Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.1003484 on March 23, 2004

Published online before print March 23, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1003484v1
76/1/48    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kropf, P.
Right arrow Articles by Müller, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kropf, P.
Right arrow Articles by Müller, I.
(Journal of Leukocyte Biology. 2004;76:48-57.)
© 2004 by Society for Leukocyte Biology

Infection of C57BL/10ScCr and C57BL/10ScNCr mice with Leishmania major reveals a role for Toll-like receptor 4 in the control of parasite replication

P. Kropf*, N. Freudenberg{dagger}, C. Kalis{ddagger}, M. Modolell{ddagger}, S. Herath*, C. Galanos{ddagger}, M. Freudenberg{ddagger} and I. Müller*,1

* Imperial College London, Faculty of Medicine, Department of Immunology, United Kingdom;
{dagger} Institute of Pathology, University Hospital Freiburg, Germany; and
{ddagger} Max-Planck-Institute for Immunbiology, Freiburg, Germany

1Correspondence: Imperial College London, Faculty of Medicine, Division of Investigative Science, Department of Immunology, Norfolk Place, London, W2 1PG, United Kingdom. E-mail: i.muller{at}imperial.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The innate immune system is essential for host defense; it senses the presence of potentially pathogenic-invading microorganisms, and the contribution of Toll-like receptors (TLRs) to this response is increasingly recognized. In the present study, we investigated the contribution of TLR4 to the course of cutaneous leishmaniasis in vivo. We used C57BL/10ScNCr (TLR40/0) and C57BL/10ScCr [TLR4/interleukin-12 (IL-12)Rß20/0] mice and compared the course of Leishmania major infection, parasite load, cell recruitment, and cytokine profile with those of wild-type C57BL/10ScSn mice. Our results confirm the importance of IL-12 receptor-mediated signaling in resistance to L. major infections. Importantly, we show that the lack of TLR4 results in an increased permissiveness for parasite growth during the innate and adaptive phase of the immune response and in delayed healing of the cutaneous lesions. The use of the tlr4 transgenic mouse strain TCr5 demonstrated unequivocally that TLR4 contributes to the efficient control of Leishmania growth in vivo.

Key Words: TLR4 • IL-12Rß2 • Leishmania


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The leishmaniases are vector-borne diseases that represent a major public health problem affecting the lives of millions of people worldwide [1 ]. The diseases are caused by more than 20 different Leishmania species, they occur in 88 countries, and the incidence of disease is still spreading. There is no defined and efficient vaccine available, and resistance to currently used chemotherapeutic agents is increasing. In humans, leishmanial infections range from self-healing, cutaneous lesions to severe, nonhealing-disseminated, cutaneous, mucocutaneous, or visceral infection [2 ]. To a large extent, the clinical manifestations of the disease reflect the efficiency of the host’s immune response to the parasite. Leishmaniasis, as leprosy, is a spectrum of diseases that presents in a range of clinical forms, and many of the clinical manifestation of human leishmaniasis are mimicked in inbred strains of mice [3 ]. Experimental infection of mice with Leishmania major is widely used as a model for resistance and susceptibility to leishmaniasis. The majority of inbred strains of mice (e.g., C57BL/6, C3H, C57BL/10) can control L. major infection, and only a few strains of mice (e.g., BALB/c) develop progressive, nonhealing disease [4 5 6 ]. It is generally accepted that the outcome of Leishmania infection is determined by the adaptive T helper (Th) cell responses and their interactions with parasitized host cells, usually macrophages. The fate of the intracellular parasites strongly depends on the induction of two enzymes in activated macrophages, inducible nitric oxide synthase (iNOS) and arginase. iNOS oxidizes L-arginine in a two-step process into NO, and NO synthesis correlates with parasite killing [7 8 9 ]. The alternative metabolic pathway of L-arginine is catalyzed by arginase and converts L-arginine to urea and L-ornithine, the main intracellular source for the synthesis of polyamines, which are essential for the growth of the parasites [10 11 12 ]. Although macrophages are the primary host cells for the intracellular Leishmania parasites, polymorphonuclear granulocytes (PMNs) are able to internalize the parasites in the early phase of infection [13 ], and fibroblasts have been identified as important host cells in the chronic phase of infection [14 ]. Th1 responses are associated with healing and parasite killing, whereas Th2 responses are associated with nonhealing disease and uncontrolled parasite growth [4 5 6 ]. Although the adaptive immune responses to these intracellular pathogens are well characterized, the mechanisms leading to these polarized Th cell responses are still not fully understood. Several reports indicate that the outcome of L. major infection may be determined in the very early phase of infection [15 16 17 18 ]. In contrast to the well-characterized adaptive immune response, the interactions of Leishmania parasites with the innate immune system and consequent implications of these interactions on the adaptive immune response are only starting to be investigated.

The innate immune system is essential for host defense; it senses the presence of potentially pathogenic-invading microorganisms, and the contribution of Toll-like receptors (TLRs) to this response is increasingly recognized [19 20 21 22 ]. TLRs recognize pathogen-associated molecular patterns (PAMPs), which are characteristic of various groups of pathogens. TLRs are expressed on many cell types, including macrophages and dendritic cells (DCs), and the activation of these receptors initiates signaling pathways that result in the up-regulation of costimulatory molecules, the production of proinflammatory cytokines, and anti-microbial compounds [23 ]. Mammalian TLRs are a family of at least 10 pattern-recognition receptors, all of which share an intracellular domain, Toll/interleukin-1 receptor (IL-1R), and signal through the MyD88 adaptor protein [24 25 26 ]. TLRs recognize a wide spectrum of ligands including lipopolysaccarides (LPS), bacterial lipoproteins, proteins, and nucleic acids [22 ]. In contrast to the ample evidence of recognition of bacterial, fungal, and viral PAMPs, very little work has been done on the role of TLRs in recognition of parasites. Recently, glycosylphosphatidylinositol anchors and glycoinositolphospholipids from Trypanosoma cruzi were shown to activate TLR2 [27 ], and lipophosphoglycan (LPG) from L. major was reported to activate natural killer (NK) cells through TLR2 [28 ]. Indirect evidence for the involvement of TLRs in the host response to infections with Leishmania parasites originates from studies with genetically resistant mice lacking the MyD88 adaptor protein; these mice develop progressive lesions and a polarized Th2 response [29 , 30 ].

Our previous work has shown that C57BL/10ScCr mice, which carry a null mutation in tlr4 corresponding to a 74-kb genomic deletion, removing all three exons [31 ], were unable to heal L. major infections [32 ]. In contrast, TLR4-competent controls (C57BL/10ScSn) could resolve the cutaneous lesions and control parasite growth [32 ]. However, it cannot be concluded from our previous study that the observed, nonhealing L. major infections in the C57BL/10ScCr mice are a result of the lack of TLR4, as these mice not only lack tlr4, they also exhibit an impaired interferon-{gamma} (IFN-{gamma}) response to bacteria and parasites [33 34 35 ]. The defective IFN-{gamma} response in the TLR4-deficient C57BL/10ScCr mice is caused by an inability to respond to IL-12 [36 ]. The genetic defect underlying this inability to respond to IL-12 was shown to be a result of a point mutation in the IL-12Rß2 gene, resulting in a defective IL-12Rß2 subunit [37 ]. However, they are not completely defective in their ability to produce IFN-{gamma}, as they were capable of producing this cytokine when stimulated with anti-CD3 monoclonal antibodies (mAb) or the T cell mitogen concanavalin A [32 33 34 35 ].

