The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 8446-8453
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gaur, U.
Right arrow Articles by Wilson, M. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gaur, U.
Right arrow Articles by Wilson, M. E.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Medline Plus Health Information
*Leishmaniasis
Hazardous Substances DB
*NITRIC OXIDE

An Effect of Parasite-Encoded Arginase on the Outcome of Murine Cutaneous Leishmaniasis1

Upasna Gaur*,{dagger}, Sigrid C. Roberts2,||, Rahul P. Dalvi*, Inés Corraliza#, Buddy Ullman|| and Mary E. Wilson3,*,{ddagger},§,¶

* Department of Internal Medicine, {dagger} Department of Biochemistry, {ddagger} Department of Microbiology, and § Department of Epidemiology, University of Iowa, and Veterans Affairs Medical Center, Iowa City, IA 52242; || Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239; and # Department of Biochemistry and Molecular Biology, Universidad de Extremadura, Cáceres, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Classical activation of macrophages infected with Leishmania species results in expression and activation of inducible NO synthase (iNOS) leading to intracellular parasite killing. Macrophages can contrastingly undergo alternative activation with increased arginase activity, metabolism of arginine along the polyamine pathway, and consequent parasite survival. An active role for parasite-encoded arginase in host microbicidal responses has not previously been documented. To test the hypothesis that parasite-encoded arginase can influence macrophage responses to intracellular Leishmania, a comparative genetic approach featuring arginase-deficient mutants of L. mexicana lacking both alleles of the gene encoding arginase ({Delta}arg), as well as wild-type and complemented {Delta}arg controls ({Delta}arg[pArg]), was implemented. The studies showed: 1) the absence of parasite arginase resulted in a significantly attenuated infection of mice (p < 0.05); 2) poorer survival of {Delta}arg in mouse macrophages than controls correlated with greater NO generation; 3) the difference between {Delta}arg or control intracellular survival was abrogated in iNOS-deficient macrophages, suggesting iNOS activity was responsible for increased {Delta}arg killing; 4) consistently, immunohistochemistry showed enhanced nitrotyrosine modifications in tissues of mice infected with {Delta}arg compared with control parasites. Furthermore, 5) in the face of decreased parasite survival, lymph node cells draining cutaneous lesions of {Delta}arg parasites produced more IFN-{gamma} and less IL-4 and IL-10 than controls. These data intimate that parasite-encoded arginase of Leishmania mexicana subverts macrophage microbicidal activity by diverting arginine away from iNOS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Leishmania spp. are dimorphic parasitic protozoa that cause an array of disfiguring or fatal human diseases. The obligate intracellular amastigote stage of the parasite resides within the phagolysosome of mammalian mononuclear phagocytes including macrophages. Genetic susceptibility or resistance to Leishmania infection varies among different strains of mice. Resistance is almost uniformly accompanied by expansion of type 1 CD4+ (Th1) cells that produce the cytokine IFN-{gamma} in response to parasite Ag (1, 2, 3). In contrast, the immunological response of mice susceptible to infection by different species of Leishmania is highly variable. Susceptibility to Leishmania major is mediated through expansion of a strong type 2 immune response against a predominant Ag (Leishmania homologue of receptors for activated C-kinase) leading to IL-4 and IL-13 expression, whereas suppression of a type 1 response without Th2 expansion is characteristic of other species (3, 4). TGF-β is a major factor suppressing the type 1 immune response during infection with Leishmania chagasi (5).

A remarkable feature of Leishmania is its ability to survive and replicate in the phagolysosome of infected mammalian macrophages, a hostile environment that is lethal to many microbes (6). Interestingly, the amino acid arginine appears to play a key role in the mechanism by which Leishmania survive intracellularly in the mammalian host (7). Macrophages harbor two competing pathways for arginine metabolism initiated by the enzymes inducible NO synthase (iNOS)4 and arginase, respectively (8, 9). The first pathway involves IFN-{gamma} activation of NOS2 and produces its protein product iNOS. iNOS catalyzes the two-step NADPH-dependent conversion of arginine through N{omega}-hydroxy-L-arginine (NOHA) to citrulline and NO·, the latter a potent inorganic microbicidal molecule. This iNOS pathway is used by classically activated murine macrophages to kill intracellular Leishmania (10, 11, 12). In contrast, macrophages can be alternatively activated by other stimuli including IL-4 or IL-13 and express an arginase I activity that hydrolyzes arginine to urea and ornithine (8). Ornithine is a key intermediate in the synthesis of glutamine, proline, and polyamines in mammalian cells (13). However, in Leishmania promastigotes the sole role of ornithine is the production of polyamines, which are ubiquitous essential cations that play critical roles in a variety of cellular processes needed by proliferating cells (14, 15).

Previous studies have demonstrated that the balance between iNOS and arginase activities is competitively regulated by the type 1 and type 2 cytokines (16). Specifically, IFN-{gamma} enhances expression of iNOS, whereas IL-4 induces both increased expression of the arginase protein and increased arginase activity (17). Furthermore, just as the type 1 and type 2 cytokines are mutually inhibitory, the induction of iNOS or arginase is also regulated in a reciprocal fashion (13). iNOS is regulated both at the level of activity and gene expression as well as by substrate (arginine) availability (17). It has been demonstrated that the local availability of arginine is an important determinant of NO·-mediated killing of Trypanosoma brucei (18) and that host cell NO· production is regulated by the scavenging of arginine by Helicobacter pylori arginase (19).

Using arginase inhibitors, Iniesta et al. (7, 16) showed that arginase activity is necessary for the survival and growth of both L. major and Leishmania infantum in murine macrophages. However, because the host macrophage and the parasite cells each express functional arginase enzymes, it is not clear whether one or both arginases are needed for parasite survival. The generation of Leishmania mexicana gene deletion mutants deficient in parasite arginase activity enables a dissection of the relative contributions of parasite and host cell arginases to parasite survival both in vitro in the host macrophage and in vivo within the host (14). The results suggest that parasite-derived arginase presents a novel defense mechanism that enhances parasite survival through local depletion of the iNOS substrate arginine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female BALB/c mice (4–6 wk old) were purchased from Harlan Breeders; iNOS knockout mice in a C57BL/6 background and wild-type C57BL/6 mice were purchased from The Jackson Laboratory. Studies using mice were approved by the Animal Care and Use Committees of the University of Iowa and the Iowa City Veterans Affairs Medical Center.

