither a substrate nor an inhibitor of NOS (28,29). Thus, in these conditions, the results could be interpreted straightforwardly since only L-arginine metabolism through arginase would be affected.

The results are presented in Table 1. As can be seen, nor-LOHA was able to inhibit all the growth achieved in the presence of IL-4, IL-10 and TGF-β. Furthermore, nor-LOHA was also effective in reducing parasite growth in control infected cells, due to the fact that Leishmania parasites have arginase activity (16), while it did not produce any increments of NO in culture supernatants, demonstrating that the inhibition of the host arginase is sufficient to revert the growth achieved in the presence of these cytokines. Thus, the results clearly demonstrate that arginase I induction in macrophages is necessary and sufficient to achieve intracellular parasite growth.

Table 1.  Effect of nor-LOHA on intracellular parasite growth in L. major-infected BALB/c BMMφ
TreatmentAmastigotes per 100 cellsNitrites (µm)
NoneNor-LOHANoneNor-LOHA
Control122 ± 20·446 ± 10·71·24 ± 0·151·21 ± 0·26
IL-4431 ± 37·975 ± 8·61·45 ± 0121·30 ± 0·08
IL-10245 ± 27·360 ± 15·21·17 ± 0·091·22 ± 0·22
TGF-β289 ± 18·271 ± 5·81·32 ± 0161·28 ± 0·12

L-ornithine and putrescine increase the growth of intracellular amastigotes in macrophages from the resistant C57BL/6 mice

Finally, to better assess the involvement of arginase I induction in the growth of Leishmania, we added L-ornithine, the product of the arginase reaction, and prutrescine, the product of ornithine decarboxylase (ODC), to infected cultures of macrophages from resistant C57BL/6 mice. We proposed that the growth of Leishmania via arginase I induction is achieved by generation of polyamines essential for parasite proliferation (30).

The results show that the number of intracellular parasites is dose-dependently increased by both L-ornithine and putrescine(Figure 5).

image

Figure 5. Both L-ornithine and putrescine proportionally increase the number of intracellular amastigotes in bone marrow-derived macrophages. L. major infected cells from C57BL/6 mice were treated with increasing amounts of L-ornithine and putrescine for 48 h. The results represent the number of intracellular parasites per 100 cells.

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This is the first experimental evidence showing that the growth of intracellular Leishmania amastigotes in vitro is dependent on L-ornithine and putrescine. The requirements of both molecules for the growth of Leishmania have been studied extensively. Parasites have their own ornithine decarboxilase that, in contrast with the mammalian enzyme, is much more stable (1). Moreover, polyamine metabolism by parasitic protozoa has attracted considerable attention because it is an important target for drug action, and difluoromethylornithine, an inhibitor of ODC, is being used for the treatment of leishmaniasis (31).

From the present findings, we believe that specific arginase inhibitors may represent good candidates for controlling protozoan infections. Additionally, we suggest that vaccines that mitigate against an unbalanced Th2 response could prevent the induction of arginase in macrophages and therefore impair the spread of parasites.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

V. Iniesta and L. C. Gómez-Nieto contributed equally to this work. This work was supported by grants from Consejería de Sanidad y Consumo, Junta de Extremadura (00/23) and Fondo Europeo de Desarrollo Regional grant 1FD97-0630-C02-02.

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  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
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