To answer whether TLR4 contributes to innate and adaptive immune responses to L. major, we used the progenitor strain of the C57BL/10ScCr mice, C57BL/10/ScNCr, which carries an identical null mutation in tlr4 but has an intact IL-12 responsiveness, a functional IL-12Rß2 chain, and normal IFN-{gamma} production [36 ]. In the present study, we investigated the course of L. major infection in two different strains of TLR4-deficient mice, C57BL/10ScCr and C57BL10/ScNCr, and compared it with infections in the TLR4-competent C57BL/10ScSn mice. The results of our study show that although the second defect in the IL-12Rß2 is undoubtedly a major factor in the nonhealing L. major infections, the lack of TLR4 in the IL-12Rß2 intact mice results in increased parasite growth in the innate phase of infection and a delayed healing of L. major infection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
C57BL/10ScSn (ScSn, wild-type), C57BL/10ScNCr (ScN, TLR40/0), and C57BL/10ScCr (ScCr, TLR4/IL-12Rß20/0) mice were bred under specific, pathogen-free conditions in the animal facilities at the Max-Planck-Institut für Immunbiologie (Freiburg, Germany). C57BL/10ScN mice have a homozygous deletion of 74,723 bp at the tlr4 locus, removing all three exons [38 ]. In addition to the TLR4 mutation, C57BL/10ScCr mice carry a point mutation in the IL-12Rß2 gene, which results in a truncated ß2 chain [37 ]. The tlr4 transgenic mouse strain TCr-5 (C57BL/10ScCr background) was generated as described [39 ] and was mated with C57BL/10ScN mice to obtain the TCr5/ScN mice (C57BL/10 tlr4T/0; IL-12Rß2n/n) used in this study. To discriminate between the wild-type and mutated IL-12Rß2 allele, the specific polymerase chain reaction primers (IL-12Rß2 wild-type) sense, TCC AGC TAC CTA CGG ATA AT; antisense, CTC TGC TTT CTA GCA CCT TG, and (IL-12Rß2 mutant) sense, TCC AGC TAC CTA CGG ATA AT; antisense, CTC TGC TTT CTA GCA CCT TC, were used at an annealing temperature of 57°C.

Six- to 12-week-old animals of both sexes were used in this study.

Parasites and infection
L. major LV39 (MRHO/SU/59/P strain) was maintained in a virulent state by monthly passage in mice [40 ]. For infections, 2 x 106 stationary-phase parasites were injected subcutaneously (s.c.) into the hind footpad. The lesions were measured by determining the increase in the footpad thickness compared with the uninfected contralateral footpad using a dial gauge calliper (Kröplin Schnelltaster, Schlüchtern, Germany).

Determination of parasite load
The number of living L. major parasites in infected tissues was determined using the parasite-limiting dilution assay [40 , 41 ]. Briefly, serial dilutions of the footpad homogenate were distributed in replicate wells, and the plates were incubated at 26°C. After 10–14 days, the assay was read microscopically, and the number of viable parasites in the tissue was determined as described [40 ].

Macrophages
Bone marrow was obtained by flushing the femurs of naïve mice. Bone marrow precursor cells were cultured as described previously [32 ] in hydrophobic Teflon bags (Biofolie 25, Heraeus, Hanau, Germany) in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal calf serum, 5% horse serum, and the supernatant of L929 fibroblasts at a final concentration of 15% (v/v) as a source of colony-stimulating factors, which drive the cell proliferation toward a pure population of bone marrow-derived macrophages. After 9–10 days of culture, macrophages were harvested, and 5 x 105 cells ml–1 were plated and stimulated in the presence or in the absence of 20 U ml–1 IFN-{gamma}, 200 U ml–1 tumor necrosis factor {alpha} (TNF-{alpha}), 20 U ml–1 IL-4, 1 µg ml–1 LPS, and 25 x 105 ml–1 L. major parasites.

Nitrite determination
NO2– accumulation was used as an indicator of NO production and measured by using the Griess reagent [9 , 42 ]. Culture supernatants were collected after 48 h, and equal volumes of macrophage culture supernatants and Griess reagent [1% sulphanilamide/0.1% N-(1-naphthyl)ethylenediamine dihydrochloride/2.5% H3PO4] were mixed and incubated for 10 min at room temperature. Absorbance was measured at 540 nm in a microscope plate reader (Molecular Devices, Ismaning, Germany). Nitrite concentration was determined using NaNO2 as standard.

Determination of arginase activity
Arginase activity was measured in macrophage lysates as described previously [12 ]. Briefly, cells were lysed with 100 µl 0.1% Triton X-100. After 30 min on a shaker, 100 µl 25 mM Tris-HCl was added. To 100 µl of this lysate, 10 µl 10 mM MnCl2 was added, and the enzyme was activated by heating for 10 min at 56°C. Arginine hydrolysis was conducted by incubating the lysate with 100 µl 0.5 M L-arginine (pH 9.7) at 37°C for 15–20 min. The reaction was stopped with 800 µl H2SO4 (96%)/H3PO4 (85%)/H2O (1/3/7, v/v/v). The urea concentration was measured at 540 nm after addition of 40 µl {alpha}-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 catalyzes the formation of 1 µmol urea per min.

Cytokine measurements
Cells (5x106 ml–1) from the lymph nodes draining the lesions of individual mice were restimulated with 1 x 106 ml–1 L. major promastigotes. Forty-eight hours later, the culture supernatants were harvested, and cytokines were determined by enzyme-linked immunosorbent assay (ELISA) according to the suppliers’ protocols. Detection limits were 1 U ml–1 for IFN-{gamma} and 7 pg ml–1 for IL-4.

Cell recruitment to the site of parasite inoculation
The footpads of ScSn, ScN, and ScCr mice were homogenized in Tenbroeck glass homogenizers 1 day after infection. A Ficoll gradient was performed to eliminate debris, and the recovered viable cell suspension was washed and preincubated with 1 µg rat anti-mouse mAb CD32/CD16 (Fc receptor for immunoglobulin G II/III), PharMingen, San Diego, CA] in a final volume of 50 µl in staining buffer (phosphate-buffered saline, Sigma Chemical Co., St. Louis, MO) supplemented with 3% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY) and 0.1% NaN3 (Sigma Chemical Co.) for 5 min to reduce nonspecific binding. Rat anti-mouse CD49b mAb (clone DX5, PharMingen) and rat anti-mouse CD11b mAb (clone M1/70, PharMingen) were directly added to the cells in a final volume of 1 µl and incubated for 20 min on ice. Cells were washed and resuspended in 200 µl fluorescein-activated cell sorter medium, cell-surface markers were determined by flow cytometry (EPICS XL, Coulter, Luton, UK), and the data were analyzed using the Expo32 software (Beckman Coulter, Fullerton, CA).

Histology
Following formaldehyde fixation, the footpads underwent an EDTA decalcification; they then were cut in two parts following their longitudinal axis and embedded in paraffin using standard protocols. Histological sections (5 µm-thick) were prepared of the entire cut surfaces from both parts of each footpad and stained with haematoxylin and eosin. The histological evaluation was performed under a light microscope using magnifications from ten- to 400-fold.