Parasite culture

All genetically modified lines were derived from wild-type MNYC/BZ/62/M379 L. mexicana. The creation and characterization of the {Delta}arg knockout and the {Delta}arg[pArg] cell lines has been reported (14). The {Delta}arg strain was isolated after two rounds of targeted gene replacement, whereas the {Delta}arg[pArg] parasites possess a {Delta}arg chromosomal background and harbor a multicopy episomal plasmid encompassing the arginase gene (14). All L. mexicana lines were cultivated in DME-L, a completely defined DMEM (14). The growth medium for the genetically manipulated L. mexicana contained the following additions: {Delta}arg knockout parasites were supplemented with 50 µg/ml phleomycin, 50 µg/ml hygromycin, and 100 µM putrescine, and episomally complemented {Delta}arg[pArg] parasites were supplemented with 50 µg/ml phleomycin, 50 µg/ml hygromycin, and 20 µg/ml G418. Some parasite cultures were tested for growth in the presence of no supplements, 200 µM putrescine, or 200 µM ornithine.

For limiting dilution assays to quantify parasite infections in mice, serial 4-fold dilutions of footpad lysates were cultivated in 96-well plates in Schneider’s medium to which 20% FCS and 20 µg/ml gentamicin were added.

Cellular arginase assays

Parasite arginase activity was assayed by measuring the conversion of L-[14C(U)]arginine to [14C]urea and [14C]ornithine over 48 h (20, 21, 22). Briefly, [14C]arginine (NEN-specific activity 360 mCi/mM) at 0.25 µM was added to 96-well plates each containing 106 parasites in 200 µl of growth medium. After 48 h at 26°C, the assay was terminated by the addition of 150 µl of cell culture supernatant to 0.8 ml of a solution of 250 mM acetic acid, 100 mM urea, 10 mM arginine (pH 4.5). Remaining [14C]arginine and [14C]ornithine in supernatants was removed by binding to Dowex HCR-W2 cation-exchange resin (Sigma-Aldrich) and, after centrifugation, the [14C]urea reaction product in 0.5 ml was counted by liquid scintillation. The limits of assay detection were 0.005–2.5 nM [14C]arginine conversion to urea.

Bone marrow macrophages (BMMs)

Bone marrow cells (BMMs) obtained from BALB/c mouse femurs were cultured at 37°C, 5% CO2 in RP-10 medium (10% heat-inactivated FCS, 2 mM L-glutamine, 100 U of penicillin/ml, and 50 µg of streptomycin/ml in RPMI 1640; Invitrogen Life Technologies) containing 20% cell culture supernatant from L929 cells (American Type Culture Collection) as a source of M-CSF. After 7–9 days, differentiated adherent macrophages were detached from the petri dish with 2.5 mg/ml trypsin containing 1 mM EDTA (Invitrogen Life Technologies) (23). A total of 5 x 105 macrophages were then allowed to adhere to coverslips in 24-well plates and infected with promastigotes at a multiplicity of infection (MOI) of 5:1. The infection was synchronized by centrifugation (3 min, 330 x g, 4°C), and infected macrophages were incubated in 5% CO2 at 37°C. Extracellular parasites were removed by rinsing macrophages 30 min postinfection.

In another experiment, the macrophage growth medium was supplemented with increasing amounts of ornithine (A) or putrescine (B). After 48 h, triplicate coverslips were fixed, Wright-Giemsa stained, and macrophages and amastigotes were enumerated. Parasite intracellular growth was compared with that of wild-type parasites, and p values were calculated by the Student t test (paired test).

Nitrite measurements

To measure NO· generated by cellular iNOS, nitrite, which forms readily from NO· in the presence of oxygen (24), was measured using the colorimetric Griess assay (25). Briefly, 50 µl of the culture supernatants were added to 96-well plates containing 100 µl of freshly prepared Griess reagent (0.1% N-1-napthylenediamine-HCl in water and 1% sulfanilamide in 2.5% H3PO4). After a 15-min incubation at room temperature, absorbance at 550 nm was determined on an ELISA plate reader.

Footpad model of L. mexicana infection

BALB/c mice were infected in the right hind footpad with a single injection of 1 x 106 stationary phase wild-type, {Delta}arg, or {Delta}arg[pArg] L. mexicana promastigotes. Footpad thickness was evaluated with a Mitutoyo digital caliper. At the termination of the experiment the foot pads were excised, and parasite loads were quantified by limiting dilution as described (26).

Lymph node cell culture and cytokine production

The lymph node draining cutaneous lesions were removed 4 wk after infection. Pooled cells from five mice in each group were seeded at 0.2 x 106 cells/well and were restimulated with 1 x 106 L. mexicana promastigotes. After 48 h, culture supernatants were harvested, and cytokines were quantified on a Bioplex system (Bio-Rad) with a LINCOplex Mouse Cytokine Panel kit (Linco Research). In each experiment, a standard curve was run in parallel. The detection limits of the assay ranged from 0.3 to 20 pg/ml.

TGF-β bioassay

Mink lung fibroblasts (MvLu) stably expressing a luciferase construct under control of the TGF-β-responsive promoter for the plasminogen activator inhibitor were provided by D. Rifkin (New York University, New York, NY). These were used to assess bioactive and total TGF-β in culture supernatants as described (27, 28). Cells from lymph nodes of mice infected with wild-type, {Delta}arg, or {Delta}arg[pArg] parasites were cultivated in serum-free medium for 48 h. MvLu cells were incubated in either regular growth medium in the absence or presence of various concentrations of TGF-β for use as a standard curve (1–3000 pg/ml) or with culture supernatants. Luciferase activity was assayed with a luciferase kit from Promega. Control wells contained medium. All conditions were tested in triplicate. The detection limits of the assay were 30–3000 pg/ml.

Histopathology and immunohistochemistry

NO· can initiate covalent modification of proteins particularly on tyrosine residues. Thus, the presence of nitrosylated tyrosine is a stable marker for NO·-mediated cellular damage. Footpads from 4-wk-infected BALB/c mice were incubated overnight in 30% sucrose-PBS as a cryoprotectant, freeze dried, and embedded in Tissue-Tek OCT Compound (Sakura Finetek) in liquid nitrogen. Five- or 10-µm sections were sliced from frozen tissues, fixed in paraformaldehyde, blocked in a PBS solution containing 5% milk, and incubated with a 1/100 rabbit polyclonal anti-nitrotyrosine Ab (Upstate Biotechnology) in PBS-0.3% BSA overnight at 4°C. Sections were then incubated for 1 h in 1/200 Alexa Fluor 546-labeled goat anti-rabbit Ig followed by 5 min in TOPRO-3 nuclear stain diluted 1/3000. After rinsing and mounting with Vectashield H-1000 (Vector Laboratories), slides were examined on a Zeiss 510 laser confocal microscope (www.zeiss.com/micro) and images were captured on a laser scanning microscope (LSM) 510 version 3.2 software. Confocal optical sections were further analyzed using the LSM 5 image browser. Slides were stained with H&E after alcohol dehydration and a distilled water rinse and then mounted in xylene-based mounting medium.