Statistical analyses
Experimental results were analyzed with PRISMTM version 2.0 (GraphPad, San Diego, CA) using a Student’s t-test, and differences were considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The absence of TLR4 signaling results in a delayed healing of the cutaneous lesions
We have previously shown that C57BL/10 ScCr (TLR4/IL-12Rß2) mice cannot resolve L. major infection; they develop progressive, nonhealing lesions and cannot control the parasite replication [32 ]. The results presented in Figure 1A confirm our earlier work and show that TLR4-competent ScSn mice were able to restrict parasite multiplication, whereas the TLR4/IL-12Rß2 double-deficient ScCr mice were unable to control the parasite multiplication. The crucial role of IL-12 in the control of cutaneous leishmaniasis has been documented extensively [43 44 45 46 ]; therefore, to determine whether the absence of TLR4 in the ScCr mice also plays a role in L. major infection, we compared the lesion development of C57BL/10ScSn (ScSn), C57BL/10ScNCr (ScN), and C57BL/10ScCr (ScCr) mice. As presented in Figure 1B , ScCr mice develop nonhealing lesions, which started to ulcerate after 33 days, and 39 days postinfection, the mice had to be killed as a result of the severity of the lesions. It is of interest to note that there was a delay in the onset of the lesion development in the ScCr mice; during the first 4 weeks of infection, they had significantly smaller lesions than the wild-type ScSn mice (P<0.05). The ScSn and ScN mice display a similar lesion development during the first 3 weeks; however, 4 weeks postinfection, at a time when the ScSn mice started to resolve their lesions, the ScN mice displayed significantly larger lesions (P<0.05). In addition, although the ScN mice eventually healed, they needed much longer to resolve their cutaneous lesions. The lesions persisted about one-third longer as compared with the ScSn mice (Fig. 1B) . These results suggest that signaling through TLR4 and IL-12Rß2 is necessary for the efficient control of L. major replication.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 1. (A) Parasite load in the footpads of ScSn and ScCr mice during the course of L. major infection. Groups of ScSn and ScCr mice (n=4) were infected with 2 x 106 L. major in the footpads. At the time-points indicated, the numbers of viable parasites were determined in the infected footpads by limiting-dilution assay (LDA). The values represent the average of four individual mice per group ± SEM. Data show the results of one representative experiment out of four. N.D., Not detectable; *, not applicable. (B) Lesion development in ScSn, ScN, and ScCr mice during the course of L. major infection. Groups of ScSn, ScN, and ScCr mice (n=4) were infected with 2 x 106 L. major in the footpads. The values represent the average of four individual mice per group per time-point ± SEM. Data show the results of one representative experiment out of three. *, P < 0.05.

 
C57BL/10 ScN and ScCr mice have significantly more parasites in their lesions
The pathology developing at the site of parasite inoculation is partially caused by the immune response against the invading parasite, and the local swelling does not necessarily correlate with the parasite load [47 ]. Therefore, we performed limiting dilution analysis to determine the number of viable parasites at the site of infection in the ScSn, ScN, and ScCr mice 1 day and 4 weeks postinfection. As shown in Figure 2 , as early as 24 h postinfection, ScN and ScCr mice had significantly more parasites in their footpads as compared with the wild-type ScSn mice (P<0.05). Four weeks later, the parasite load started to decline in the ScSn mice, but it was still increasing in the ScN and in the ScCr mice. In addition, the parasite burden was significantly higher in the latter groups of mice (P<0.05). The ScCr mice had to be killed 10 days later as a result of the severity of the ulceration. Ten weeks postinfection, no parasites were detectable in the footpads of the ScSn mice, but an average of 1086 parasites per lesion was still present in the footpads of the ScN mice, showing that in addition to the delayed resolution of the lesions, these mice have an impaired capacity to clear L. major parasites [48 ]. These results extend our earlier findings [32 , 48 ] and demonstrate that ScN and ScCr mice have an impaired capacity to control the parasite replication.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 2. (A) Parasite load in the footpads of ScSn, ScN, and ScCr mice 1 and 28 days postinfection. Groups of ScSn, ScN, and ScCr mice (n=4) were infected with 2 x 106 L. major in the footpads. At the time-points indicated, the numbers of viable parasites were determined in the infected footpads by LDA. The values represent the average of four individual mice per group ± SEM. Data show the results of one representative experiment out of four. (B) Activation of NO synthesis in macrophages from ScSn, ScN, and ScCr mice. Bone marrow macrophages (5x105 ml–1) from naïve, TLR4-competent ScSn control (solid bars), ScN (open bars), and ScCr (hatched bars) mice were cultured in the presence and/or in the absence of 25 x 105 ml–1 L. major promastigotes and the indicated combinations of LPS (1 µg ml–1), IFN-{gamma} (20 U ml–1), and TNF-{alpha} (200 U ml–1). Culture supernatants were collected after 48 h and tested for NO2–. Data show the results of one representative experiment out of four. (C) Induction of arginase activity in macrophages from ScSn, ScN, and ScCr mice. Bone marrow macrophages (5x105 ml–1) from naïve, TLR4-competent ScSn control (solid bars), ScN (open bars), and ScCr (hatched bars) mice were cultured in the presence and/or in the absence of 25 x 105 ml–1 L. major promastigotes and IL-4 (20 U ml–1). After 48 h of culture, the macrophages were lysed, and the arginase activity was measured.

 
Impact of TLR4-mediated signaling on macrophage effector functions
The fate of the intracellular L. major parasites is dependent on the activities of iNOS and arginase in parasitized cells [10 , 49 ]. Therefore, it was determined whether the absence of TLR4 signaling influences the production of NO or the arginase activity in macrophages derived from ScSn, ScN, and ScCr mice. The results presented in Figure 2B show that NO production could not be detected in response to stimulation with LPS, LPS and L. major, or LPS and IFN-{gamma} by macrophages derived from the TLR mutant strains. Stimulation with LPS, IFN-{gamma}, and L. major resulted in a marked reduction of the NO production by the TLR4-deficient macrophages from ScN and ScCr mice as compared with those elicited in macrophages derived from TLR4-competent ScSn mice (Fig. 2B) . It is important to note that the NO pathway is not defective in macrophages from both TLR4-deficient strains of mice, as stimulation with IFN-{gamma}, TNF-{alpha}, and L. major resulted in a strong production of NO in all three strains of mice (Fig. 2B) .

Determination of the arginase activity showed that infection of macrophages from all three strains of mice with L. major parasites did not result in the induction of arginase (Fig. 2C) , whereas activation with IL-4 alone resulted in marginally higher arginase activity in macrophages from the TLR4-deficient mice (Fig. 2C) . It is interesting that activation of parasitized macrophages with IL-4 clearly resulted in the induction of a more pronounced arginase activity in macrophages from both strains of TLR4-deficient mice (Fig. 2C) . These results demonstrate that in the absence of TLR4-mediated signaling, alternatively activated L. major-infected macrophages express higher arginase activity, which could contribute to the enhanced parasite growth by promoting the polyamine synthesis.

Antigen-specific Th1 and Th2 cytokine production
It has been shown previously that ScCr mice display an impaired IFN-{gamma} response to bacteria and parasites [32 33 34 35 ]. To determine whether the absence of TLR4 signaling has an influence on the Th phenotype, we measured the production of antigen-specific IFN-{gamma} and IL-4 by the lymph node cells from all three strains of L. major-infected mice (Fig. 3 ). There was a slightly higher level of IFN-{gamma} produced by the cells from the ScN mice compared with the control ScSn mice (P>0.05). As expected, the lymph node cells from the ScCr mice produced low levels of IFN-{gamma} and high levels of IL-4. No IL-4 was detectable by ELISA in the supernatants from the ScSn and the ScN mice. These results suggest that in contrast to the defect in the IL-12Rß2 chain, the absence of TLR4 signaling does notinfluence the production of antigen-specific IFN-{gamma} or IL-4 by the lymph node cells.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 3. Antigen-specific production of IFN-{gamma} and IL-4. Groups of ScSn, ScN, and ScCr mice (n=4) were infected with 2 x 106 L. major in the footpads. Four weeks post infection, the lymph nodes draining the lesions were harvested and stimulated with L. major. Forty-eight hours later, the supernatants were harvested and tested for their content of cytokines by ELISA. The values represent the average of four individual mice per group ± SEM. Data show the results of one representative experiment out of two.

 
Histological analysis of the footpads from L. major-infected ScSn, ScN, and ScCr mice
To assess whether the L. major infection of the three strains of mice (ScSn, ScN, and ScCr) used in this study was associated with a different cell recruitment to the site of parasite inoculation, we investigated the lesions histologically 10 h postinfection and compared it with those of naïve mice. All uninfected animals showed normal histology of the footpads without any pathological, cellular infiltration (Fig. 4A , Table 1 ). Ten hours postinfection, a mild cellular infiltration was observed in the corium of the footpad from the ScSn mice, showing predominantly mononuclear cells (Fig. 4B , Table 1 ). In addition, in two out of five animals, a mild focal accumulation of granulocytes was observed. The footpad of the ScN mice showed in four out of five cases a marked granulocytic infiltration 10 h postinfection, which could be observed in the lower corium, often reaching until the muscular layer (Fig. 4C , Table 1 ). One animal developed only a moderate mononuclear infiltrate. The infected ScCr mice also showed a marked granulocytic infiltration, and two animals revealed a moderate infiltration of mononuclear cells (Table 1) . In addition to the histological analysis, cells infiltrating the local site of parasite inoculation were also determined by flow cytometry in the three strains of mice. Compared with the control-infected wild-type mice, there was a clearly higher infiltration of Mac1+ cells, which comprise granulocytes, macrophages, and myeloid-derived DCs and of NK cells into the footpad of ScN and ScCr mice (Table 2 ), confirming the histopathological results. These results show that the lack of TLR4 or TLR4 and IL-12Rß2 results in distinct cell recruitments at the site of infection, characterized by marked granulocytic infiltrates.



View larger version (127K):
[in this window]
[in a new window]
  		Figure 4. Histological analysis of the footpads from ScSn, ScN, and ScCr mice. Groups of ScSn, ScN, and ScCr mice (n=3–5) were infected with 2 x 106 L. major in the footpads. Ten hours later, the footpads were processed as described in Materials and Methods. (A) Footpad section from a naïve ScSn mouse. Hematoxylin and eosin (H&E): original primary magnification, x200. (B) Footpad section from L. major-infected ScSn mouse 10 h postinfection. H&E: original primary magnification, x200. A mild mixed infiltration of mononuclear cells and granulocytes is seen in the corium. (C) Footpad section from L. major-infected ScN mouse 10 h postinfection. H&E: original primary magnification, x200. A marked confluent infiltration of granulocytes can be seen in the corium.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Semiquantitative Histopathological Analysis of Footpads from Naive and L. major-Infected ScSn, ScN, and ScCr Mice

 

View this table:
[in this window]
[in a new window]
  		Table 2. Lack of TLR4 and TLR4/IL-12Rß2 Signaling Alters the Cellular Infiltration at the Site of Infection

 
TLR4 transgenic ScN (TCr5/ScN) mice recover their ability to control parasite replication efficiently
We have shown that ScN mice cannot control the replication of L. major parasites efficiently (Fig. 2) . To demonstrate that the impaired capacity to eliminate the parasites was a result of the absence of TLR4, we used TLR4 transgenic TCr5/ScN mice, which have an intact IL-12Rß2 as well as a complemented TLR4 trangene [39 ]. We determined the parasite load 24 h and 2 weeks postinfection. As presented in Figure 5 , there is a statistically significant higher parasite load in the lesions of ScN mice as compared with the Sn mice (an average of 1173 and 3901 viable parasite/lesion, respectively, P<0.05). Noticeably, the TLR4 transgenic ScN mice (TCr5/ScN) harbored a similar parasite burden as compared with the ScSn mice (average of 934 and 1173 viable parasites/lesion, respectively, P>0.05). Similar results were obtained 2 weeks postinfection: There was a 9.2-fold increase in the parasite load of the ScN mice, as compared with the wild-type ScSn mice (P<0.05); however, the number of viable parasites in the lesions of the TCr5/ScN was similar to that of the ScSn mice (an average 461,606 and 220,140, respectively; P>0.05). These results demonstrate that the absence of TLR4 in the ScN mice is responsible for their impaired capacity to control parasite replication efficiently.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 5. Parasite load in the footpads of ScSn, ScN, and TCr5/ScN mice. Groups of ScSn, ScN, and TCr5/ScN mice (n=4) were infected with 2 x 106 L. major in the footpads. At the time-points indicated, the numbers of viable parasites were determined in the infected footpads by LDA. The values represent the average of four individual mice ± SEM.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we used two different genetically resistant strains of mice which carry natural mutations in tlr4 as well as the tlr4 transgenic strain TCr5 to investigate the contribution of TLR4 to the course of cutaneous leishmaniasis in vivo. Our results show that genetically resistant C57BL/10ScCr mice, which carry two independent mutations, a null mutation in tlr4 and a point mutation in the IL-12Rß2 gene, develop nonhealing L. major infections, whereas the genetically closely related, TLR4-intact C57BL/10ScSn mice were able to clear the parasites in vivo. These results confirm and extend our previous work [32 ], which was published before it was shown that lps and tlr4 are identical [38 ] and before the second mutation in the IL-12Rß2 chain was recognized [36 , 37 ]. The crucial role of IL-12- and IL-12R-mediated events in the polarization of T cells into Th1 effector cells and in the control of cutaneous leishmaniasis is firmly established [43 , 46 , 50 , 51 ]. An intact, heterodimeric IL-12R, composed of the IL-12Rß1 and IL-12Rß2 chain, is required for the functioning of the bioactive form of IL-12 (IL-12p70), which consists of the IL-12p35 and the IL-12p40 subunit; the latter one is also used by IL-23 [52 ]. The IL-12Rß2 chain is the IL-12-specific component of the IL-12R; therefore, the point mutation in the IL-12Rß2 gene, resulting in a truncated IL-12Rß2 chain and an inability to respond to IL-12 [36 , 37 ], explains the nonhealing phenotype of the SnCr mice to L. major infections [45 , 46 , 53 , 54 ]. The unrestricted parasite growth in ScCr mice correlated with a polarized Th2 cytokine profile in these mice 4 weeks after L. major infection; lymph node cells draining the lesions produced IL-4 but little IFN-{gamma}. Thus, the results we obtained with L. major-infected SnCr mice confirm a role for IL-12 in the development of Th1 responses and in resistance to L. major infections. Similar results have been reported recently using L. major-infected IL-12Rß2 knockout (KO) mice on the C57BL/6 background; these mice developed large lesions, which ulcerated earlier than those of susceptible BALB/c mice, and displayed a Th2 cytokine profile and were also severely compromised in their ability to secrete IFN-{gamma}, a finding consistent with a lack of Th1 responses [51 ].