Statistical analysis

Statistical analyses was performed using either one-way ANOVA, Kruskal-Wallis one-way ANOVA on ranks, or paired t test algorithms using Sigma Stat software (SPSS).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Arginase-deficient and -complemented parasites

To verify the functional consequences of the gene replacements in {Delta}arg parasites, arginase activity was measured in wild-type, {Delta}arg, and {Delta}arg[pArg] parasites. Whereas significant arginase activity was detected in both wild-type and {Delta}arg[pArg] L. mexicana, arginase activity in {Delta}arg parasites was essentially undetectable.

Although knockout organisms were unable to proliferate in unsupplemented growth medium, the growth of {Delta}arg L. mexicana promastigotes (14) or amastigotes (data not shown) in axenic culture was restored by the addition of either 200 µM putrescine or 200 µM ornithine.

Decreased survival of {Delta}arg L. mexicana in macrophages

To investigate the role of parasite-encoded arginase on tissue amastigote proliferation, the abilities of wild-type, {Delta}arg, or {Delta}arg[pArg] parasites to survive and maintain an infection in BMMs was analyzed. Initial parasite entry into macrophages, quantified 2 h postinfection, did not differ significantly among the wild-type, {Delta}arg, and {Delta}arg[pArg] strains (70.3 ± 17.6, 71.5 ± 15.8, 74.8 ± 12.8 parasites/100 BMMs, respectively). In contrast, the number of {Delta}arg parasites that survived in BMM cultures 24 or 48 h after infection was significantly lower than those obtained for either wild-type or {Delta}arg[pArg] parasites (Fig. 1). This was not due to significant difference in the enhanced total macrophage arginase activity in macrophages infected with mutant compared with wild-type parasites (Table I). Arginase activity due to the parasite itself was only a fraction of that observed in the total macrophage (see Table I). However, the local arginase activity influencing arginine availability in the parasitophorous vacuole surrounding the intracellular parasite could be significantly influenced by parasite enzyme activity at a level not detectable in an assay of total macrophage arginase.


Figure 1
View larger version (36K):
[in this window]
[in a new window]

  		FIGURE 1. Survival of {Delta}arg L. mexicana in BMMs. Macrophages were infected with opsonized parasites (5:1 MOI) for 30 min before removing extracellular parasites. At appropriate time points, samples were collected and infection levels were quantified using light microscopy. Values represent the mean ± SE for three experiments, each with duplicate or triplicate conditions. A, The percent of infected macrophages; B, the number of parasites counted per 100 macrophages. C and D, Macrophages were infected in the presence of exogenous putrescine (C) or ornithine (D). Parasite loads were assessed microscopically 48 h after infection was initiated. Statistical analyses were done using Kruskal-Wallis one-way ANOVA on ranks. Asterisks refer to comparisons between knockout mutants with add-back mutants; significance levels comparing knockout to wild type were similar.

 

View this table:
[in this window]
[in a new window]

  		Table I. Arginase activity in infected macrophages,a uninfected macrophages,a or parasites aloneb

 
To evaluate whether the decreased intracellular growth of the {Delta}arg parasites in macrophages could be ascribed to a lack of salvageable polyamines, the growth medium of infected bone marrow-derived macrophages was supplemented with ornithine and putrescine after removal of extracellular parasites. The addition of either ornithine or putrescine increased the number of amastigotes of both wild-type and {Delta}arg cells (Fig. 1, C and D), implying that intracellular L. mexicana amastigotes are capable of salvaging both ornithine and putrescine from the macrophage and extracellular medium. These findings confirm previous observations that the availability of increased amounts of exogenous polyamines favors intracellular parasite proliferation (16). Putrescine consistently stimulated parasite proliferation more efficiently than ornithine (Fig. 1C) consistent with prior findings (14).

In contrast with the studies of axenic cultures described above, the exogenous addition of either putrescine or ornithine to infected macrophages did not augment the intracellular growth of {Delta}arg to the level of wild-type parasites (see Fig. 1, C and D). There was a statistically significant increase in intracellular growth when either ornithine or putrescine was added to cultured wild-type {Delta}arg or {Delta}arg[pArg] parasites, compared with the same parasite strain with no addition. Nonetheless, there continued to be significantly slower growth of intracellular {Delta}arg compared with either intracellular wild-type or {Delta}arg[pArg] L. mexicana at each ornithine or putrescine concentration. These data suggest that, although the growth defect in axenic culture can be attributed to the lack of polyamines, an alternate mechanism, such as nutrient deficiency or microbicidal function, may also be inhibiting the intracellular growth of {Delta}arg parasites in macrophages. These observations led us to investigate other potential consequences of the lack of parasite arginase and accumulation of its substrate arginine in the infected cell.

Enhanced NO production by BMMs infected with {Delta}arg parasites

It has been reported that the intracellular concentration of arginine is rate-limiting for iNOS activity in vivo (19). We hypothesized that parasite arginase could deplete host arginine supplies that might otherwise be available to iNOS as a substrate. If this is the case, then eliminating parasite arginase should enhance the metabolic flux through iNOS in the infected host cell macrophage. To test this conjecture, the amount of cellular NO· released into culture supernatants of BMMs infected with wild-type, {Delta}arg, or {Delta}arg[pArg] L. mexicana was assessed by measuring the amount of nitrite ion produced. Because IFN-{gamma} is necessary for leishmanicidal activity and transcription of iNOS (29, 30), some of the cells were preincubated for 24 h with 100 U recombinant murine IFN-{gamma}/ml. Forty-eight hours after infection was initiated, nitrite was significantly increased in IFN-{gamma}-primed BMMs infected with the {Delta}arg knockout compared with IFN-{gamma}-primed BMMs infected with either wild-type or {Delta}arg[pArg] parasites (Fig. 2). No significant differences were observed among the three strains in the absence of IFN-{gamma} priming. These findings support the hypothesis that parasite arginase may reduce host cell arginine pools as a mechanism by which iNOS activity can be regulated, even in the presence of the type 1 cytokine IFN-{gamma}.


Figure 2
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 2. NO· production by infected macrophages. Macrophages were infected with opsonized parasites using a 10:1 MOI. Culture supernatants were harvested 72 h later, and NO· accumulation was assessed using the Griess assay. Shown is the mean ± SE for three experiments, each with triplicate conditions. Statistical analyses were performed using Kruskal-Wallis one-way ANOVA on ranks.