Although the findings discussed above confirm a role for IL-12 in L. major infections, they do not answer whether the lack of TLR4 affects L. major infections. To answer this question, we used the progenitor strain of the ScCr mice, the ScN mice, which also lack TLR4 but have a normal ability to respond to IL-12 [36 ]. In the present study, we show that the lack of TLR4 in IL-12Rß2 intact C57BL/10ScNCr mice resulted in an increased permissiveness for parasite growth during the innate and the adaptive phase of the immune response and in significantly delayed healing of the cutaneous lesions as compared with the TLR4-competent control mice. The enhanced intracellular survival and multiplication of Leishmania parasites in their main host cells, the macrophages, correlated with increased arginase activity and reduced NO synthesis in bone marrow macrophages from TLR4-deficient mice. It is well documented that the induction of the iNOS pathway and the synthesis of NO are essential for parasite killing [7 , 9 , 49 ]; thus, reduced NO levels will result in less-efficient parasite killing. In contrast, the growth of the intracellular parasites is dependent on the availability of polyamines, and arginase catalyses the hydrolysis of L-arginine into urea and L-ornithine, an amino acid essential for the synthesis of polyamines used for the proliferation of Leishmania parasites [10 , 11 ]. Several studies in mice indicate that the balance between arginase and iNOS represents an important mechanism in controlling macrophage function [12 , 55 , 56 ]. Based on the reduced synthesis of NO and the increased arginase activity in alternatively activated L. major-infected macrophages from TLR4-deficient mice, it is tempting to speculate that TLR4 signaling influences the regulation of the L-arginine metabolism in infected macrophages. We conclude from the increased parasite growth in the absence of TLR4 in L. major-infected mice that signaling through this innate recognition receptor contributes to the control of parasite growth in the early as well as in the chronic phase of L. major infection. The underlying molecular mechanisms are not yet identified and are a major focus of our current research. The ligand activating TLR4 after L. major infection has not yet been identified, and as TLR4 not only interacts with pathogen-associated PAMPs but can also recognize endogenous ligands such as heat shock proteins [57 ] and components of the extracellular matrix [58 ], infection-induced endogenous host factors could also play a role. Furthermore, it is also possible that cooperation between different TLRs or between TLR4 and parasite-specific receptors is required [59 , 60 ]. Independently of the nature of the ligand-activating TLR4 after L. major infection in vivo, the use of TLR4-transgenic TCr5 mice demonstrated unequivocally that TLR4 contributes to the efficient control of Leishmania growth in vivo.

Immediately after infection with L. major, a local inflammatory process is initiated, and the very early events after parasite entry, the recruitment of cells into the site of lesion, may be essential for the outcome of disease. Indeed, investigation of the early inflammatory response at the cutaneous site of infection in genetically resistant C57BL/6 and in susceptible BALB/c mice has shown that the early, inflammatory response in nonhealer mice is characterized by more pronounced features of acute inflammation and by a more pronounced recruitment of PMNs as well as by a higher proportion of infected PMNs [13 , 61 , 62 ]. The cellular recruitment to the site of parasite inoculation early after L. major infection was strikingly different between TLR4-competent controls and the two strains of TLR4 mutant mice used in the present study. The marked infiltrate of granulocytes in the lesions of TLR4-deficient and TLR4/IL-12Rß2 double-deficient mice could contribute to the observed differences in healing and parasites clearance. PMNs have been associated with efficient innate defense mechanisms in different experimental models of bacterial, fungal, and parasitic infections, and neutrophils are able to phagocytose Leishmania promastigotes [63 ]. Although PMNs are considered to be primary effector cells, which are involved in the first line of defense, they can also enhance parasite survival [62 , 63 ]. Neutrophils have a very short lifespan; they spend only 6–10 h in circulation before they die. It is interesting that internalization of living L. major parasites can delay spontaneous apoptosis of neutrophil granulocytes in vivo and extend the lifespan of these cells [13 , 18 ]. The delayed apopotosis of infected neutrophils could result in increased survival of the intracellular parasites in the first few days of infection. Macrophages, the main host cells of Leishmania, are not present at high numbers at the very early time of infection; they are recruited later into the lesions. Therefore, phagocytosis of Leishmania by granulocytes at the site of lesion, combined with the ability of Leishmania parasites to delay the apoptosis of infected PMNs [13 ], could represent an escape mechanism resulting in increased parasite survival in the initial phase of infection. Moreover, the uptake of apoptotic cells does not activate antimicrobial effector mechanism in macrophages [64 ]. Therefore, as suggested recently [13 ], it is possible that the uptake of Leishmania-infected neutrophils could help the parasites to enter the macrophages silently and avoid the activation of defense strategies in macrophages. Furthermore, a role for the early wave of infiltrating PMNs in the instruction of Th2 responses in BALB/c mice has been reported [62 ], and depletion of PMNs shortly before L. major infection of BALB/c mice prevented the early IL-4 burst, decreased Th2 responses, and resulted in partial inhibition of lesion progression and parasite growth [62 ]. Thus, the increased recruitment of PMNs to the site of parasite entry could contribute to the enhanced multiplication of Leishmania parasites in TLR4-deficient mice.

In contrast to the breadth of existing knowledge on the regulation and modulation of adaptive immune response to L. major, the understanding of the innate immune responses and the contribution of TLR-mediated functions to these responses are still very limited. Indirect evidence for a role of TLRs in L. major infections was obtained recently by using MyD88 KO mice on a genetically resistant C57BL/6 background. L. major activates IL-1{alpha} in macrophages through a MyD88-dependent pathway in vitro [65 ], and MyD88 KO mice failed to develop a protective response after L. major infection in vivo; they displayed large, cutaneous lesions and a polarized Th2 response, similarly to susceptible BALB/c mice [29 , 30 ]. These results show clearly that the MyD88 protein is crucial for the efficient clearance of L. major parasites and for the induction of protective Th1 responses [29 , 30 ], but they do not provide direct evidence that TLRs are activated in vivo after L. major infection. The cytoplasmic MyD88 adaptor protein is one of the key mediators of the signal transduction for all TLRs; however, it is not exclusive for TLR signaling pathways, and it is also common to IL-1R, IL-18, and IL-1R-associated protein kinase signaling pathways.