 
Intracellular proliferation of wild-type and {Delta}arg parasites in the absence of host macrophage iNOS

Mice lacking both alleles of the NOS2 gene encoding iNOS are significantly more vulnerable to Leishmania spp. infection than wild-type mice (31). Macrophages from NOS2 knockout mice on a C57BL/6 background were exploited to further explore the hypothesis that enhanced killing of {Delta}arg parasites by macrophages is due to increased availability of arginine and consequent increased iNOS activity. If the hypothesis is correct, the enhanced killing of {Delta}arg parasites should be ameliorated in the absence of host cell iNOS. The in vitro survival of wild-type, {Delta}arg, and {Delta}arg[pArg] parasites in BMMs from iNOS-deficient mice were compared with that in BMMs from wild-type C57BL/6 mice (Fig. 3). No significant differences among entry rates of any of the parasite strains were observed between macrophages of wild-type C57BL/6 or iNOS knockout mice (data not shown; paired t test). Similar to data obtained with BALB/c BMMs shown in Fig. 1, the {Delta}arg parasites did not survive as well as wild-type or {Delta}arg[pArg] parasites in C57BL/6 macrophages. However, the absence of host cell iNOS in BMMs from NOS2 gene-deficient mice resulted in a significantly enhanced survival of {Delta}arg parasites. Differences among the intracellular survival of wild-type, {Delta}arg, or {Delta}arg[pArg] parasites in iNOS knockout BMMs were statistically insignificant. These data suggest that the increased sensitivity of {Delta}arg parasites to macrophage killing is dependent on the presence of host iNOS.


Figure 3
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 3. Survival of {Delta}arg L. mexicana in BMMs derived from iNOS KO mice. Macrophages were infected with opsonized parasites (5:1 MOI) for 30 min before removing extracellular parasites. Forty-eight hours postinfection, samples were collected and infection levels were quantified using light microscopy. Values represent the mean ± SE of two experiments, each performed in triplicate. Statistical analyses within BMM types, comparing WT to {Delta}arg to {Delta}arg[pArg], were conducted using Kruskal-Wallis one-way ANOVA on ranks. NS denotes a lack of significant difference.

 
In vivo infection with control vs {Delta}arg L. mexicana

To assess the effects of a genetic lack of parasite arginase on virulence in an animal model, BALB/c mice were infected in one hind footpad with wild-type, {Delta}arg, or {Delta}arg[pArg] stationary phase promastigotes (Fig. 4). Although footpad swelling was similar among the three parasite strains during the first 4 wk of infection, the lesion size in mice infected with wild-type L. mexicana increased more rapidly than {Delta}arg- or {Delta}arg[pArg]-induced lesions in the ensuing weeks (Fig. 4, left panel). Lesions from {Delta}arg[pArg] parasites were initially smaller than those caused by wild-type parasites, but they eventually developed to the same size as the wild-type controls. In contrast, mice infected with {Delta}arg parasites developed lesions more slowly than mice infected with the other strains. Limiting dilution assay of parasites recovered from each footpad at 4 and 16 wk postinfection suggested the smaller size lesions in mice infected with {Delta}arg parasites could be ascribed to lower parasite numbers in these lesions (Fig. 4, right panel). Statistically significant differences in lesion size between footpads infected with wild-type vs {Delta}arg parasites occurred at all time points between 8 and 17 wk postinfection with the exception of week 13 (p < 0.05).


Figure 4
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 4. Arginase activity correlates with footpad lesion size. Left panel, Mice (n = 5) were infected with 106 stationary phase L. mexicana promastigotes, and lesion sizes were measured as described in Materials and Methods. The data presented are representative of three independent experiments. Values are the means ± SD lesion size in five mice per condition. *, Values for {Delta}arg mice were significantly different from wild-type controls at all time points after 7 wk except week 13 (p < 0.01, Student’s t test). Right panel, Eight BALB/c mice were infected in the footpad with wild-type, {Delta}arg, or {Delta}arg[pArg] L. mexicana. Four or 16 wk later, footpads were excised and parasites quantified by limiting dilution. Shown are the mean ± SE parasite loads. Statistics were done using ANOVA.

 
Immune responses to infection with wild-type, {Delta}arg, or {Delta}arg[pArg] parasites

The changes in NO· levels elicited by the three parasite strains reflect shifts in the microbicidal activity of the macrophage. To determine whether there was an associated adjustment in the adaptive immune response, we examined the variations in type 1, 2, and 3 cytokine levels produced by draining lymph node cells of infected animals after restimulation in vitro with live promastigote Ag (Fig. 5). The decreased lesion sizes of mice infected with {Delta}arg organisms was correlated with significantly increased IFN-{gamma} and decreased IL-4 levels compared with lymph node cells derived from either wild-type or {Delta}arg[pArg] parasites. The amount of IL-10 in lymph node cells from mice infected with the null mutant was also diminished compared with the wild-type and {Delta}arg[pArg] controls, although the differences were within the statistical margins of error.


Figure 5
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 5. Cytokine responses in mice infected with wild-type or mutant parasites. BALB/c mice were injected with either 106 L. mexicana wild-type (WT), {Delta}arg, or {Delta}arg[pArg] parasites in a hind footpad. Five mice from each group were euthanized four weeks after infection. Single-cell suspensions from the draining lymph nodes were cultured in the absence or presence of live L. mexicana Ag. Shown are the levels of (left panel) IFN-{gamma} (nanograms per milliliter), (middle panel) IL-4 (picograms per milliliter), and (right panel) IL-10 (nanograms per milliliter) released into supernatants after 72 h stimulation with Ag. Statistical analyses were done using Kruskal-Wallis one-way ANOVA on ranks.

 
Lack of a detectable disease-exacerbating role for TGF-β

Susceptibility of BALB/c mice to L. chagasi correlates with increased local production of TGF-β (28), a macrophage-deactivating cytokine that is known to induce arginase expression in mammalian macrophages (32). Due to the inverse relationship between TGF-β and IFN-{gamma}, we investigated whether TGF-β activity might be lower in mice infected with {Delta}arg than with wild-type or {Delta}arg[pArg] parasites, correlating with their decreased intracellular survival. Because ELISAs detect both inactive and active TGF-β whereas bioassays are more sensitive indicators of TGF-β activity, an assay using a transfected cell line expressing luciferase under control of the TGF-β-responsive plasminogen activator inhibitor promoter was used (27). Infection with wild-type L. mexicana did not lead to detectable bioactive TGF-β activity, although inactive TGF-β was present. Surprisingly, mice infected with {Delta}arg L. mexicana showed significant increases in both bioactive and total TGF-β levels compared with mice infected with wild-type or add-back parasites (Fig. 6). Thus, the lower level of infection with {Delta}arg L. mexicana cannot be ascribed to a decrease in the suppressor cytokine TGF-β. The mechanism leading to increased TGF-β during {Delta}arg infection and whether this is a compensatory response to other changes in the local tissue cannot be discerned from these data.