It is interesting that LPG, which has no counterpart in other eukaryotes, has recently been identified as a ligand for TLR2 [28 29 30 ]. LPG, a complex glycophospholipid, is one of the major surface molecules of Leishmania parasites [66 , 67 ] and contributes to the virulence of the parasites [68 , 69 ]. LPG expression is developmentally regulated, and stage-specific changes occur during metacyclogenesis, when the parasite differentiates from a procyclic promastigote to an infective metacyclic promastigote [70 71 72 ]. LPG from L. major has been shown in an in vitro transfection system to activate TLR2 signaling [30 ]. LPG is recognized by TLR2 expressed on human NK cells, and the LPG-TLR2 interaction results in an up-regulation of TLR2 message and cell-surface expression and the activation of NK cells to secrete IFN-{gamma} and TNF-{alpha} [28 ]. The extent of the NK cells activation was dependent on the developmental changes that occur in the carbohydrate residues of the phosphosaccharide repeat units of the LPG molecule during metacyclogenesis, and LPG purified from metacyclic promastigotes was a more potent stimulator than LPG from procyclic parasites [28 ].

In summary, our results presented here confirm the important role of IL-12R-mediated signals for the development of protective immunity, and furthermore, they identify TLR4 as a contributor in the complex host parasite interplay in the early phase of L. major infection. It is likely that complex organisms such as Leishmania parasites express several PAMPs and that a cooperation of different innate recognition receptors is required for the effective host defense against these eukaryotic parasites. The present study indicates that TLR4 is one of the innate recognition receptors that contributes to the innate response against L. major infection.


   ACKNOWLEDGEMENTS
 
The authors thank N. Nazari, R. Dorin, and A. Ross for their excellent technical assistance. This investigation received financial support from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR), The Wellcome Trust, The St. Mary’s Development Trust, and die Deutsche Forschungsgemeinschaft.