Figure 6
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 6. TGF-β production during infection with L. mexicana. Draining lymph node cells from lesions of BALB/c mice (n = 5) infected for 4 wk with wild-type, {Delta}arg, or {Delta}arg[pArg] parasites were cultivated for 48 h in the absence of serum. Total and bioactive TGF-β released into supernatant was measured using the MvLu-plasminogen activator inhibitor luciferase assay. Results were quantified by comparison to a standard curve of rTGF-β. Shown is the mean ± SD of one representative experiment from three total assays, each performed in triplicate. Statistical analyses were completed using one-way ANOVA.

 
Other suppressive mechanisms might be active as well within tissue. IL-10 and IL-4 were not found to increase but rather to diminish in cultured cells from mice infected with {Delta}arg parasites (Fig. 5). Other factors that could influence the response were not measured, such as PGs. TGF-β is only one of several immunosuppressive molecules that would be expected to influence the course of disease.

Histopathology in infected animals

NO· generation can lead to nitration of tyrosyl residues in proteins. These nitrotyrosines can be used as a marker of the local toxic effects of NO (33). To determine whether there is increased NO· generation in vivo during infection with {Delta}arg parasites, frozen sections were prepared from the footpads of mice infected with wild-type, {Delta}arg, or {Delta}arg[pArg] parasites and stained with Ab to nitrotyrosine residues (Fig. 7, A–C). There was an increase in nitrosylated tyrosine staining in the foot pads of mice infected with {Delta}arg compared with either wild-type or {Delta}arg[pArg] parasites. H&E staining of morphology revealed the presence of amastigotes in the dermis and epidermis from the region of increased staining (Fig. 7, D–F).


Figure 7
View larger version (121K):
[in this window]
[in a new window]

  		FIGURE 7. Immunohistochemical detection of nitrosylated tyrosine in infected footpads. Enhanced nitrotyrosine modifications were detected in footpad sections of mice infected with {Delta}arg parasites. A–C, Sections from infected animals were probed with TOPRO-3 nuclear stain and with rabbit polyclonal anti-nitrotyrosine (followed by Alexa Fluor 546-labeled goat anti-rabbit Ig and mounted with Vectashield. The slides were examined on a Zeiss confocal. Micrographs are representative of three repeats with similar findings. D–F, H & E staining of similar section of OCT embedded footpad. Due to the small size of amastigotes, a higher magnification is shown in B compared with A. Magnified regions in H & E stains correspond to the base of the epidermis. Scale bar, 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The manifestations of leishmaniasis result from a balance between host microbicidal and parasite defense mechanisms. Leishmania spp. are obligate intracellular parasites residing primarily in host macrophages. One of the most important antimicrobial effector molecules in murine macrophages is NO· (9, 10, 34), and a role for NO· in human macrophage microbicidal function has been documented in a few instances (29, 35, 36). NO· is produced from arginine by iNOS, the product of the NOS2 gene. Arginase competes for the arginine substrate of iNOS by converting the amino acid to ornithine and liberating urea. Ornithine, in turn, is the immediate precursor for the synthesis of polyamines, essential growth factors for Leishmania (13). Thus, the balance between iNOS and arginase activities may determine whether the intracellular environment within the macrophage is microbicidal or supportive of Leishmania survival and multiplication.

The outcome of Leishmania infection in mice is dependent on whether the host develops an adaptive type 1 immune response resulting in production of IFN-{gamma} and consequent microbicidal activity, or whether the type 1 response is suppressed by type 2 cytokines (e.g., IL-4) or other factors such as TGF-β (2, 3). The immune response is determined in part by the parasite species initiating infection and in part by host factors. The course of murine infection with L. major has been shown to be under genetically determined immunoregulatory controls that are different from those associated with L. mexicana infection (37, 38, 39). Whereas a majority of inbred mouse strains develop self-healing lesions when infected s.c. with L. major, virtually all mice develop rapidly growing large nonhealing lesions full of parasites following s.c. L. mexicana infection (40, 41). BALB/c mice are an exception in that both parasites cause progressive disease (3, 4, 40, 41).

There is an extensive body of literature indicating that the type 2 cytokines (IL-4, IL-9, IL-13) are associated with murine susceptibility to L. major infection (reviewed in Refs. 1 and 3) However, the contribution of type 2 cytokines to progressive infection with other Leishmania species is variable. For instance, during L. chagasi infection the type 1 response is inhibited by TGF-β (42), whereas type 1 immunity is inhibited by other non-IL-4 factors during infection with L. mexicana and Leishmania donovani (4, 43).

Leishmania promote their intracellular survival within macrophages through mechanisms that enable them to resist or inactivate reactive oxygen species generated by reactions of the NADPH oxidase and iNOS enzymes (44, 45, 46). It is becoming apparent that Leishmania also have mechanisms by which they can manipulate the infected host cell such that microbicidal responses are blunted or fail to develop. Examples include the ability of the parasite to suppress macrophage protein kinase C activation, MHC class II expression, and IFN-{gamma} pathway signaling (47, 48, 49).

Previous studies have demonstrated that functional arginase is important for Leishmania spp. survival. The growth of both L. major and L. infantum in BALB/c macrophages is inhibited by N{omega}-hydroxy-L-arginine (called nor-NOHA or LOHA in different publications), and this effect is reversed by inhibiting iNOS with N-monomethyl-L-arginine (L-NMMA) (16). Arginase I protein levels progressively increase during lesion development in susceptible BALB/c mice, whereas a low protein level is present in self-healing resistant C57BL/6 mice infected with L. major (7).

Arginase inhibitors used in the above studies are active against both host and parasite arginase. During the current investigation, the specific role of leishmanial arginase as a virulence factor that acts directly on host cell was analyzed. A genetic strategy exploiting a {Delta}arg null mutant of L. mexicana with wild-type and complemented {Delta}arg controls was implemented (14). The {Delta}arg knockout was impaired in its ability to survive in vitro in macrophages and in vivo in mice. The growth defect of the mutant in axenic culture was reversed by the addition of exogenous putrescine or ornithine, which provided precursors for polyamine synthesis. However, the intracellular growth defect in the macrophage was not reversed by putrescine or ornithine despite evidence that the parasites were able to scavenge these compounds when added extracellularly. Therefore, we hypothesized that the accumulation of the arginase substrate, rather than merely the deficiency of the arginase product, contributed to the defective intracellular growth.

Consistent with the above hypothesis, the {Delta}arg elicited increased NO· production by infected macrophages. Furthermore, the differences observed between {Delta}arg and wild-type or {Delta}arg[pArg] survival were abrogated in host macrophages that lack iNOS, suggesting that survival of {Delta}arg was impaired at least in part because host iNOS activity was greater in these macrophages compared with macrophages infected with wild-type parasites. An increase in nitrosylated tyrosine staining in {Delta}arg-infected mouse tissues supported this hypothesis.

NO· synthesis is regulated to a great extent at the level of transcription of the NOS2 gene encoding iNOS. IFN-{gamma}, a type 1 cytokine, up-regulates NOS2 transcription and in so doing enhances macrophage microbicidal capacity (50). Not as well-recognized but equally as important, iNOS activity has been shown to be regulated at the level of substrate availability. Arginine depletion can occur by IL-4- or IL-13-mediated up-regulation of arginase (17), or pathogen arginase can scavenge arginine. For example, wild-type but not arginase-deficient H. pylori consume arginine and inhibit NO· production by eukaryotic cells (19). According to the data reported herein, we hypothesize that L. mexicana arginase acts similarly to consume local arginine supplies and thus diminish iNOS activity in murine macrophages.

Mouse infection with {Delta}arg parasites lacking arginase developed a significantly different adaptive immune response from those infected with wild-type or arginase "add-back" controls. Ag-specific cellular responses in lymph nodes draining lesions caused by {Delta}arg parasites were characterized by a type 1 response with increased IFN-{gamma} and decreased IL-4 and IL-10 compared with lesions caused by wild-type or {Delta}arg[pArg] parasites. We cannot discern from these data whether the phenotype observed in mice infected with {Delta}arg was due to poor establishment of infection or preferential induction of a type 1 immune response, although we favor the former possibility. These data suggest that parasite arginase plays a crucial role in directing host macrophage microbicidal activity through iNOS substrate depletion, and that infection with the parasite favors a type 1 response.

In contrast to our observations using {Delta}arg L. mexicana, pharmacological inhibition of arginase did not blunt the type 2 immune response to L. major (15). The difference between the two discrepant findings could be attributable to different roles of arginase between the two Leishmania spp., or to the different effects of partial vs total abrogation of arginase activity on the immune response possibly due to a more efficient attenuation of virulence in the {Delta}arg mutant.

Leishmania arginase plays a critical role in the synthesis of the polyamines putrescine and spermidine. The auxotrophy conferred by the {Delta}arg null mutation in vitro can be bypassed in promastigotes by either low concentrations of putrescine, high concentrations of ornithine or spermidine, or episomal complementation (14). Even though the virulence of {Delta}arg L. mexicana is diminished in mice and in the infected macrophage, slow proliferation of footpad lesions induced by {Delta}arg parasites was detectable and parasites were recovered after 16 wk. This suggests that the mutant parasites were able to scavenge ornithine or polyamines from the host. The decreased virulence of {Delta}arg compared with wild-type and {Delta}arg[pArg] L. mexicana could reflect the combined effects of 1) enhanced iNOS activity due to increased availability of arginine, 2) lower levels of trypanothione in the absence of arginase-derived spermidine, making the parasite more vulnerable to NO·-mediated toxicity; 3) enhanced development of cells producing the type 1 cytokine IFN-{gamma}; and 4) decreased endogenous parasite polyamines (51).

Only a few studies document a role for parasite-encoded molecules in directing the host microbicidal activities (49, 52). Data presented in this report suggest that parasite-encoded arginase can be added to the list of parasite-derived molecules that actively modify infection through its influence on the microbicidal function of the macrophage infected with L. mexicana.


   Acknowledgment
 
We are grateful to Melissa Miller for her technical expertise and support. We thank Jian Shao and the University of Iowa Central Microscopy facility for training and help with microscopy. We also thank Paul Reimann for help in preparation of illustrations.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI45540, AI067874, and AI048822 (to M.E.W.), AI10096 (to S.C.R.), and AI41622 (to B.U.), and a Merit Review and Persian Gulf grant from the Department of Veterans Affairs (to M.E.W.). The work was performed while U.G. served as a fellow on National Institutes of Health T32 AI07511. Back

2 Current address: Pacific University, School of Pharmacy, Hillsboro, OR 97123. Back

3 Address correspondence and reprint requests to Dr. Mary E. Wilson, University of Iowa, SW34-GH, 200 Hawkins Drive, Iowa City, IA 52242. E-mail address: mary-wilson{at}uiowa.edu Back

4 Abbreviations used in this paper: iNOS, inducible NO synthase; BMM, bone marrow macrophage; MOI, multiplicity of infection; MvLu, mink lung fibroblast. Back