Received October 17, 2003; revised January 29, 2004; accepted February 9, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sacks, D., Sher, A. (2002) Evasion of innate immunity by parasitic protozoa Nat. Immunol. 3,1041-1047[CrossRef][Medline]
  2. Carvalho, E. M., Barral-Neto, M., Barral, A., Broskyn, C. I., Bacellar, O. (1994) Immunoregulation in Leishmaniasis Cienc. Cult. 46,441-445
  3. Turk, J. L., Bryceson, A. D. M. (1971) Immunological phenomena in leprosy and related diseases Adv. Immunol. 13,209-266[Medline]
  4. Sacks, D. L., Noben-Trauth, N. (2002) The immunology of susceptibility and resistance to Leishmania major in mice Nat. Rev. Immunol. 2,845-858[CrossRef][Medline]
  5. Reiner, S. L., Locksley, R. M. (1995) The regulation of immunity to Leishmania major Annu. Rev. Immunol. 13,151-177[CrossRef][Medline]
  6. Etges, R., Müller, I. (1998) Progressive disease or protective immunity to Leishmania major infection: the result of a network of stimulatory and inhibitory interactions J. Mol. Med. 76,372-390[CrossRef][Medline]
  7. Liew, F. Y., Millot, S., Parkinson, C., Palmer, R. M. J., Moncada, S. (1990) Macrophage killing of Leishmania parasite in vivo is mediated by nitric oxide from arginine J. Immunol. 144,4794-4797[Abstract]
  8. Green, S. J., Nacy, C. A., Meltzer, M. S. (1991) Cytokine-induced synthesis of nitrogen oxides in macrophages: a protective host response to Leishmania and other intracellular pathogens J. Leukoc. Biol. 50,93-103[Medline]
  9. Mauel, J., Ransijn, A., Buchmüller-Rouiller, Y. (1991) Killing of Leishmania parasites in activated murine macrophages is based on an L-arginine-dependent process that produces nitrogen derivatives J. Leukoc. Biol. 49,73-82[Abstract]
  10. Iniesta, V., Gomez-Nieto, L. C., Corraliza, I. (2001) The inhibition of arginase by N{omega}-hydroxy-L-arginine controls the growths of Leishmania inside macrophages J. Exp. Med. 193,777-783[Abstract/Free Full Text]
  11. Iniesta, V., Gomez-Nieto, L. C., Molano, I., Mohedano, A., Carcelen, J., Miron, C., Alonso, C., Corraliza, I. (2002) Arginase I induction in macrophages, triggered by Th2-type cytokines supports the growth of intracellular Leishmania parasites Parasite Immunol. 24,113-118[CrossRef][Medline]
  12. Munder, M., Eichmann, K., Modolell, M. (1998) Alternative metabolic states in murine macrophages reflected by the nitric oxide synthase/arginase balance: competitive regulation by CD4+ T cells correlates with Th1/Th2 phenotype J. Immunol. 160,5347-5354[Abstract/Free Full Text]
  13. Aga, E., Katschinski, D. M., van Zandbergen, G., Laufs, H., Hansen, B., Müller, K., Solbach, W., Laskay, T. (2002) Inhibition of spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major J. Immunol. 169,898-905[Abstract/Free Full Text]
  14. Bogdan, C., Donhauser, N., Döring, R., Röllinghoff, M., Diefenbach, A., Rittig, M. G. (2000) Fibroblasts as host cells in latent leishmaniasis J. Exp. Med. 191,2121-2129[Abstract/Free Full Text]
  15. Launois, P., Ohteki, T., Swihart, K., MacDonald, H. R., Louis, J. A. (1995) In susceptible mice, Leishmania major induce very rapid interleukin-4 production by CD4+ T cells which are NK1.1– Eur. J. Immunol. 25,3298-3307[Medline]
  16. Launois, P., Maillard, I., Pingel, S., Swihart, K. G., Xenarios, I., Acha-Orbea, H., Diggelmann, H., Locksley, R. M., MacDonald, R., Louis, J. A. (1997) IL-4 rapidly produced by Vß4V{alpha}8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice Immunity 6,541-549[CrossRef][Medline]
  17. Diefenbach, A., Schindler, H., Donhauser, N., Lorenz, E., Laskay, T., MacMicking, J., Röllinghoff, M., Gresser, I., Bogdan, C. (1998) Type 1 interferon (IFN{alpha}/ß) and type 2 nitric oxide synthase regulate the innate immune response to a protozoan parasite Immunity 8,77-87[CrossRef][Medline]
  18. Laskay, T., van Zandbergen, G., Solbach, W. (2003) Neutrophil granulocytes—Trojan horses for Leishmania major and other intracellular microbes? Trends Microbiol. 11,210-214[Medline]
  19. Medzhitov, R., Janeway, C. A. (1997) Innate immunity: the virtues of a nonclonal system of recognition Cell 91,295-298[CrossRef][Medline]
  20. Medzhitov, R., Janeway, C. (2000) The Toll receptor family and microbial recognition Trends Microbiol. 8,452-456[CrossRef][Medline]
  21. Akira, A., Takeda, K., Kaisho, T. (2001) Toll-like receptors: critical proteins linking innate and acquired immunity Nat. Immunol. 2,675-680[CrossRef][Medline]
  22. Underhill, D. M. (2003) Toll-like receptors: networking for success Eur. J. Immunol. 33,1767-1775[CrossRef][Medline]
  23. Kaisho, T., Akira, S. (2001) Dendritic cell function in Toll-like receptor and MyD88 knockout mice Trends Immunol. 22,78-83[CrossRef][Medline]
  24. Rock, F. L., Hardiman, G., Timans, J. C., Kastelein, R. A., Bazan, J. F. (1998) A family of human receptors structurally related to Drosophila Toll Proc. Natl. Acad. Sci. USA 95,588-593[Abstract/Free Full Text]
  25. Xu, Y., Tao, X., Shen, B., Horng, T., Medzhitov, R., Manley, J. L., Tong, L. (2000) Structural basis for signal transduction by the Toll/interleukin-1 receptor domains Nature 408,111-115[CrossRef][Medline]
  26. Medzhitov, R., Preston-Hurlburt, P., Kopp, E., Stadlen, A., Chen, C., Ghosh, S., Janeway, C. (1998) MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways Mol. Cell 2,253-258[CrossRef][Medline]
  27. Campos, M. A. S., Almeida, I. C., Takeuchi, O., Akira, S., Valente, E. P., Procopio, D. O., Travassos, L. R., Smith, J. A., Golenbock, D. T., Gazzinelli, R. T. (2001) Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite J. Immunol. 167,416-423[Abstract/Free Full Text]
  28. Becker, I., Salaiza, N., Aguirre, M., Delgado, J., Carrillo-Carrasco, N., Gutierres-Kobeh, L., Ruiz, A., Cervantes, R., Perez-Torres, A., Cabrera, N. G. A. M. C., Isibasi, A. (2003) Leishmania lipophosphoglycan (LPG) activates NK cells through Toll-like receptor 2 Mol. Biochem. Parasitol. 130,65-74[CrossRef][Medline]
  29. Muraille, E., De Trez, C., Brait, M., De Baetselier, P., Leo, O., Carlier, Y. (2003) Genetically resistant mice lacking MyD88-adapter protein display a high susceptiblity to Leishmania major infection associated with a polarized Th2 response J. Immunol. 170,4237-4241[Abstract/Free Full Text]
  30. de Veer, M. J., Curtis, J. M., Baldwin, T. M., DiDonato, J. A., Sexton, A., McConville, M. J., Handman, E., Schofield, L. (2003) MyD88 is essential for clearance of Leishmania major: possible role for lipophosphoglycan and Toll-like rececptor 2 signaling Eur. J. Immunol. 33,2822-2831[CrossRef][Medline]
  31. Poltorak, A., Smirnova, I., Clisch, R., Beutler, B. (2000) Limits of a deletion spanning Tlr4 in C57BL/10ScCr mice J. Endotoxin Res. 6,51-56[Medline]
  32. Müller, I., Freudenberg, M., Kropf, P., Kiderlen, A., Galanos, C. (1997) Leishmania major infection in C57BL/10 mice differing at the Lps locus: a new non-healing phenotype Med. Microbiol. Immunol. (Berl.) 186,75-81[CrossRef][Medline]
  33. Katschinski, T., Galanos, C., Coumbos, A., Freudenberg, M. A. (1992) {gamma} Interferon mediates Propionibacterium acnes-induced hypersensitivity to lipopolysaccharide in mice Infect. Immun. 60,1994-2001[Abstract/Free Full Text]
  34. Yaegashi, Y., Nielsen, P., Sing, A., Galanos, C., Freudenberg, M. A. (1995) Interferon ß, a cofactor in the interferon {gamma} production induced by gram-negative bacteria in mice J. Exp. Med. 181,953-960[Abstract/Free Full Text]
  35. Freudenberg, M. A., Kumazawa, Y., Meding, S., Langhorne, J., Galanos, C. (1991) {gamma} Interferon production in endotoxin-responder and -nonresponder mice during infection Infect. Immun. 59,3484-3491[Abstract/Free Full Text]
  36. Merlin, T., Sing, A., Nielsen, P. J., Galanos, C., Freudenberg, M. A. (2001) Inherited IL-12 unresponsiveness contributes to the high LPS resistance of the Lpsd C57BL/10ScCr mouse J. Immunol. 166,566-573[Abstract/Free Full Text]
  37. Poltorak, A., Merlin, T., Nielsen, P. J., Sandra, O., Smirnova, I., Schupp, I., Boehm, T., Galanos, C., Freudenberg, M. A. (2001) A point mutation in the IL-12Rß2 gene underlies the IL-12 unresponsiveness of Lps-defective C57BL/10ScCr mice J. Immunol. 167,2106-2111[Abstract/Free Full Text]
  38. Poltorak, A., He, X., Smirnova, I., Liu, M-Y., van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B., Beutler, B. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene Science 282,2085-2088[Abstract/Free Full Text]