Received for publication November 1, 2006. Accepted for publication October 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Sacks, D., N. Noben-Trauth. 2002. The immunology of susceptibility and resistance to Leishmania major in mice. Nat. Rev. Immunol. 2: 845-858. [Medline]
  2. Wilson, M. E., S. M. B. Jeronimo, R. D. Pearson. 2005. Immunopathogenesis of infection with the visceralizing Leishmania species. Microb. Pathog. 38: 147-160. [Medline]
  3. Scott, P.. 2003. Development and regulation of cell-mediated immunity in experimental leishmaniasis. Immunol. Res. 27: 489-498. [Medline]
  4. Jones, D. E., L. U. Buxbaum, P. Scott. 2000. IL-4 independent inhibition of IL-12 responsiveness during Leishmania amazonensis infection. J. Immunol. 165: 364-372. [Abstract/Free Full Text]
  5. Wilson, M. E., B. M. Young, B. L. Davidson, K. A. Mente, S. E. McGowan. 1998. The importance of transforming growth factor-β in murine visceral leishmaniasis. J. Immunol. 161: 6148-6155. [Abstract/Free Full Text]
  6. Chang, K.-P., G. Chaudhuri. 1990. Molecular determinants of Leishmania virulence. Annu. Rev. Microbiol. 44: 499-529. [Medline]
  7. Iniesta, V., J. Carcelen, L. Molano, P. M. V. Peixoto, E. Redondo, P. Parra, M. Mangas, I. Monroy, M. L. Campo, C. G. Nieto, I. Corraliza. 2005. Arginase I induction during Leishmania major infection mediates the development of disease. Infect. Immun. 73: 6085-6090. [Abstract/Free Full Text]
  8. Mosser, D. M.. 2003. The many faces of macrophage activation. J. Leukocyte Biol. 73: 209-212. [Free Full Text]
  9. Mauel, J., A. Ransijn, Y. Buchmuller-Rouiller. 1991. Killing of Leishmania parasites in activated murine macrophages is based on an L-arginine-dependent process that produces nitrogen derivatives. J. Leukocyte Biol. 49: 73-82. [Abstract]
  10. Liew, F. Y., X.-Q. Wei, L. Proudfoot. 1997. Cytokines and nitric oxide as effector molecules against parasitic infections. Philos. Trans. R. Soc. Lond. 352: 1311-1315. [Abstract/Free Full Text]
  11. Diefenbach, A., H. Schindler, N. Donhauser, E. Lorenz, T. Laskay, J. MacMicking, M. Rollinghoff, I. Gresser, C. Bogdan. 1998. Type 1 interferon (IFN{alpha}/β) and type 2 nitric oxide synthase regulate the innate immune response to a protozoan parasite. Immunity 8: 77-87. [Medline]
  12. Wei, X.-Q., I. G. Charles, A. Smith, J. Ure, G.-J. Feng, F.-P. Huang, D. Xu, W. Muller, S. Moncada, F. Y. Liew. 1995. Altered immune responses in mice lacking inducible nitric oxide synthase. Nature 375: 408-411. [Medline]
  13. Vincendeau, P., A. P. Gobert, S. Dalouede, D. Moynet, M. D. Mossalayi. 2003. Arginases in parasitic diseases. Trends Parasitol. 19: 9-12. [Medline]
  14. Roberts, S. C., M. J. Tancer, M. R. Polinsky, K. M. Gibson, O. Heby, B. Ullman. 2004. Arginase plays a pivotal role in polyamine precursor metabolism in Leishmania: characterization of gene deletion mutants. J. Biol. Chem. 279: 23668-23678. [Abstract/Free Full Text]
  15. Kropf, P., J. M. Fuentes, E. Fahnrich, L. Arpa, S. Herath, V. Weber, G. Soler, A. Celada, M. Modolell, I. Muller. 2005. Arginase and polyamine synthesis are key factors in the regulation of experimental leishmaniasis in vivo. FASEB J. 19: 1000-1002. [Abstract/Free Full Text]
  16. Iniesta, V., L. C. Gomez-Nieto, I. Corraliza. 2001. The inhibition of arginase by N-hydroxy-L-arginine controls the growth of Leishmania inside macrophages. J. Exp. Med. 193: 777-783. [Abstract/Free Full Text]
  17. Rutschman, R., R. Lang, M. Hesse, J. N. Ihle, T. A. Wynn, P. J. Murray. 2001. Cutting edge: Stat6-dependent substrate depletion regulates nitric oxide production. J. Immunol. 166: 2173-2177. [Abstract/Free Full Text]
  18. Gobert, A. P., S. Daulouede, M. Lepoivre, J. L. Boucher, B. Bouteille, A. Buguet, R. Cespublio, B. Veyret, P. Vincendeau. 2000. L-arginine availability modulates local nitric oxide production and parasite killing in experimental trypanosomiasis. Infect. Immun. 68: 4653-4657. [Abstract/Free Full Text]
  19. Gobert, A. P., D. J. McGee, M. Akhtar, G. L. Mendz, J. C. Newton, Y. Cheng, H. L. T. Mobley, K. T. Wilson. 2001. Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells: a strategy for bacterial survival. Proc. Natl. Acad. Sci. USA 98: 13844-13849. [Abstract/Free Full Text]
  20. Rueesll, A. S., U. T. Ruegg. 1980. Arginase production by peritoneal macrophages: a new assay. J. Immunol. Methods 32: 375-382. [Medline]
  21. Corraliza, I. M., G. Soler, K. Eichmann, M. Modolell. 1995. Arginase induction by suppressors of nitric oxide synthesis (IL-4, IL-10 and PGE2) in murine bone marrow-derived macrophages. Biochem. Biophys. Res. Commun. 206: 667-673. [Medline]
  22. Rodriguez, N. E., H. K. Chang, M. E. Wilson. 2004. A novel program of macrophage gene expression induced by phagocytosis of Leishmania chagasi. Infect. Immun. 72: 2111-2112. [Abstract/Free Full Text]
  23. Coligan, J. E., A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober. 1991. Current Protocols in Immunology John Wiley and Sons, New York.
  24. Liew, F. Y., Y. Li, D. Moss, C. Parkinson, M. V. Rogers, S. Moncada. 1991. Resistance to Leishmania major infection correlates with the induction of nitric oxide synthase in murine macrophages. Eur. J. Immunol. 21: 3009-3014. [Medline]
  25. Tracey, W. R.. 1992. Spectrophotometric detection of nitrogen oxides. Neuroprotocols: Comp. Methods Neurosci. 1: 125-131.
  26. Buffet, P. A., A. Sulahian, Y. J. F. Garin, N. Nassar, F. Derouin. 1995. Culture microtitration: a sensitive method for quantifying Leishmania infantum in tissues of infected mice. Antimicrob. Agents Chemother. 39: 2167-2168. [Abstract]
  27. Abe, M., J. G. Harpel, C. N. Metz, I. Nunes, D. J. Loskutoff, D. B. Rifkin. 2000. An assay for transforming growth factor-β using cells transfected with a plasminogen activator inhibitor-1 promoter luciferase construct. Anal. Biochem. 216: 276-284.
  28. Gantt, K. R., S. Schultz-Cherry, N. Rodriguez, S. M. B. Jeronimo, E. T. Nascimento, T. L. Goldman, T. J. Recker, M. A. Miller, M. E. Wilson. 2003. Activation of TGF-β by Leishmania chagasi: importance for parasite survival in macrophages. J. Immunol. 170: 2613-2620. [Abstract/Free Full Text]