  39. Kalis, C., Kanzler, B., Lembo, A., Poltorak, A., Galanos, C., Freudenberg, M. A. (2003) Toll-like receptor 4 expression levels determine the degree of LPS-susceptibility in mice Eur. J. Immunol. 33,798-805[CrossRef][Medline]
  40. Kropf, P., Brunson, K., Etges, R., Müller, I. (1998) The Leishmaniasis model Immunology of Infection 25,419-458 Academic San Diego, CA.
  41. Titus, R. G., Marchand, M., Boon, T., Louis, J. A. (1985) A limiting dilution assay for quantifying Leishmania major in tissues of infected mice Parasite Immunol. 7,545-555[Medline]
  42. Ding, A. H., Nathan, C. F., Stuehr, D. J. (1988) Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production J. Immunol. 141,2407-2412[Abstract]
  43. Sypek, J. P., Chung, C. L., Mayor, S. E. H., Subramanyam, J. M., Goldman, S. J., Sieburth, D. S., Wolf, S. F., Schaub, R. G. (1993) Resolution of cutaneous leishmaniasis: interleukin 12 initiates a protective T helper type 1 immune response J. Exp. Med. 177,1797-1802[Abstract/Free Full Text]
  44. Heinzel, F. P., Schoenhaut, D. S., Rerko, R. M., Rosser, L. E., Gately, M. K. (1993) Recombinant interleukin 12 cures mice infected with Leishmania major J. Exp. Med. 177,1505-1509[Abstract/Free Full Text]
  45. Mattner, F., Magram, J., Ferrante, J., Launois, P., Di Padova, K., Behin, R., Gately, M. K., Louis, J. A., Alber, G. (1996) Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response Eur. J. Immunol. 26,1553-1559[Medline]
  46. Mattner, F., Di Padova, K., Alber, G. (1997) Interleukin-12 is indispensable for protective immunity against Leishmania major Infect. Immun. 65,4378-4383[Abstract]
  47. Lima, H. C., Bleyenberg, J. A., Titus, R. G. (1997) A simple method for quantifying Leishmania in tissues of infected animals Parasitol. Today 13,80-82[CrossRef][Medline]
  48. Kropf, P., Freudenberg, M. A., Modolell, M., Price, H. P., Herath, S., Antoniazi, S., Galanos, C., Smith, D. F., Müller, I. (2004) Toll-like receptor 4 contributes to the efficient control of infection with the protozoan parasite Leishmania major Infect. Immun. 72,1920-1922[Abstract/Free Full Text]
  49. Wei, X., Charles, I. G., Smith, A., Ure, J., Feng, G., Huang, F., Xu, D., Müller, W., Moncada, S., Liew, F. Y. (1995) Altered immune responses in mice lacking inducible nitric oxide synthase Nature 375,408-411[CrossRef][Medline]
  50. Kropf, P., Etges, R., Schopf, L., Chung, C., Sypek, J., Müller, I. (1997) Characterization of T cell-mediated responses in nonhealing and healing Leishmania major infections in the absence of endogenous interleukin-4 J. Immunol. 159,3434-3443[Abstract]
  51. Chakir, H., Campos-Neto, A., Mojibian, M., Webb, J. R. (2003) IL-12Rß2-deficient mice of a genetically resistant background are susceptible to Leishmania major infecton and develop a parasite-specific Th2 response Microbes Infect. 5,241-249[CrossRef][Medline]
  52. Oppmann, B., Lesley, R., Blom, B., Timans, J. C., Xu, Y., Hunte, B., Vega, F., Yu, N., Wnag, J., Singh, K., Zonin, F., Vaisberg, E., Churakova, T., Liu, M., Gorman, D., Wagner, J., Zurawski, S., Liu, Y-J., Abrams, J. S., Moore, K. W., Rennick, D. (2000) Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distant from IL-12 Immunity 13,715-725[CrossRef][Medline]
  53. Hsieh, C-S., Macatonia, S. E., Tripp, C. S., Wolf, S. F., O’Garra, A., Murphy, K. M. (1993) Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages Science 260,547-549[Abstract/Free Full Text]
  54. Germann, T., Gately, M. K., Schoenhaut, D. S., Lohoff, M., Mattner, F., Jin, S-C., Schmitt, E., Rüde, E. (1993) Interleukin-12/T cell stimulating factor, a cytokine with multiple effects on T helper type 1 (Th1) but not on Th2 cells Eur. J. Immunol. 23,1762-1770[Medline]
  55. Gordon, S. (2003) Alternative activation of macrophages Nat. Rev. Immunol. 3,23-35[CrossRef][Medline]
  56. Camargo, P. E., Coelho, J. A., Moraes, G., Figueiredo, E. N. (1978) Trypanosoma spp., Leishmania spp. and Leptomonas spp.: enzymes of ornithine-arginine metabolism Exp. Parasitol. 46,141-144[CrossRef][Medline]
  57. Ohashi, K., Burkart, V., Flohe, S., Kolb, H. (2000) Heat shock protein 60 is a putative endogenous ligand of the Toll-like receptor-4 complex J. Immunol. 164,558-561[Abstract/Free Full Text]
  58. Termeer, C., Bendix, F., Sleemann, K., Fieber, C., Voith, U., Ahrens, T., Miyake, K., Freudenberg, M., Galanos, C., Simon, J. C. (2002) Oligosaccharides of hyaluronan activates dendritic cells via Toll-like receptor 4 J. Exp. Med. 195,99-111[Abstract/Free Full Text]
  59. Brown, G. D., Herre, J., Williams, D. L., Willment, J. A., Marshall, A. S. J., Gordon, S. (2003) Dectin-1 mediates biological effects of ß-glucans J. Exp. Med. 197,1119-1124[Abstract/Free Full Text]
  60. Gantner, B., Simmons, R. M., Çanavera, S. J., Akira, S., Underhill, D. M. (2003) Collaborative induction of inflammatory response by dectin-1 and Toll-like receptor 2 J. Exp. Med. 197,1107-1117[Abstract/Free Full Text]
  61. Beil, W. J., Meinardus-Hager, G., Neugebauer, D. C., Sorg, C. (1992) Differences in the onset of the inflammatory response to cutaneous leishmaniasis in resistant and suceptible mice J. Leukoc. Biol. 52,135-142[Abstract]
  62. Tacchini-Cottier, F., Zweifel, C., Belkaid, Y., Mukankundiye, C., Vasei, M., Launois, P., Milon, G., Louis, J. A. (2000) An immunomodulatory function for neutrophils during the induction of a CD4+ Th2 response in BALB/c mice infected with Leishmania major J. Immunol. 165,2628-2636[Abstract/Free Full Text]
  63. Laufs, H., Müller, K., Fleischer, J., Reiling, N., Jahnke, N. J. J. C., Solbach, W., Laskay, T. (2002) Intracellular survival of Leishmania major in neutrophil granulocytes after uptake in the absence of heat-labile serum factors Infect. Immun. 70,826-835[Abstract/Free Full Text]
  64. Aderem, A., Underhill, D. M. (1999) Mechanisms of phagocytosis in macrophages Annu. Rev. Immunol. 17,593-623[CrossRef][Medline]
  65. Hawn, T. R., Ozinsky, A., Underhill, D. M., Buckner, F. S., Akira, S., Aderem, A. (2002) Leishmania major activates IL-1{alpha} expression in macrophages through a MyD88-dependent pathway Microbes Infect. 4,763-771[CrossRef][Medline]
  66. Turco, S. J. (1988) The lipophosphoglycan of Leishmania Parasitol. Today 4,255-257
  67. Turco, S., Descoteaux, A. (1992) The lipophosphoglycan of Leishmania parasites Annu. Rev. Microbiol. 46,65-94[CrossRef][Medline]
  68. Sacks, D. L., Modi, G., Rowton, E., Späth, G., Epstein, L., Turco, S. J., Beverley, S. M. (2000) The role of phosphoglycans in Leishmania-sand fly interactions Proc. Natl. Acad. Sci. USA 97,406-411[Abstract/Free Full Text]
  69. Späth, G. F., Lye, L-F., Segawa, H., Sacks, D. L., Turco, S. J., Beverely, S. M. (2003) Persistence without pathoglogy in phosphoglycan-deficient Leishmania major Science 301,1241-1243[Abstract/Free Full Text]
  70. Sacks, D. L., Hieny, S., Sher, A. (1985) Identification of cell surface carbohydrate and antigenic changes between noninfective and infective developmental stages of Leishmania major promastigotes J. Immunol. 135,564-569[Abstract]
  71. Sacks, D. L., Da Silva, R. P. (1987) The generation of infective stage Leishmania major promastigotes is associated with the cell-surface expression and release of a developmentally regulated glycolipid J. Immunol. 139,3099-3106[Abstract]
  72. DaSilva, R., Sacks, D. L. (1987) Metacyclogenesis is a major determinant of Leishmania promastigote virulence and attenuation Infect. Immun. 55,2802-2806[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Infect. Immun.Home page
F. F. Tuon, V. S. Amato, H. A. Bacha, T. AlMusawi, M. I. Duarte, and V. Amato Neto
Toll-Like Receptors and Leishmaniasis
Infect. Immun., March 1, 2008; 76(3): 866 - 872.
[Full Text] [PDF]

Home page
 J. Immunol.Home page
F. L. Ribeiro-Gomes, M. C. A. Moniz-de-Souza, M. S. Alexandre-Moreira, W. B. Dias, M. F. Lopes, M. P. Nunes, G. Lungarella, and G. A. DosReis
Neutrophils Activate Macrophages for Intracellular Killing of Leishmania major through Recruitment of TLR4 by Neutrophil Elastase
J. Immunol., September 15, 2007; 179(6): 3988 - 3994.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
S. Divanovic, A. Trompette, L. K. Petiniot, J. L. Allen, L. M. Flick, Y. Belkaid, R. Madan, J. J. Haky, and C. L. Karp
Regulation of TLR4 signaling and the host interface with pathogens and danger: the role of RP105
J. Leukoc. Biol., August 1, 2007; 82(2): 265 - 271.
[Abstract] [Full Text]</