  29. Gantt, K. R., T. L. Goldman, M. A. Miller, M. L. McCormick, S. M. B. Jeronimo, E. T. Nascimento, B. E. Britigan, M. E. Wilson. 2001. Oxidative responses of human and murine macrophages during phagocytosis of Leishmania chagasi. J. Immunol. 167: 893-901. [Abstract/Free Full Text]
  30. Proudfoot, L., A. V. Nikolaev, G.-J. Feng, X.-Q. Wei, M. A. Ferguson, J. S. Brimacombe, F. Y. Liew. 1996. Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmania lipophosphoglycan in murine macrophages. Proc. Natl. Acad. Sci. USA 93: 10984-10989. [Abstract/Free Full Text]
  31. Murray, H. W., C. F. Nathan. 1999. Macrophage microbicidal mechanisms in vivo: reactive nitrogen versus oxygen intermediates in the killing of intracellular visceral Leishmania donovani. J. Exp. Med. 189: 741-746. [Abstract/Free Full Text]
  32. Boutard, V., R. Havouis, B. Fouqueray, C. Philippe, J.-P. Moulinoux, L. Baud. 1995. Transforming growth factor-β stimulates arginase activity in macrophages. J. Immunol. 155: 2077-2084. [Abstract]
  33. Radi, R.. 2004. Nitric oxide, oxidants, and protein tyrosine nitration. Proc. Natl. Acad. Sci. USA 101: 4003-4008. [Abstract/Free Full Text]
  34. Green, S. J., M. S. Meltzer, J. B. Hibbs, Jr, C. A. Nacy. 1990. Activated macrophages destroy intracellular Leishmania major amastigotes by an L-arginine-dependent killing mechanism. J. Immunol. 144: 278-283. [Abstract]
  35. Nicholson, S., G. Bonecini-Almeida Mda, J. R. Lapa e Silva, C. Nathan, Q. W. Xie, R. Mumford, J. R. Weidner, J. Calaycay, J. Geng, N. Boechat. 1996. Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis. J. Exp. Med. 183: 2293-2302. [Abstract/Free Full Text]
  36. Vouldoukis, I., P.-A. Becherel, V. Riveros-Moreno, M. Arock, O. da Silva, P. Debre, D. Mazier, M. D. Mossalayi. 1997. Interleukin-10 and interleukin-4 inhibit intracellular killing of Leishmania infantum and Leishmania major by human macrophages by decreasing nitric oxide generation. Eur. J. Immunol. 27: 860-865. [Medline]
  37. Alexander, J., P. M. Kaye. 1985. Immunoregulatory pathways in murine leishmaniasis: different regulatory control during Leishmania mexicana and Leishmania major infections. Clin. Exp. Allergy 61: 674-682.
  38. Roberts, M., J. Alexander, J. M. Blackwell. 1990. Genetic analysis of Leishmania mexicana infection in mice: single gene (Scl-2) controlled predisposition to cutaneous lesion development. J. Immunogenet. 17: 89-100. [Medline]
  39. Roberts, M., B. A. Mock, J. M. Blackwell. 1993. Mapping of genes controlling Leishmania major infection in CXS recombinant inbred mice. Eur. J. Immunogenet. 20: 349-362. [Medline]
  40. Ji, J., J. Sun, L. Soong. 2003. Impaired expression of inflammatory cytokines and chemokines at early stages of infection with Leishmania amazonensis. Infect. Immun. 71: 4278-4288. [Abstract/Free Full Text]
  41. Buxbaum, L. U., H. Denise, G. H. Coombs, J. Alexander, J. C. Mottram, P. Scott. 2003. Cysteine protease B of Leishmania mexicana inhibits host Th1 responses and protective immunity. J. Immunol. 171: 3711-3717. [Abstract/Free Full Text]
  42. Wilson, M. E., M. Sandor, A. M. Blum, B. M. Young, A. Metwali, D. Elliott, R. G. Lynch, J. V. Weinstock. 1996. Local suppression of IFN-{gamma} in hepatic granulomas correlates with tissue-specific replication of Leishmania chagasi. J. Immunol. 156: 2231-2239. [Abstract]
  43. Ji, J., J. Sun, H. Qi, L. Soong. 2002. Analysis of T helper cell responses during infection with Leishmania amazonensis. Am. J. Trop. Med. Hyg. 66: 338-345. [Abstract]
  44. Miller, M. A., S. E. McGowan, K. R. Gantt, M. Champion, S. Novick, K. A. Andersen, C. J. Bacchi, N. Yarlett, B. E. Britigan, M. E. Wilson. 2000. Inducible resistance to oxidant stress in the protozoan Leishmania chagasi. J. Biol. Chem. 275: 33883-33889. [Abstract/Free Full Text]
  45. Levick, M. P., E. Tetaud, A. H. Fairlamb, J. M. Blackwell. 1998. Identification and characterisation of a functional peroxidoxin from Leishmania major. Mol. Biochem. Parasitol. 96: 125-137. [Medline]
  46. Paramchuk, W. J., S. O. Ismail, A. Bhatia, L. Gedamu. 1997. Cloning, characterization and overexpression of two iron superoxide dismutase cDNAs from Leishmania chagasi: role in pathogenesis. Mol. Biochem. Parasitol. 90: 203-221. [Medline]
  47. Descoteaux, A., S. J. Turco. 1993. The lipophosphoglycan of Leishmania and macrophage protein kinase C. Parasitol. Today 9: 468-471. [Medline]
  48. Reiner, N. E., W. Ng, W. R. McMaster. 1987. Parasite-accessory cell interactions in murine leishmaniasis. II. Leishmania donovani suppresses macrophage expression of class I and class II major histocompatibility complex gene products. J. Immunol. 138: 1926-1932. [Abstract]
  49. Nandan, D., T. Yi, M. Lopez, C. Lai, N. E. Reiner. 2002. Leishmania EF-1{alpha} activates the Src homology 2 domain containing tyrosine phosphatase SHP-1 leading to macrophage deactivation. J. Biol. Chem. 277: 50190-50197. [Abstract/Free Full Text]
  50. Spink, J., T. Evans. 1997. Binding of the transcription factor interferon regulatory factor-1 to the inducible nitric-oxide synthase promoter. J. Biol. Chem. 272: 24417-24425. [Abstract/Free Full Text]
  51. Romao, P. R. T., J. Tovar, S. G. Fonseca, R. H. Moraes, A. K. Cruz, J. S. Hothersall, A. A. Noronha-Dutra, S. H. Ferreira, F. Q. Cunha. 2006. Gluthathione and the redox control system trypanthione/trypanothione reudcatse are involved in the protection of Leishmania spp. against nitrosothiol-induced cytotoxicity. Brazilian J. Med. Biol. Res. 39: 355-363. [Medline]
  52. Olivier, M., D. J. Gregory, G. Forget. 2005. Subversion mechanisms by which Leishmania parasites can escape the host immune response: a signaling point of view. Clin. Microbiol. Rev. 18: 293-305. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Infect. Immun.Home page
J. M. Boitz, P. A. Yates, C. Kline, U. Gaur, M. E. Wilson, B. Ullman, and S. C. Roberts
Leishmania donovani Ornithine Decarboxylase Is Indispensable for Parasite Survival in the Mammalian Host
Infect. Immun., February 1, 2009; 77(2): 756 - 763.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gaur, U.
Right arrow Articles by Wilson, M. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gaur, U.
Right arrow Articles by Wilson, M. E.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Medline Plus Health Information
*Leishmaniasis
Hazardous Substances DB
*NITRIC OXIDE

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS